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Introduction

Diabetic retinopathy (DR), an ocular microvascular complication of diabetes, is a leading cause of visual impairment and blindness among working-age individuals.¹ An estimated 103 million individuals are currently living with DR worldwide, and this number is projected to rise to 160 million by 2045, driven by an aging population and improved survival rates among individuals with diabetes.² Between 1990 and 2015, DR-related blindness increased from 200,000 to 400,000 cases and visual impairment from 1.4 million to 2.6 million, reflecting a steady increase in global prevalence that poses a critical public health challenge.³ Treatment decisions for DR are often complex, as patients face a variety of options, including retinal laser photocoagulation, anti-vascular endothelial growth factor (anti-VEGF) drugs, corticosteroids, and pars plana vitrectomy.⁴ These treatment options differ in their efficacy, risks, economic burden, and impact on daily life.⁵ Improper decision-making may lead to severe consequences, including significant vision loss, irreversible blindness, and other serious secondary complications.^5,6 Evaluating these complex, high-stakes trade-offs presents significant challenges to decision-making for patients with DR.

The World Health Organization (WHO) advocates for patient involvement in clinical decision-making to uphold patients’ rights to participate in their treatment plans and maximize treatment benefits.⁷ Previous studies have demonstrated that patient participation in treatment decision-making can not only improve treatment adherence and reduce medical visits but also enhance the doctor-patient relationship and improve health outcomes.^8,9 Therefore, involving DR patients and incorporating their preferences is essential. However, little is known about patient involvement in DR treatment decisions, especially in the context of the Chinese healthcare system. Compared to many Western countries, Chinese medical culture has traditionally been characterized by a physician-centered model, in which physicians are regarded as authorities and patients often adopt a passive role, with compliance considered optimal.¹⁰ Thus, a systematic exploration of the factors influencing patient involvement in DR decision-making in China is warranted to enhance clinical decision quality.

Previous studies suggest that factors such as age, gender, economic status, educational level, health literacy, and social support may influence participation in decision-making.^11,12 However, several factors remain controversial. Two studies found that older patients tended to play a passive role in treatment decision-making,^13,14 whereas one study reported no significant age-related effects.¹⁵ One study found that female patients, compared to men, reported a greater preference for a collaborative role and a lesser preference for a passive role in decision-making,¹⁶ while another study demonstrated the opposite conclusion.¹⁷ A study indicated that high social support was associated with increased patient participation in surgical decision-making.¹⁸ In contrast, a qualitative study revealed that family support, a component of social support, sometimes hindered patient involvement and, in some cases, led to family members making decisions on the patient’s behalf.¹⁹ These contradictory findings underscore the necessity for a more systematic and theoretically grounded approach to understanding patient involvement.

To fully capture the influencing factors, a comprehensive theoretical framework is essential. The Capability, Opportunity, Motivation – Behavior (COM-B) model offers such a framework.²⁰ Widely recognized for its comprehensive and systematic approach, the COM-B model has been extensively applied to understand a range of patient health behaviors.^21–23 The model posits that an individual’s behavior is a result of their capability, opportunity, and motivation, with capability and opportunity also affecting behavior both directly and indirectly through their impact on motivation.²⁰

Through literature review and group discussions, we identified a set of potential determinants which were then mapped onto the COM-B framework. Capability is defined as an individual’s psychological and physical ability to perform a behavior. Health literacy reflects the patient’s ability to access, comprehend, and utilize health information.²⁴ This ability allows patients to understand their treatment options, evaluate risks and benefits, and communicate their preferences meaningfully. In this study, capability was conceptualized as health literacy. Opportunity encompasses physical and social factors that facilitate or prompt a behavior. After seeking medical attention, physicians often serve as the primary source of medical information for patients in China.²⁵ Research has shown that physician support is a crucial facilitator for patient involvement in treatment decisions.²⁶ Furthermore, social support from interpersonal networks, including family members and friends, can enhance patients’ psychological resilience and mitigate decision-making pressure.²⁷ For this study, we conceptualized ophthalmologist facilitation of patient involvement and social support as opportunity. Motivation refers to the internal brain processes that energize and direct behavior, including both reflective and automatic mechanisms. Patients with higher decision self-efficacy are more likely to seek information, express their preferences, and play a more engaged role in the treatment process.²⁸ Additionally, the need for decision-making involvement reflects patients’ intrinsic desire or preference for participation. In this study, we measured motivation through decision self-efficacy and the need for decision-making involvement. Figure 1 illustrates the conceptual framework that guided our study.

Given that constructs such as health literacy, social support, and need for decision-making involvement are complex and multifaceted, we conducted our analysis on their respective sub-dimensions. For example, we analyzed the functional, communicative, and critical sub-dimensions of health literacy. This approach enabled us to more comprehensively understand how different layers of capability, opportunity, and motivation collectively influenced patient decision-making behavior. The aims of this study were to investigate the current status of actual involvement roles in treatment decision-making among patients with DR and to analyze the influencing factors. The research results will provide a valuable reference for developing measures to promote patient involvement in decision-making.

Method

Study Design and Participants

This cross-sectional study was conducted at the ophthalmology center of a large public hospital in Shanghai, China, from August 2024 to January 2025. The institution serves as a regional referral center for a broad geographic area encompassing Shanghai municipality and the surrounding provinces of Jiangsu, Zhejiang, and Anhui. The study participants were recruited using a convenience sampling method. Participants meeting the following criteria were included: (1) age 18 years and above; (2) diagnosed with DR stages III to VI; (3) voluntary participation in the study and signed informed consent. Patients with mental illness, intellectual disability, or verbal communication disorders, as well as severe cardiac, hepatic, or renal dysfunction, respiratory failure, or critical illness, were excluded from the study. According to the Kendall sample size estimation method, which is calculated based on the principle that the sample size should be at least 5 to 10 times the number of variables.²⁹ Through a literature review, this study included a total of 24 predictive influencing variables, comprising 13 sociodemographic and disease characteristics and 11 variables from five scales. Considering a 10% attrition rate, the calculated total sample size of this study ranged from 134 to 267 cases. Ultimately, the study obtained 336 valid samples. This study was reported using the STROBE guidelines.

Ethical Considerations

This study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was granted by the Ethics Review Committee of Shanghai General Hospital (Approval No. 2024–098). All participants provided written informed consent and retained the right to withdraw at any time. All data were collected and analyzed anonymously to guarantee confidentiality.

Measurements

Sociodemographic and Clinical Information

The questionnaire was designed based on a literature review and consultation with ophthalmology experts. It includes the following information: gender, age, marital status, educational level, monthly per capita household income, method of healthcare payment, duration of disease diagnosis, DR stage, and comorbidities.

Control Preference Scale (CPS)

The CPS is used to assess actual roles in treatment decision-making among patients with DR. The scale was originally developed by Degner and later adapted and revised by Nolan.³⁰ Chinese scholar Xu Xiaolin and colleagues translated and revised the scale, and the Cronbach’s α coefficient of the Chinese version is 0.899.³¹ The CPS is a unidimensional scale consisting of five options to characterize the types of patient involvement in treatment decision-making. Options 1 and 2 represent the active type, option 3 represents the collaborative type, and options 4 and 5 represent the passive type.

All Aspects of Health Literacy Scale (AAHLS)

The AAHLS is used to assess patients’ health literacy levels. The scale was developed by Chinn in 2013,²⁴ and translated and revised by Wu in 2016.³² It consists of 11 items across three dimensions: functional health literacy, communicative health literacy, and critical health literacy. Each item is scored on a 3-point Likert scale (1 = rarely, 2 = sometimes, 3 = often). The total score ranges from 11 to 33, with higher values indicating greater health literacy. In this study, the Cronbach’s α coefficient of the scale was 0.834.

Social Support Rating Scale (SSRS)

The SSRS, developed by Xiao, is used to assess patients’ social support levels.³³ It includes 10 items across three dimensions: objective support, subjective support, and utilization of social support. The total score ranges from 8 to 44, with higher scores indicating higher social support. In this study, the Cronbach’s α coefficient of the scale was 0.800.

Facilitation of Patient Involvement Scale (FPIS)

The FPIS is used to measure the extent to which patients perceive that their healthcare professionals involve them in their healthcare, developed by Martin.³⁴ The Chinese version was translated and revised by Wu in 2015.³² It is a unidimensional scale with nine items. Each item is scored on a 6-point Likert scale ranging from 1 (never) to 6 (always). The scale yields a total score ranging from 9 to 54, with higher values reflecting greater healthcare providers’ facilitation of patient involvement in treatment decisions. In this study, the Cronbach’s α coefficient of the scale was 0.830.

Decision Self-Efficacy Scale (DSES)

The DSES is used to assess patients’ confidence in making treatment decisions for themselves and was developed by O’Conner.³⁵ It is a unidimensional scale comprising eleven items, each rated on a 5-point Likert scale from 0 (not at all confident) to 4 (very confident). The total score is calculated by averaging the sum of 11 items and then multiplying by 25 to convert it to a 0–100 scale. Higher scores reflect greater patient self-efficacy in treatment decision-making. In this study, the Cronbach’s α coefficient of the scale was 0.864.

Patient Expectation for Participation in Medical Decision‐Making Scale (PEPMDS)

The PEPMDS, developed by Xu, is used to assess patients’ need for participation in treatment decisions.³⁶ This scale consists of 12 items in three dimensions: need for information, need for deliberation, and need for decisional control. Items are rated on a 5-point Likert scale (1 = strongly disagree to 5 = strongly agree). The total score ranges from 12 to 60, with higher scores indicating greater patient need for involvement in treatment decisions. In this study, the Cronbach’s α coefficient of the scale was 0.846.

Data Collection

Before conducting the questionnaire survey, all research team members received standardized training to ensure the consistency and uniformity of terminology and procedures in the survey. During the distribution of the questionnaires, researchers provided participants who met the inclusion criteria with a detailed explanation of the study’s purpose, content, and procedures, and obtained their written informed consent. Participants’ medical information was collected via the hospital’s electronic medical record system. Questionnaires were distributed and collected on-site, with immediate checks to ensure the completeness and accuracy of data collection. For participants who were unable to complete the questionnaire independently due to visual impairment, the researchers administered the questionnaire via dictation based on their verbal responses.

Statistical Analysis

Data were entered independently by two investigators using EpiData 3.1 and exported to SPSS 26.0 for analysis after verification. Descriptive analyses were performed for all included variables. Frequencies and percentages were used for categorical variables, means and standard deviations (M±SD) were used for normally distributed continuous variables, and medians and interquartile ranges (IQR) were used for non-normally distributed continuous variables. The χ² test was used to analyze the differences in the types of patient involvement in treatment decision-making between categorical variables. One-way ANOVA or the non-parametric Kruskal–Wallis H-test was used to analyze continuous variables. Variables with a P<0.05 in univariate analysis were subsequently included in the unordered multinomial logistic regression analysis to determine independent factors associated with the types of patient involvement in treatment decision-making. The Variance Inflation Factor (VIF) was used to analyze whether the variables in the model have multicollinearity. A two-sided test was used, and a P-value <0.05 was considered statistically significant.

Results

Participant Characteristics

A total of 362 questionnaires were distributed in this study. Following the exclusion of 26 unqualified questionnaires, 336 valid questionnaires were collected, yielding an effective response rate of 92.8%. The mean age of the participants was 53.74±13.01 years. Among the participants, 52.1% were male, and 40.2% had attained a junior high school education or below. The largest proportion of patients was diagnosed with DR Stage IV, accounting for 47.3%. Patients who received retinal photocoagulation accounted for the largest treatment group, at 30.0%. Furthermore, 56.5% of patients presented with ocular comorbidities, and 62.5% had systemic comorbidities. The sociodemographic and clinical characteristics of the patients are shown in Table 1.

Table 1 Comparison of Sociodemographic and Disease Characteristics Among Patients with Diabetic Retinopathy in Different Decision-Making Involvement Roles (N = 336)

Descriptive Statistics of Patient Involvement in Treatment Decision-Making for Diabetic Retinopathy

Regarding actual patient involvement in treatment decision-making roles, 21.1% of patients reported being active, 30.7% reported being collaborative, and the largest proportion, 48.2%, reported being passive.

Univariate Analysis

The mean scores for the 336 patients with DR were AAHLS (24.84±3.53), SSRS (43.59±5.51), FPIS (40.39±4.78), DSES (72.03±11.44), and PEPMDS (47.89±5.39). Univariate analysis revealed significant differences in actual involvement in decision-making roles among patients with DR across several factors, including: age, educational level, monthly per capita household income, health literacy, social support, ophthalmologist facilitation of patient involvement, decision self-efficacy, and need for decision-making involvement (P<0.05). See Table 1 and Table 2.

Table 2 Comparison of COM-B Factors Among Patients with Diabetic Retinopathy in Different Decision-Making Involvement Roles (N = 336)

Multinomial Logistic Regression Analysis

An unordered multinomial logistic regression analysis was conducted to identify the independent factors influencing patient involvement in treatment decision-making for diabetic retinopathy. Prior to conducting the multinomial logistic regression, multicollinearity was assessed among all independent variables. The aggregate variables social support and need for decision-making involvement demonstrated perfect collinearity with their respective sub-dimensional variables, as indicated by a tolerance value of 0.000. The variable health literacy also exhibited significant collinearity, with a VIF of 11.381. Since the simultaneous inclusion of these aggregate variables and their subdimensions would have rendered the model unstable and statistically un-fittable, they were excluded in accordance with established statistical principles. Consequently, only the sub-dimensional variables were retained in the final model. This approach ensured the robustness of the model while enabling evaluation of the impact of specific components within each theoretical construct on decision-making behavior. After refitting, all remaining variables had VIF values below 4, indicating no substantial multicollinearity. The following variables were retained and subsequently entered into the multinomial logistic regression analysis: age, educational level, monthly per capita household income, functional health literacy, critical health literacy, objective support, subjective support, utilization of social support, need for information, need for deliberation, need for decisional control, ophthalmologist facilitation of patient involvement, and decision self-efficacy. The assigned values of the included independent variables are shown in Table 3.

Table 3 Assignment of Independent Variables

The likelihood ratio test indicated that the final model provided a significantly better fit than a null model (χ² = 272.002, df = 32, P < 0.001). This conclusion was bolstered by the pseudo R² values (Cox & Snell R² = 0.555; Nagelkerke R² = 0.634), which collectively suggest that the model captures a substantial proportion of the outcome’s variability and signals a good overall fit. The results identified age, monthly per capita household income, critical health literacy, ophthalmologist facilitation of patient involvement, objective support, and need for deliberation as significant independent predictors of patient decision-making roles. Compared to passive roles, patients adopting active roles tended to be younger, have higher income, report lower ophthalmologist facilitation, and express higher need for deliberation. Conversely, compared to passive roles, collaborative roles were associated with higher income, greater critical health literacy, stronger ophthalmologist facilitation, and elevated need for deliberation. Furthermore, when collaborative roles were compared to active roles, patients adopting the former were significantly older, reported higher objective support, and experienced greater ophthalmologist facilitation. The detailed results are shown in Table 4.

Table 4 Multinomial Logistic Regression Analysis of Factors Influencing the Actual Involvement in Decision-Making Roles Among Patients with Diabetic Retinopathy (n=336)

Discussion

To our knowledge, this is the first study to examine actual treatment decision-making involvement among Chinese patients with DR and explore its influencing factors using the COM-B framework. Overall, the passive role was the most common pattern observed. Furthermore, our analysis identified several key factors significantly influencing patient involvement: age, monthly per capita household income, critical health literacy, ophthalmologist facilitation of patient involvement, objective support, and need for deliberation.

Current Status of Patient Involvement in Treatment Decision-Making for Diabetic Retinopathy

The results revealed that 48.2% of patients reported passive roles, 30.7% reported collaborative roles, and 21.1% reported active roles. The proportion of passive role was relatively higher in patients with DR than in those with inflammatory bowel disease (32.12%),³⁷ gynecologic cancer (29.9%),³⁸ or atrial fibrillation (40.3%).²⁶ These differences may be attributed to distinct study populations and research settings. Specifically, DR has one of the lowest levels of public awareness compared with other ophthalmic diseases, which is likely due to the high professional barriers inherent in its diagnosis and treatment.³⁹ This low awareness is evidenced by a Chinese study where only 1.2% of diabetic patients could correctly identify symptoms of DR.⁴⁰ Furthermore, the combination of diverse treatment options, prognostic uncertainty, and the older demographics of the patient cohort may collectively impair patients’ understanding of the disease and therapeutic alternatives, thus predisposing them to passive decision-making.

Capability and Patient Involvement

Previous studies have pointed out that health literacy, encompassing various dimensions, is necessary for patient involvement in decision-making.^41,42 Successful patient involvement is predicated on patients possessing practical communication skills, the ability to acquire, comprehend, and communicate relevant information about disease and treatment from healthcare professionals, and critical evaluation skills.⁴¹ However, few studies have explored the distinct impacts of these different health literacy dimensions on patients’ actual involvement in decision-making. Our findings indicate that patients with higher critical health literacy are more likely to adopt collaborative decision-making roles. This result is consistent with a study conducted in the Netherlands, which found that critical health literacy was more important for patient participation than its functional and communicative counterparts.⁴³ In contrast, a French study reported a positive correlation for functional and communicative health literacy with patient involvement but no association for critical health literacy.⁴⁴ This discrepancy may be attributable to the relatively homogeneous scores for functional and communicative health literacy in our cohort, which exhibited limited variability. Our research suggests that the ability to simply acquire information and communicate with healthcare professionals is insufficient for meaningful participation in medical decision-making. Therefore, future interventions should focus on enhancing the critical health literacy of patients with DR, empowering them to analyze, evaluate, and question health information to make informed decisions that align with their personal values and preferences.

Opportunity and Patient Involvement

Our study revealed that increased ophthalmologist facilitation is significantly associated with patients adopting a collaborative role over either a passive or an active one. This suggests that when ophthalmologists proactively provide information, encourage questions, and respect patient concerns, they effectively bridge the inherent information and power asymmetry. Such empowerment fosters a climate of equitable dialogue, promoting patient engagement to collaborate rather than simply shifting the decision-making burden onto them. This finding aligns with a qualitative meta-summary which highlighted that clear information delivery, active listening, and trust-building are critical facilitators for patient engagement.⁴⁵ Furthermore, our results showed that greater ophthalmologist facilitation decreased the likelihood of an active role. A potential explanation is that highly facilitative ophthalmologists build a strong foundation of trust, engendering a sense of security and understanding in patients. Consequently, the perceived need for patients to assume a solely autonomous, active role diminishes. This perspective is supported by Kraetschmer’s research, which established that an active role is associated with low trust, a passive role with blind trust, and a collaborative role with high but not excessive trust.⁴⁶ Conversely, when patients perceive a lack of support or transparency, their trust may diminish, compelling them to adopt an active, patient-dominated role as a compensatory strategy to regain a sense of control.⁴⁷ Physician support, therefore, bridges the information and power gap, enabling true shared decision-making rather than pushing patients toward extremes.⁴⁸ This is further corroborated by a cross-sectional study which showed that physicians’ facilitative communication predicted greater patient participation.⁴⁹ Consequently, future interventions should focus on implementing training programs for ophthalmologists to enhance communication skills, integrate appropriate decision aids into practice, and foster patient empowerment and collaboration.

Visual impairment caused by DR leads to significant consequences, including disrupted family functioning, increased social isolation and dependence, and economic constraints, and inadequate social support is common in patients with DR.⁵⁰ Previous studies have established that social support promotes greater patient participation in decision-making by providing financial, emotional, and informational resources that reduce psychological stress and decision-making conflicts.^51–53 However, few studies have specifically evaluated the distinct impacts of different dimensions of social support on patient involvement in decision-making. Our study found that higher objective support was significantly associated with the adoption of a collaborative role in treatment decision-making. Objective support, in this context, refers to the tangible, visible, and practical assistance that patients receive from their social network, such as family, relatives and friends. Our study suggests that objective support is a more robust predictor of a patient’s decision-making role than are subjective support and the utilization of social support. Objective support provides the concrete resources necessary for shared information exchange, preference clarification, and joint deliberation.⁵⁴ This association is particularly salient in cultural contexts with strong family involvement and collectivist values, where health decisions are often regarded as a shared family responsibility.⁵⁵ In such settings, adequate objective support helps mitigate unilateral physician dominance while also alleviating the burden of solitary patient decision-making, thereby promoting a collaborative approach. Therefore, healthcare professionals should systematically assess the objective support levels of patients with diabetic retinopathy. Interventions should simultaneously focus on encouraging patients to strengthen their ties with social networks, such as family, relatives, and friends, and guide family members to become engaged partners in the patient’s treatment journey, thereby bolstering the decision-making support available to them.

Motivation and Patient Involvement

Need for decision-making reflects an individual’s intrinsic motivation to control the process and outcome of medical decisions.²⁸ Patients with a strong need for decision-making typically desire in-depth disease information, weighing pros and cons, participating in discussions, and having more personal control over treatment decisions. Charles et al categorized the treatment decision-making process into three components: information exchange, deliberation, and decision-making control.⁵⁶ The three dimensions of the PEPMDS correspond to the treatment decision-making process.⁵⁷ The univariate analysis results in our study indicate that all three dimensions are related to the patient’s decision-making role. In the multinomial logistic regression model, only the need for deliberation remained statistically significant. Our research suggests that the need for deliberation is a better predictor of patients taking a collaborative or active role in decision-making than the need for information and decision control. Logically, patients necessarily need access to sufficient medical information if they are to deliberate, and crucially, the process of deliberation often serves as a means for patients to strive for or achieve decision control. Therefore, when the need for deliberation is included in the model, it may have already captured most of the variation explained by the need for information and decision control. This finding suggests that patients, even if well-informed and possessing control, may still choose passive decision-making if they do not have the will to deliberate. Rather than just passively providing information or asking the patient about their willingness to take control of the decision, it is more important to identify whether patients are willing to think deeply, weigh the pros and cons, and discuss with healthcare professionals.

Other Factors Associated with Patient Involvement

Our research indicated that as patients age, those with diabetic retinopathy were more likely to adopt a passive or collaborative role in decision-making. This finding aligns with Salm’s study,⁵⁸ while it conflicts with the null results reported by Xie.¹⁵ Several contextual factors might explain this discrepancy. In contrast to Xie’s research, which involved undergraduate students and community-dwelling older adults in the United States and focused on general health decision-making scenarios, our work specifically examined patients with sight-threatening diabetic retinopathy. These patients face complex, urgent decisions, such as choosing intravitreal injections or laser surgery that directly affect their vision. For older adults, who may experience age-related cognitive decline or feel overwhelmed by technical medical information, the perceived complexity and high stakes of these decisions likely diminish their capacity to adopt proactive decision-making.⁵⁹ Furthermore, in the cultural context of our study, younger patients, compared with older patients influenced by traditional paternalistic norms in the doctor-patient relationship, tend to place greater emphasis on asserting personal autonomy and maintaining control over medical decisions.⁶⁰ Therefore, the effect of age may not be a simple main effect but is likely moderated by the disease context, the nature of the treatment decisions, and the cultural environment. We found no statistically significant association between gender and decision-making roles, a finding that stands in contrast to studies which concluded that females were more participatory or males were more involved.^16,17 This divergence suggests that the purported effects of gender may not be a stable, universal phenomenon but are likely contingent upon specific contextual moderators. Overall, there was no consistent evidence to support associations between decision-making roles and gender. Our findings presented that patients with a monthly per capita household income of ≤3000 yuan and 3001–5000 yuan were more inclined to take a passive role in actual decision-making for diabetic retinopathy compared to those with higher incomes. Similar to our results, Wang et al reported that patients with a monthly per capita household income of 5000–7999 yuan exhibited a greater tendency for collaborative decision-making than low-income patients.³⁷ This socioeconomic disparity in decision-making involvement is particularly consequential, given that diabetic retinopathy requires lifelong management involving significant ongoing costs. Treatments such as anti-VEGF therapy, while effective, are relatively expensive. In China, access to these therapies is further constrained by medical insurance policies requiring strict clinical indications and imposing limits on the cumulative reimbursable doses. Consequently, patients with lower household incomes often face substantial financial toxicity, which forces them to weigh treatment costs against potential efficacy and experience heightened psychological distress. This economic pressure frequently leads them to adopt a passive stance, accepting the more economical options recommended by physicians.⁶¹ In contrast, patients with higher incomes are less burdened by cost considerations. Their relative financial security, coupled with potentially greater access to comprehensive health information and support services, reduces the perceived burden of decision-making and fosters greater participation in choosing their treatment options. These observed disparities underscore the critical need for reforms in the national medical insurance payment system. Such reforms should aim to alleviate the financial barriers that currently prevent equitable access to preferred treatments and hinder the full participation of socioeconomically disadvantaged patients in treatment decision-making.

Strengths and Limitations

The study has two primary strengths. First, its theory-driven approach, underpinned by the COM-B model, allowed for a systematic and integrated analysis of the determinants of patient involvement, offering clear targets for future interventions. Second, its comprehensive assessment of key variables within the specific, high-stakes context of diabetic retinopathy enhanced the clinical relevance and applicability of the findings. This study has several limitations. First, the generalizability of our findings is limited by the sample, which was not only relatively small in size but also drawn from a single tertiary hospital in Shanghai. This specific context may not be representative of broader populations, particularly those in community clinics or rural settings where patient demographics and healthcare resources differ considerably. Second, the cross-sectional design cannot establish causal relationships between variables. Longitudinal studies are necessary to untangle these complex temporal dynamics and verify the directionality of these associations. Third, the reliance on self-reported data may introduce social desirability and recall biases, potentially affecting measurement validity. Future studies could incorporate objective measures, such as audio recordings of clinical encounters, to improve accuracy. Finally, our analytical strategy of using sub-dimensions limited our ability to quantify the overall impact of the integrated COM-B constructs. Given these limitations, the findings should be interpreted as exploratory and require validation in larger, more diverse cohorts. Future large-scale studies will also be better equipped to employ advanced statistical modeling techniques to further elucidate these complex relationships.

Conclusions

This study, the first to apply the COM-B model to decision-making involvement among DR patients in China, revealed a predominance of passive involvement among patients. Our findings suggested several potential determinants consistent with the COM-B framework, including capability factors such as critical health literacy, opportunity factors including ophthalmologist facilitation of patient involvement and objective support, and motivation factors like the need for deliberation, in addition to demographic variables such as age and income. These findings underscore the necessity for multifaceted interventions, including tailored patient education programs, clinician communication training, and accessible decision aids, to promote shared decision-making. However, given the constraints of the cross-sectional design and limited sample size, these conclusions should be considered exploratory. Future research should focus on large-scale longitudinal studies to track changes in patient decision-making roles over time, as well as intervention trials assessing the effectiveness of COM-B-based strategies in promoting shared decision-making.

Abbreviations

DR, diabetic retinopathy; COM-B, capability, opportunity, motivation, and behavior; CPS, control preference scale; AAHLS, all aspects of health literacy scale; SSRS, social support rating scale; FPIS, facilitation of patient involvement scale; DSES, decision self-efficacy scale; PEPMDS, patient expectation for participation in medical decision-making scale.

Data Sharing Statement

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai General Hospital (Approval No. 2024-098). Written informed consent was obtained from all participants.

Acknowledgments

The authors wish to thank all the patients and staff who participated in this study.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

The research did not get any dedicated financial funding from public, commercial, or not-for-profit funding organizations.

Disclosure

The authors report no conflicts of interest in this work.

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Introduction

Background

Acute pancreatitis (AP) is characterized by the dysfunction of pancreatic cellular pathways and organelles due to various etiologies, with gallstones and alcohol abuse being the most prevalent.¹ This condition ultimately results in the death of pancreatic acinar cells.² In severe instances, AP may arise as a consequence of significant complications, including local and systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF).³ AP has acute onset, severe condition and many complications, and its incidence is increasing year by year.⁴ A study indicated that the annual global incidence of AP is 34 cases per 100,000 individuals,⁵ with an overall mortality rate of approximately 5%. In cases of severe acute pancreatitis (SAP), the mortality rate may approach 20%. Currently, there is no established clinical treatment for AP. The primary pathological response in AP is the premature activation of trypsinogen, resulting in damage and death of acinar cells. Recent research has identified necroptosis as a form of regulated cell death (RCD).⁶ It is crucial in the mechanism of acinar cell death and the premature activation of trypsinogen.⁷

Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3). The activation of the RIPK3/mixed lineage kinase like (MLKL) pathway exhibits characteristics of both necrosis and apoptosis. Specifically, necroptosis is actively regulated by various genes and occurs in an orderly manner through the activation of specific death pathways.^8–10 Salidroside (Sal) has a wide range of pharmacological activities, including Anti-inflammatory, anti-aging, antioxidant, and anti-tumor, etc.^11–14 Additionally, it has been shown to decrease the activity of pancreatic enzymes during the initial stages of SAP.^15,16

Aim of the Study

Based on the aforementioned evidence, we hypothesized that Sal might alleviate AP by modulating necroptosis. To test this hypothesis, the present study was designed to achieve the following specific aims: First, to investigate the therapeutic effects of Sal on pancreatic injury and systemic inflammation in a rat model of sodium taurocholate-induced AP; Second, to determine whether the protective effects of Sal are associated with the inhibition of the RIPK1/RIPK3/MLKL necroptosis pathway in both in vivo and in vitro (cerulein-stimulated AR42J cells) AP models; Finally, to explore the functional interaction between Sal and Nec-1 (a specific RIPK1 inhibitor) in order to elucidate the potential mechanism of action of Sal.

Materials and Methods

Experimental Animals and Cells

The Medical Laboratory Animal Center of Lanzhou University supplied 18 male Wistar rats with weights ranging from 250 to 280 grams. The animals were maintained in an environment characterized by alternating 12-hour light and dark cycles, with unrestricted access to food and water provided. All the rat experiments were conducted in accordance with the “Regulations on the Administration of Experimental Animals” (Lanzhou University) and were approved by the Ethics Committee of Lanzhou University Second Hospital (Approval No.D2024-410). At the same time,all methods are reported in accordance with ARRIVE guidelines.

Rat pancreatic exocrine cells-AR42J (Cellverse, iCell-r002) were cultured in AR42J specialized medium containing 20% Foetal Bovine Serum (FBS) (Procell, CM-0025) at 37°C, 5% CO₂.

AP Rat Model

Firstly, the AP rat model was established by retrograde injection of sodium taurocholate solution. All rats were fasted and water-deprived overnight before the operation, and then anesthetized by intraperitoneal injection of 0.3% pentobarbital sodium (0.2 mL/10 g). Next, the rats were randomly divided into three groups (n=6): (1) Sham surgery group: only underwent laparotomy and closure sham surgery; (2) AP group: Induction of pancreatic injury by retrograde injection of 3.5% sodium taurocholate (1mL/kg, injection rate 0.1 mL/min) into the pancreaticobiliary duct. Observation after approximately 10 minutes showed congestion, edema, local bleeding, and necrosis in the pancreatic tissue, confirming the establishment of the model^17,18 (3) Sal group: Based on our previous experimental results, Sal (60 mg/kg) (Medchemexpress, HY-N0109) was injected intraperitoneally 2 hours after AP modeling.¹⁵ And all animals were euthanized for sample collection at 24h post-modeling (Pentobarbital sodium, 200 mg/kg), followed by blood sample collection and serum separation through centrifugation. The pancreatic tissue was divided into three parts for pathological analysis, Western blot, and TEM detection. Finally, the animal carcasses were uniformly sent to the Gansu Province Hazardous Waste Disposal Center for processing. The final analysis included only those Wistar rats that successfully underwent the AP model induction surgery and survived the intended postoperative observation period. Rats that died during anesthesia or surgery (prior to model completion) were excluded from the analysis. Furthermore, any rat that did not exhibit a significant elevation in serum amylase levels at the predetermined time point post-modeling was also excluded, as it was deemed an induction failure. No animals were excluded from the final analysis based on these criteria.

Histopathology and Molecular Analysis

After fixation with 4% paraformaldehyde, pancreatic tissue was embedded in paraffin and sectioned (5 μm). Hematoxylin-eosin (H&E) staining was used to assess tissue pathological damage (edema, inflammatory infiltration, acinar necrosis), with a semi-quantitative scoring system on a 0–3 scale.¹⁹ The anti-p-MLKL antibody (Affinity, AF7420, diluted 1:200) was used in immunohistochemistry (IHC) to locate p-MLKL expression, followed by DAB staining and microscopic observation. Transmission electron microscopy samples were fixed with 2.5% glutaraldehyde and ultrathin sections were observed under an electron microscope to examine the ultrastructure of mitochondria.

Biochemical and Inflammatory Factor Testing

Serum and cell culture supernatant levels of AMY were quantified using a commercial kit (Yuanye, R22037) strictly according to the manufacturer’s instructions. Briefly, the assay involves the enzymatic hydrolysis of a defined substrate, and the resulting product is measured spectrophotometrically at a wavelength of 660 nm using a microplate reader. AMY activity is expressed in U/L. All samples were measured in duplicate. The concentrations of the inflammatory cytokines IL-6, IL-1β, and TNF-α were quantified using commercially available ELISA kits (Jonlnbio, catalog numbers JL20896, JL20884, and JL13202, respectively). The sensitivities of the assays were 1.11 pg/mL, 1.51 pg/mL, and 1.75 pg/mL for IL-6, IL-1β, and TNF-α, respectively. The detection ranges for all three cytokines were 3.12–200 pg/mL. Both intra- and inter-assay coefficients of variation were less than 10% for all assays, indicating high reproducibility and precision.

Western Blot Analysis

In Western blot analysis, tissues or cells are lysed with RIPA lysis buffer (containing protease/phosphatase inhibitors), and protein concentration is determined using the BCA method. 30 μg of protein was loaded into each well, separated by SDS-PAGE, and transferred to a PVDF membrane. After blocking with 5% BSA, the membrane was incubated with primary antibodies (RIPK1: Proteintech, 17519-1-AP, 1:1000; RIPK3: Bioss, bs-3551R, 1:1000; MLKL: Proteintech, 66675-1-Ig, 1:10000; p-MLKL: Affinity, AF7420, 1:1000; GAPDH: Selleck, F0003, 1:10000) and secondary antibodies. Protein expression levels were quantified by measuring the integrated density of the immunoreactive bands. Blots were imaged using a ECL chemiluminescence detection and analyzed with ImageJ software. The intensity of each target band was measured. To correct for potential variations in sample loading, the intensity of each target protein was normalized to that of the internal control (eg, GAPDH or MLKL) from the same sample. Data are presented in the bar graphs as the Mean ± SD of 4 independent experiments (due to tissue allocation constraints for protein extraction, Western blot analysis was performed on a subset of n=4 randomly selected samples per group).

AR42J Cell Experiment

First, the Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability to determine the optimal drug concentrations of Sal and the RIPK1 inhibitor Necrostatin-1 (Nec-1): Cells were seeded in a 96-well plate (2×10⁴/well), allowed to adhere overnight, and then incubated with the corresponding drugs at gradient concentrations for 8 hours. CCK-8 reagent (10 μL/well) was added, and the incubation continued at 37°C for 4 hours. The optical density (OD) at 450 nm was measured using a microplate reader to calculate cell viability. Next, AR42J cells were pre-treated with Salidroside (50 μM) or Nec-1 (10 μM) for 4 hours prior to a 4-hour stimulation with cerulein (100 nM), resulting in a total intervention period of 8 hours.²⁰ Mitochondrial membrane potential was detected using the JC-1 dye (Beyotime, C2003S) and observed under a confocal microscope by measuring the red/green fluorescence ratio (excitation wavelength 490/525 nm, emission wavelength 530/590 nm). A decrease in the ratio indicates mitochondrial membrane potential depolarization. In the immunofluorescence experiment, after fixation and permeabilization of the cells, the anti-p-MLKL antibody (1:200) was co-stained with DAPI, and the subcellular localization of p-MLKL was observed using a confocal microscope. Only AR42J cell cultures with >95% viability were used. Any culture wells showing signs of bacterial or fungal contamination at the start of the experiment were excluded from the analysis.

Instrumentation and Equipment

The following key instruments were used in this study: Tissue FAXS PLUS microscope (Tissue FAXS PLUS, AUT), Electron microscope (Hitachi HT7800, Japan), Microplate reader (BioTek Synergy H1, USA), Confocal microscope (Zeiss LSM880, GER); ECL chemiluminescence detection (Bio-Rad ChemiDoc, SG).

Statistical Analysis

Data are expressed as mean ± standard deviation (Mean ± SD). Comparisons among multiple groups were performed using one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison test for post-hoc analysis. All analyses were conducted using GraphPad Prism software (Version 10). A P-value of less than 0.05 (p < 0.05) was considered statistically significant. In the figures, significance levels are denoted as follows: ns (not significant, p > 0.05), *p < 0.05, **p < 0.01, and **p < 0.001.

Results

Sal Alleviates Pancreatic Injury in Rats with AP

The changes in serum AMY and inflammatory cytokine levels indicate that Sal exerts significant anti-inflammatory effects in AP. One-way ANOVA indicated a significant difference in serum AMY levels among the groups (Figure 1A, F (2, 15) = 27.13, p < 0.001). Post-hoc Tukey’s test revealed that compared to the Sham group, AMY levels were significantly elevated in the AP model group, whereas Sal treatment reduced AMY levels by 37.4% (p < 0.01). Similarly, serum levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were markedly increased in the AP group (Figure 1B; one-way ANOVA, TNF-α: F (2, 15) = 97.37, p < 0.001; IL-6: F (2, 15) = 239.6, p < 0.001; IL-1β: F (2, 15) = 99.17, p < 0.001). Sal intervention significantly reduced these cytokines by 27.6%, 23.9%, and 45.3%, respectively (all p < 0.001). H&E showed extensive pancreatic edema, inflammatory cell infiltration, and fat necrosis in the AP model group (Figure 1C). In contrast, the Sal-treated group exhibited a marked reduction in pathological damage, with a 40% decrease in histopathological scores compared to the model group (Figure 1D, p < 0.001).

Sal Inhibits the Activation of the Necroptosis Pathway in Pancreatic Tissue

Since Sal alleviated pancreatic damage and inflammation in AP, we further investigated whether its protective effects depend on the necroptosis pathway. Western blot analysis revealed a significant upregulation of RIPK1, RIPK3, and p-MLKL protein expression in the pancreatic tissue of the AP group (Figure 2A–C). One-way ANOVA indicated statistically significant differences among the groups for all three proteins (RIPK1: F (2, 9) = 21.70, p < 0.001; RIPK3: F (2, 9) = 29.85, p < 0.001; p-MLKL: F (2, 9) = 39.33, p < 0.001). Post hoc analysis showed that Sal treatment significantly reduced their expression levels by 21.2%, 26.1%, and 18.7%, respectively (all p < 0.05). Consistent with these results, IHC staining demonstrated strong p-MLKL immunopositivity (brown) in the perimembranous region of pancreatic cells in the AP group, which was markedly attenuated in the Sal-treated group (Figure 2D and E). TEM was employed to evaluate the ultrastructural consequences of necroptotic signaling, with a focus on mitochondrial integrity. Pancreatic acinar cells from AP model rats exhibited severe organellar damage, most notably in the form of swollen mitochondria with vacuolation and disrupted cristae (Figure 2F, middle panel), which is a characteristic manifestation of ongoing necroptosis. Treatment with Sal markedly preserved mitochondrial morphology, presenting with only mild swelling and intact cristae (Figure 2F, right panel).

Figure 2 Sal inhibits the activation of the necroptosis pathway in pancreatic tissue. (A) Representative Western blot images showing protein levels of RIPK1, RIPK3, MLKL, and p-MLKL in pancreatic tissues from different experimental groups. (B) Quantitative analysis of RIPK1 and RIPK3 protein expression normalized to GAPDH (n=4). (C) Quantitative analysis of p-MLKL protein expression normalized to total MLKL (n=4). (D) Representative immunohistochemical staining of p-MLKL in pancreatic tissues (scale bar: 50 μm). Brown granules indicate positive signals. The AP group shows extensive p-MLKL expression localized around pancreatic acinar cell membranes. (E) Quantitative analysis of p-MLKL immunohistochemical staining expressed as mean optical density (OD) values (n=4). (F) Transmission electron microscopy observation of mitochondrial ultrastructure (scale bar: 1 μm). The mitochondria in the AP group showed swelling and cristae rupture, while the mitochondria in the Sal group appeared nearly normal (The black arrows indicate the mitochondrial structure of each group). *p < 0.5, **p < 0.01, ***p < 0.001 compared with AP group.

Sal Reduces Inflammatory Damage in the AR42J Cell AP Model by Mitigating Necroptosis

To further validate the anti-necroptotic and anti-inflammatory effects of Sal and elucidate its underlying mechanism, we established an in vitro AP model using cerulein-stimulated AR42J cells. First, the optimal non-cytotoxic concentrations of Sal (50 μM) and Nec-1 (10 μM) were determined via CCK-8 assay (Figure 3A and B). Cerulein (100 nM) stimulation significantly increased AMY release into the supernatant by 1.4-fold (Figure 3C; one-way ANOVA, F(3, 20) = 36.74, p < 0.001), indicating successful induction of acinar cell injury. Sal treatment alone reduced AMY levels by 15.3%, while co-treatment with Sal and Nec-1 did not yield a significant additive effect compared to Sal alone. Similarly, the release of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) was markedly elevated in the AP group (Figure 3D, one-way ANOVA, TNF-α: F (3, 20) = 37.14, p < 0.001; IL-6: F (3, 20) = 48.49, p < 0.001; IL-1β: F (3, 20) = 51.72, p < 0.001), with the most potent suppression observed in the Sal + Nec-1 co-treatment group (reductions of 29.8%, 13.2%, and 14.9%, respectively). Furthermore, cerulein induction resulted in a severe loss of mitochondrial membrane potential (indicated by a 94% decrease in red/green fluorescence ratio, p < 0.001), which was significantly restored by Sal pretreatment (Figure 3E and F, one-way ANOVA, F(3, 16) = 25.08, p < 0.001). This loss was significantly attenuated by Sal pretreatment (p < 0.05). These results confirm that Sal mitigates inflammatory damage and mitochondrial dysfunction in acinar cells under AP-like conditions.

Figure 3 Sal reduces inflammatory damage in the AR42J cell AP model. (A) The effect of Sal (1–200 μM) on the viability of AR42J cells (CCK-8 assay, n=4). Choose 50 μM (where cell viability significantly increased and there was no significant toxicity) for subsequent experiments. (B) The effect of Nec-1 (1–200 μM) on cell viability (n=4). Choose 10 μM (cell viability significantly increased and no significant toxicity) for subsequent experiments. (C) AMY levels in cell supernatants (n=6). (D) TNF-α, IL-6, and IL-1β levels in cell supernatants (n=6). (E) Quantitative analysis of mitochondrial membrane potential assessed by JC-1 staining (n=5). Data are presented as the red/green fluorescence ratio. (F) Representative fluorescence images of JC-1 staining in different experimental groups (scale bar: 20 μm). Red fluorescence indicates JC-1 aggregates (high membrane potential), while green fluorescence indicates JC-1 monomers (low membrane potential). A decrease in the red/green fluorescence ratio indicates a decline in membrane potential, and Sal pretreatment partially restores the ratio. *p < 0.5, **p < 0.01, ***p < 0.001 compared with AP group.

Sal Inhibits the RIPK1/RIPK3/MLKL Pathway to Suppress Necroptosis in AR42J Cells

We further investigated whether Sal confers protection through specific inhibition of the necroptotic pathway. Western blot analysis revealed that cerulein significantly upregulated key mediators of necroptosis, increasing RIPK1, RIPK3, and p-MLKL levels by 1.34-fold, 1.77-fold, and 1.61-fold, respectively (Figure 4A–D, one-way ANOVA, RIPK1: F(3, 12) = 10.25, p = 0.001; RIPK3: F(3, 12) = 10.47, p = 0.001; p-MLKL: F(3, 12) = 7.813, p = 0.004). Sal treatment alone significantly reduced the expression of these proteins, with p-MLKL decreasing by 34.7%. The absence of an additive effect between Sal and Nec-1 suggests that both compounds likely target the same node within the pathway. Immunofluorescence staining further demonstrated robust membrane translocation of p-MLKL in AP group cells, whereas both Sal and Nec-1 treatments markedly reduced p-MLKL membrane aggregation (Figure 4E and F, other representative fields of view for each group are shown in Supplementary Figure 1). Collectively, these data strongly support our hypothesis that Sal alleviates cerulein-induced AP injury in AR42J cells by inhibiting the RIPK1/RIPK3/MLKL-mediated necroptosis pathway.

Figure 4 Sal reduces necroptosis in AR42J cells by inhibiting the RIPK1/RIPK3/MLKL pathway. (A) Representative Western blot images showing protein levels of RIPK1, RIPK3, MLKL and p-MLKL in different experimental groups. (B) Quantitative analysis of RIPK1 protein expression normalized to GAPDH (n=4). (C) Quantitative analysis of RIPK3 protein expression normalized to GAPDH (n=4). (D) Quantitative analysis of p-MLKL protein expression normalized to total MLKL (n=4). (E) Quantitative analysis of p-MLKL fluorescence intensity from immunofluorescence staining (n=4). (F) Representative immunofluorescence images of p-MLKL subcellular localization (scale bar: 50 μm). Red fluorescence represents p-MLKL signal, and blue represents DAPI nuclear staining. *p < 0.5, **p < 0.01, ***p < 0.001 compared with AP group.

Discussion

The pathological process of AP involves complex mechanisms, among which necroptosis has been widely recognized as a critical contributor to cellular damage and inflammatory responses. However, its regulatory mechanisms and potential as a therapeutic target in AP remain incompletely elucidated. This study suggests that Sal significantly alleviates pancreatic injury in both rat AP models and AR42J cells by suppressing RIPK1/RIPK3/MLKL-mediated necroptosis, providing novel experimental evidence to support the potential clinical application of Sal in the management of AP (Figure 5).

Figure 5 Sal inhibits the RIPK1/RIPK3/MLKL necroptosis pathway, prevents mitochondrial damage, and reduces inflammation in AP.

The present study indicate that Sal treatment markedly reduced serum levels of amylase (AMY) and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in AP rats (Figure 1A and B), consistent with previous studies reporting the protective effects of Sal against multi-organ injury through its anti-inflammatory and antioxidant properties.^16,21–24 For instance, Wang et al²⁵ showed that Sal inhibited furan-induced barrier damage and intestinal inflammation by suppressing TLR4/MyD88/NF-κB signaling, while Wang et al²⁶ reported Sal alleviated mitochondrial dysfunction by activating the PGC-1α/Mfn2 signaling pathway, and restrained the endoplasmic reticulum stress. However, unlike these earlier studies that focused primarily on general anti-inflammatory effects, our study provides novel mechanistic insights by specifically establishing the inhibitory effect of Sal on necroptosis. Histopathological evaluation further confirmed that Sal ameliorated pancreatic tissue edema, necrosis, and inflammatory infiltration (Figure 1C and D).

The most significant finding of this study is the identification of Sal as a regulator of necroptosis. Western blot and immunohistochemical analyses revealed that Sal significantly inhibited the expression of RIPK1, RIPK3, and p-MLKL in pancreatic tissues (Figure 2A–C). TEM further demonstrated that Sal preserved mitochondrial structural integrity (Figure 2F), suggesting that its protective effects may be closely associated with the mitigation of mitochondrial dysfunction during necroptotic stress.

The RIPK1/RIPK3/MLKL axis is a well-established pathway mediating necroptosis.^6,27 Our findings not only confirm the pivotal role of this pathway in AP but also provide new evidence for its pharmacological modulation. Sal significantly inhibited the activation of this pathway in both in vivo and in vitro AP models (Figures 2A and 4A). Notably, Nec-1 is a well-characterized and highly specific allosteric inhibitor of RIPK1. It functions by stabilizing RIPK1 in an inactive conformational state, thereby preventing its kinase activity and subsequent recruitment and phosphorylation of RIPK3. This specific inhibition blocks the initiation of the necroptotic cascade upstream of MLKL activation. In our study, the use of Nec-1 served a dual purpose: firstly, as a positive control to confirm the involvement of RIPK1-dependent necroptosis in our AP model, and secondly, as a pharmacological tool for pathway interrogation. The observation that the combination of Sal and Nec-1 did not produce a significant additive effect is a critical pharmacological clue. It suggests that Sal likely intersects with the necroptosis pathway at the level of RIPK1 or its upstream regulators, rather than acting on a parallel or downstream node.

Furthermore, Sal was found to restore mitochondrial membrane potential (Figure 3F), supporting the notion that it mitigates AP progression through a dual mechanism—inhibiting necroptosis and preserving mitochondrial function. This aligns with emerging studies emphasizing the crosstalk between necroptosis and mitochondrial dysfunction,^28–30 although the precise mechanisms require further investigation. While the inhibition of this pathway is a major mechanism, we cannot rule out contributions from other pathways. This is now framed as a key mechanism rather than the exclusive mechanism.

Despite these advances, several limitations should be acknowledged. First, this study did not directly determine the pharmacokinetic parameters of Sal in an ascites environment. The absorption and distribution characteristics of Sal need to be further clarified in future research. Second, a limitation of our in vitro study is the lack of a Nec-1 alone treatment group, which would have served as a crucial positive control to benchmark the efficacy of Sal against a known RIPK1 inhibitor. Future studies will include Nec-1 as a standalone treatment to fully validate the model and provide a direct comparison for the potency of novel inhibitors like Sal. Third, it is important to note that although the pharmacological data we obtained using Nec-1 strongly suggest that Sal acts on the RIPK1 pathway, the lack of in vivo inhibitor experiments or a rescue experiment (eg, through RIPK1 overexpression) remains a limitation. Future studies employing genetic approaches both in vitro and in vivo will be essential to conclusively validate RIPK1 as the direct target. To further solidify our conclusions, future work will involve administering Nec-1 in a rat AP model to directly compare its efficacy with that of Sal and to investigate potential synergistic effects, or transfecting AR42J cells with RIPK1 plasmids to examine whether forced RIPK1 expression can counteract the protective effects of Sal, which would provide definitive mechanistic validation. Additionally, while our TEM analysis provided clear evidence of mitochondrial damage and its prevention by Sal, future studies could aim to capture more panoramic views to document the full spectrum of necroptotic ultrastructural features, such as plasma membrane rupture. Most importantly, while pharmacological evidence strongly suggests that Sal targets the necroptosis pathway, direct binding assays and validation using genetic knockout animal models are needed to identify its precise molecular target.

From a clinical translation perspective, the pharmacokinetic profile, bioavailability, and long-term safety of Sal require systematic evaluation. Moreover, crosstalk between necroptosis and other cell death modalities (eg, apoptosis, pyroptosis^9,31,32) may influence its therapeutic efficacy. However, potential compensatory survival mechanisms resulting from excessive inhibition of cell death should be carefully monitored.

Conclusions

In conclusion, our study provides evidence that Sal alleviates AP by inhibiting necroptosis, likely through targeting the RIPK1/RIPK3/MLKL pathway. Our pharmacological data are consistent with RIPK1 being a potential target, although this requires direct genetic validation in future studies.

Abbreviations

AP, Acute pancreatitis; SAP, Severe acute pancreatitis; Sal, Salidroside; Nec-1, Necrostatin-1; AMY, Amylase; RCD, Regulated cell death; RIPK, Receptor interacting protein kinase 3; MLKL, Mixed lineage kinase like; TEM, Transmission electron microscope; H&E, Hematoxylin-eosin; IF, Immunofluorescence; IHC, Immunohistochemistry; ELISA, Enzyme-linked immunosorbent assay; IL, Interleukin; TNF, Tumor necrosis factor; CCK-8, Cell Counting Kit-8; OD, Optical density.

Data Sharing Statement

The data are available from the corresponding author upon reasonable request.

Ethics Approval Statement

All the rat experiments were conducted in accordance with the “Regulations on the Administration of Experimental Animals” (Lanzhou University) and were approved by the Ethics Committee of Lanzhou University Second Hospital (Approval No. D2024-410). At the same time,all methods are reported in accordance with ARRIVE guidelines.

Acknowledgments

We acknowledge the Cuiying Biomedical Research Center of the Lanzhou University Second Hospital for providing all the research equipments for this experiment.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was supported by the National Natural Science Foundation of China (82260135) and Science and Technology Program of Gansu Province (22YF7WA087).

Disclosure

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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