Phillygenin rescues impaired autophagy flux by modulating the PI3K/Akt/mToR signaling pathway in a rat model of severe acute pancreatitis

Abstract

To investigate the mechanism of pancreatic alveolar cell autophagy in rats with severe acute pancreatitis (SAP) by phillygenin (PHI) based on the PI3K/Akt/mToR pathway. Rats were randomly divided into control group (CON group), SAP model group (SAP group) and PHI treatment group (SAP+PHI group), with 10 rats in each group. 5% sodium taurocholate was injected retrogradely into the biliopancreatic duct to establish a SAP rat model, and PHI was injected intraperitoneally into the pancreas after successful establishment of the model. The colorimetric assay was used to determine serum amylase and lipase activity levels. Pancreatic morphology and histological changes were assessed by H&E staining. Autophagy-related indices were determined by immunohistochemistry: LC3-II, P62, LAMP. Autophagy pathway-related indices were determined by western blotting assay: p-PI3K, PI3K, p-Akt, Akt, p-mToR, mToR. Autophagy vesicle alteration. Compared with the SAP group, the SAP+PHI group showed a decrease in amylase, lipase and pathological score, an increase in the expression of LAMP-2, and a decrease in the expression of p62, p-PI3K, p-Akt and p-mToR, with a statistically significant difference (p < 0.05). Electron microscopy showed that autophagic flux was restored and accumulated autophagic vehicles were relatively reduced by PHI intervention. PHI can rescue the impaired autophagic flux by inhibiting the PI3K/Akt/mToR pathway, allowing abnormal autophagic vesicles to complete autophagy to protect the rat.

Introduction

Severe acute pancreatitis (SAP) is a serious gastrointestinal disease often associated with severe dysregulation of the internal microenvironment and organ failure. ¹ Approximately 20% of patients diagnosed with acute pancreatitis (AP) develop SAP, which can rapidly progress to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). ² This condition has an inferior prognosis, with a mortality rate of up to 30%. Timely intervention to prevent or mitigate disease progression in the early stages of development is critical in the management of SAP. ³ Tissue necrosis develops progressively after the onset of SAP. It is therefore important to act quickly to prevent or reduce its progression. Previous scientific studies have shown that several cellular and molecular events, including apoptosis, autophagy, inflammation and oxidative stress, occur frequently during the pathogenesis of SAP . ⁴ Autophagy is considered to be one of the important regulatory pathways in the development of SAP. Autophagy is the process of degrading and recycling intracellularly damaged or unwanted substances in lysosomes to maintain intracellular homeostasis. ⁵ There is considerable evidence that modulators of autophagy, such as trehalose, sitagliptin and deoxyarbutin, may be an effective strategy for preventing or alleviating SAP.^6–8 Therefore, modulation of autophagy levels may be an important target for the prevention or treatment of SAP.

Clinical treatments such as fasting and early enteral nutrition are often used to prevent further deterioration. However, these treatments do not significantly improve the overall condition of patients with SAP. ⁹ Therefore, finding safe and effective preventive or therapeutic drugs is an urgent clinical problem. In recent years, traditional Chinese medicine (TCM) has attracted attention for its unique therapeutic effects in SAP.^10–12 However, due to the complexity of the chemical composition and pharmacological effects of TCM, it is crucial to identify its effective therapeutic components for SAP intervention. Phillygenin (PHI) is a lignan chemical component found in the TCM Forsythiae Fructus. It has various pharmacological activities such as anti-inflammatory, antioxidant, anti-tumor, antiviral and immunomodulatory effects. ¹³ In addition, PHI has been shown to regulate autophagy by inhibiting lipid accumulation and inflammation. ¹⁴ Furthermore, PHI has been shown to be beneficial in the prevention and treatment of several diseases including inflammatory bowel disease, liver disease, cancer, bacterial and viral infections. ¹³ Therefore, further research on PHI may not only provide a new theoretical basis for the prevention and treatment of SAP, but also serve as a reference for the development of new drugs.

This research aims to investigate the regulatory mechanism of PHI in a rat model to concerning autophagy, which may provide new ideas for future clinical treatment of SAP.

Materials and methods

Animals

The Laboratory Animal Committee of Shanxi Medical University approved all animal experiments and methods (No. DW2023015), which were conducted by the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. Thirty healthy male Sprague-Dawley rats, weighing 200–250 g and aged 8–10 weeks, were obtained from the Laboratory Animal Centre of Shanxi Medical University. The rats were acclimatized for one week in an environment with a temperature of 18°C–22°C and a humidity of 55%–65%, with an alternating day/night cycle, and were provided with standard rodent chow and water ad libitum.

Antibodies and reagents

Phillygenin (purity >98%) was purchased from Chengdu Must Biotechnology Co., Ltd (Chengdu, China). Sodium taurocholate (purity >98%) was purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Phospho-AKT, anti-AKT and anti-mTOR antibodies were purchased from Abcam Technology Co., Ltd (Shanghai, China). Phospho-PI3K, anti-PI3K, phospho-mTOR antibody and β-actin antibody were purchased from ABclonal Technology Co., Ltd (Wuhan, China). Anti-LAMP2 antibody, anti-P62 antibody and anti-LC3 antibody were purchased from Boster Biological Technology Co., Ltd (Beijing, China). The amylase and lipase detection kit was purchased from Jiancheng Biotech Co., Ltd. (Nanjing, China).

Grouping

Thirty healthy and clean Sprague-Dawley rats were randomly assigned to three groups after 12 h of fasting: Control (CON), SAP model (SAP) and SAP treated with PHI (SAP+PHI). Each group consisted of 10 rats.

SAP modeling sodium taurocholate

Anesthesia was induced with 1% pentobarbital sodium (0.35 ml/100 g). The animal was placed head down and a 30-60° angled needle was slowly inserted into the lower abdomen. After breaking through the peritoneum, a check was made for backflow of blood or intestinal fluid before the anesthetic was injected, first rapidly and then slowly.

Prior to surgery, the rats were fasted for 12 h and deprived of water for 2 h. They were then anaesthetized by intraperitoneal injection and placed on a clean table for skin preparation, disinfection and toweling. A median abdominal incision was made to access the abdominal cavity, revealing light pink pancreatic tissue behind the stomach. The pancreaticobiliary duct was localized by spreading the duodenal omentum after temporarily clamping the common hepatic duct with a microvascular clamp. The biliopancreatic duct was punctured obliquely with a 1ml syringe needle and secured with a microvascular clamp. Then 5% sodium taurocholate was injected retrogradely into the biliopancreatic duct at a rate of 0.1 mL/min (injection dose: 0.1 mL/100 g). At the end of the injection, the puncture site was held under pressure for 5 min. The CON group underwent sham surgery in which the incision was closed after the pancreas was gently rotated. In contrast, rats in the SAP+PHI group received a single intraperitoneal injection of PHI at a dose of 30 mg/kg, administered 30 min after modelling. Meanwhile, rats in both the CON and SAP groups were injected with equivalent volumes of saline to provide a baseline comparison for the effects of PHI.

Sample collection

Rats were anaesthetized with pentobarbital sodium after 12 h and samples were collected (method as above). The mid-abdominal incision was extended downwards to locate the inferior vena cava and blood was collected. The collected blood was then centrifuged at 4000 rpm for 20 min, and the upper serum layer was stored at -80°C for later analysis. Immediately after excision, the pancreas was divided into three parts: one was refrigerated at −80°C for protein blotting and biochemical analysis, another was fixed in 4% paraformaldehyde for histological analysis, and the third was fixed in 2.5% glutaraldehyde for electron microscopic analysis.

Colorimetric assay

Serum amylase and lipase activities were determined by a colorimetric assay. For the determination of serum amylase activity, substrate buffer was added to both the test and blank tubes, which were then preheated. Then 0.1 mL of sample was added to the test tube, while the blank tube remained untreated. The contents were mixed thoroughly to initiate the reaction. Iodine solution and double-distilled water were then added to the test tube, with an equivalent volume of double-distilled water added to the blank tube. The absorbance of each reaction solution was measured at 660 nm. In the case of serum lipase activity, the spectrophotometer was initially zeroed at 420 nm. The substrate buffer was pre-warmed and 50 μL of each serum sample was added to separate tubes together with the pre-warmed substrate buffer and mixed thoroughly. The samples were then measured using the spectrophotometer at 420 nm. After this initial measurement, the tubes were re-bathed and the percentage difference in absorbance before and after the bath was calculated.

H&E staining

Pancreatic tissues were isolated, fixed in 4% paraformaldehyde solution overnight, dehydrated in ethanol and embedded in paraffin. The samples were then sectioned and stained with haematoxylin and eosin (H&E). For each section, five fields of view were randomly selected at high magnification and examined under a blinded light microscope. Schmidt et al. described four categories for scoring each field: oedema, vesicular necrosis, inflammation and peripheral vascular exudation, and hemorrhage and fatty necrosis. ¹⁵

Immunohistochemistry

For immunohistochemical staining, 3.5 μm thick paraffin sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide solution. Antigen retrieval was performed by boiling the sections in citrate buffer (pH 6.0) for 20 min. The sections were then allowed to cool naturally and sealed with goat serum for 30 min. After sealing, the sections were incubated overnight at 4°C with anti-LAMP2 antibody (1:100), anti-LC3 antibody (1:50) or anti-p62 antibody (1:100). The sections were then rinsed with PBS and incubated with peroxidase-linked secondary antibody for 30 min at room temperature. Finally, the sections were treated with diaminobenzidine (DAB) to develop color. Positive cells were identified by the presence of yellow granules in the cytoplasm and the final score was based on the product of the intensity of cellular staining and the percentage of positive cells (Table 1).

Table 1.

Immunohistochemical quantification analysis table.

Cell staining intensity	Percentage of positive cells	Score
No positive colouring (negative)	0%	0
Light yellow colour (weakly positive)	≤25%	1
Brownish yellow (positive)	26%–50%	2
Tan colour (strong positive)	51%–75%	3
	>75%	4

Western blotting assay

Sample tissues were lysed in RIPA buffer and quantified using the BCA protein concentration assay kit. Approximately 10 ug of each protein sample was separated on either 8% or 6% SDS polyacrylamide gels (selected according to the instructions for use) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were soaked in 5% skimmed milk or BSA. The membranes were then incubated with the following primary antibodies Phospho-PI3K antibody (1:1000), Phospho-AKT antibody (1:1000), Phospho-mTOR antibody (1:1000), Anti-PI3K antibody (1:1000), Anti-AKT antibody (1:5000), Anti-mTOR antibody (1:5000) and β-actin antibody (1:5000). Primary antibodies were incubated overnight at 4°C. The secondary antibody (1:20,000) was then added and incubated for 2 h at room temperature on a shaker. The bands were observed using ECL reagent and their intensity was quantified using Image J software. β-actin was used as an endogenous control for all samples.

Transmission electron microscope assay

Pancreatic tissue was fixed in 2.5% glutaraldehyde for 1 h at room temperature. They were then transferred to a 4℃ environment for overnight fixation, followed by static treatment with osmic acid. After gradient dehydration, the tissues were embedded and semi-thin sections were obtained using an ultrathin slicer. The number of autophagosomes and autophagic lysosomes was observed.

Statistical analysis

Experimental data were analyzed using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation (Mean ± SEM). One-way ANOVA was used to compare between groups, while further multiple comparisons were performed to test for normality and chi-squared variance. When ANOVA assumptions were not met, the Welch ANOVA test was used. When normality assumptions were not met, the Kruskal-Wallis test was used. A statistically significant difference was defined as p < 0.05.

Results

The rat model exhibites clear pancreatic injury as a result of sodium taurocholate

The SAP rat model was established by retrograde injection of sodium taurocholate into the pancreatic bile duct. Serum amylase and lipase levels in the SAP group were significantly higher than those in the CON group(p < 0.05, Figure 1e, f). Under the microscope using H&E staining, the pancreatic tissue in the CON group appeared structurally clear, with only a few cases of localized interlobular septal swelling and oedema. Conversely, pancreatic tissue injury was evident in the SAP group. The pancreatic tissue in the SAP group showed diffuse interlobular interval swelling as the main feature, along with partial swelling of the local interlobular space, a large number of necrotic follicular cells, fusion of necrotic follicles and inflammatory cell infiltration. Most of the pancreatic tissues were fused microabscesses with hemorrhage and fat necrosis. The pancreatic pathology score was significantly higher in the SAP group than in the CON group (p < 0.05, Figure 1a, b, d).

Figure 1.

Pancreatic tissue impairment and recovery in SAP rats. (a) H&E staining pictures in CON group of rats. (b) H&E staining pictures in SAP group of rats. (c) H&E staining in SAP+PHI group of rats. (d) Pancreatic histological scores in each group of rats. (e–f) Serological indicators in each group of rats. Data shown are means ± SEM. Compared with CON group. ****P < 0.0001; compared with SAP group, #P < 0.05, ####P < 0.0001.

SAP causes enhanced autophagy initiation but impaired completion in a rats model

Autophagy flux indices were determined by immunohistochemical detection and scoring. LC3-II, LAMP-2 and p62 were all cytoplasmic staining. Rats in the SAP group showed a larger area of expression and deeper staining of LC3-II positive cells compared to rats in the CON group (p < 0.05, Figure 2a, b). In addition, there was an increased area of expression and significantly deeper staining of p62-positive cells (p < 0.05, Figure 2a, d) in the SAP group, while the area of expression of LAMP-2-positive cells decreased with lighter staining (p < 0.05, Figure 2a, c). Transmission electron microscopy revealed a significant increase in autophagic vesicles in the SAP group compared to the CON group. These vesicles were distributed in clustered aggregates and some vesicles were surrounded by damaged mitochondria and endoplasmic reticulum (Figure 2e, f).

Figure 2.

Autophagy impairment and recovery. (a- 1-9) Immunohistochemistry analysis of LC3-II, LAMP-2 and p62 protein expression levels in each group of rats (400×). (b–d) Quantitative analysis of protein expression in each group of rats. (e) Transmission electron microscope observation in CON group of rats (15000×). (f) Transmission electron microscope observation in SAP group of rats (15000×). (g) Transmission electron microscope observation in SAP+PHI group of rats (15000×). Data shown are means ± SEM. Compared with CON group. *P < 0.05, **P < 0.01, ****P < 0.0001 ; compared with SAP group, #P < 0.05, ####P < 0.0001.

PHI facilitates pancreatic injury recovery in rats with SAP

After intraperitoneal injection of PHI into SAP rats, serum amylase levels in the SAP+PHI group were significantly lower than those in the SAP group (p < 0.05, Figure 1e, f). Microscopic observation using H&E staining revealed diffuse lobular and lobular septal swelling in the pancreatic tissue of the SAP+PHI group, some localized follicular septal swelling along with a small number of necrotic follicular cells and local infiltration of inflammatory cells. Localized fat necrosis was observed in a few cases. Pancreatic pathology scores were significantly lower in the SAP+PHI group compared to the SAP group (p < 0.05, Figure 1c, d).

PHI partially restores the impaired autophagic flux in rats with SAP

The expression area of LAMP-2-positive cells increased significantly and the staining was significantly deeper in the SAP+PHI group compared to the SAP group(p < 0.05, Figure 2a, c). The expression area of p62-positive cells decreased and the staining was significantly weaker (p < 0.05, Figure 2a, d). However, there was no significant change in the expression area and staining of LC3-II-positive cells (p > 0.05, Figure 2a, b). Transmission electron microscopy showed that the number of autophagic vesicles in the SAP+PHI group was lower than in the SAP group. The vesicles were scattered and the structure of mitochondria and endoplasmic reticulum was normal with occasional damage (Figure 2g).

PHI restores impaired autophagic flux through the PI3K/Akt/mTOR signaling pathway

The activation indicators were p-PI3K, p-Akt and p-mTOR. The protein expression levels of p-PI3K, p-Akt and p-mTOR were found to be reduced in the SAP+PHI group compared to the SAP group (p < 0.05, Figure 3a–d), as determined by protein blotting analysis. However, there was no statistically significant difference in the protein expression levels of PI3K, Akt and mTOR between the groups (p > 0.05, Figure 3a, e–g).

Figure 3.

Protein expression of the PI3K/Akt/mToR signalling pathway. (a) Western blot analysis of p-PI3K, p-Akt, p-mToR, PI3K, Akt and mToR protein expression levels in each group of rats. (b–g) Quantitative analysis of protein expression in each group of rats. Data shown are means ± SEM. Compared with CON group. **P < 0.01, ***P < 0.001. Compared with SAP group, #P < 0.05, ##P < 0.01.

Discussion

SAP is a complex clinicopathological syndrome, its specific mechanism is not yet fully understood, but its serious complications and relatively high mortality rate have already imposed a heavy burden on the family and society. ¹⁶ In recent years, the use of TCM to intervene in cellular autophagy has been emphasized by scholars at home and abroad, and PHI has gradually come into the focus of many researchers due to its multi-targeted and multi-step pharmacological effects, but the specific roles of PHI and autophagy in SAP are still not clear. In this study, we used the 5% sodium taurocholate pancreaticobiliary retrograde injection method to establish a rat SAP model to investigate the relationship between autophagy, pancreatic injury and the role of early intervention of PHI and its possible mechanism in the rat SAP model. It was confirmed that autophagy was abnormally initiated but the completion process was impaired in the rat model of SAP, and PHI could restore the impaired autophagic flux by inhibiting the PI3K/AKT/mTOR signaling pathway, thus exerting a protective effect on the pancreas.

This study found a significant increase in serum amylase and lipase indices when SAP occurred. H&E staining revealed marked pathological damage characterized by tissue oedema, high numbers of inflammatory cell infiltrations, necrosis, hemorrhage and other pathological changes. However, administration of PHI in the early intervention phase resulted in a reduction in serum amylase and lipase levels. The results of H&E staining suggested that there was a reduction in tissue oedema and local inflammatory cell infiltration, indicating a relative alleviation of damage. The damage observed was limited to tissue oedema with local inflammatory cell infiltration and occasional local fat necrosis. Taken together, these findings suggest that early intervention in pancreatic health issues significantly protects pancreatic tissue in rats with SAP. Further investigation into the mechanism by which PHI affects autophagy may provide novel therapeutic targets or approaches, given the growing consensus among researchers regarding the relationship between SAP and autophagy.^17,18

SAP is associated with impaired autophagic flux, which is primarily characterized by abnormal autophagosome formation or lysosomal damage. ¹⁹ Autophagic flux encompasses the entire autophagic process, starting with autophagy initiation, followed by autophagosome formation, and culminating in the degradation of substances. ²⁰ The initiation of autophagy and the production of relevant proteins are stimulated by various autophagic signals. Subsequently, autophagic vesicles form and expand their membranes to engulf cytoplasmic components in a closed loop. These vesicles then fuse with lysosomes, releasing hydrolytic enzymes that degrade both the membranes and the enclosed substances. This process ultimately facilitates the recycling of degraded products. Previous studies have shown that autophagy in both experimental and human cases of pancreatitis is characterized by the accumulation of autophagic vesicles and lysosomes, together with increased expression of LC3-II and p62, and decreased expression of LAMP-2. ²⁰ LC3-II, an alternative form of LC3, is critical for the formation of autophagic vesicles. After autophagy, LC3-I is converted to LC3-II and is preferentially translocated to the autophagosome membrane. After fusion with lysosomes, LC3-II is degraded by lysosomal enzymes.^21–24 Therefore, the expression of LC3-II serves as an indicator of autophagic activity and positively correlates with the amount of autophagic vesicles. P62 acts as a substrate in autophagic vesicle formation, binding to LC3-II before entering lysosomes with LC3-II for degradation.^25–28 Accumulation of p62 is a critical signaling mechanism indicating inhibited autophagic flux resulting from impaired substrate degradation. In addition, lysosome-associated membrane protein-2 (LAMP-2) stabilizes lysosomal membranes, protecting the cytoplasm from acidic protease damage and providing insight into lysosomal functionality.^29–31 Therefore, the evaluation of LC3-II, p62 and LAMP as autophagic markers provides a comprehensive assessment of autophagic flux in SAP rats and helps to identify sites of impaired autophagic flux.

In the SAP rat model, pancreatic injury activated autophagy in pancreatic follicular cells. However, completion of autophagy was inhibited, resulting in exacerbation of pathological damage in pancreatic cells due to impaired repair processes. Specifically, an increase in LC3-II levels indicated the formation of autophagosomes, but their degradation was hindered, leading to pathological accumulation. In addition, the increase in p62 levels and the decrease in LAMP-2 levels indicate a hindrance in the fusion of autophagosomes with lysosomes, which in turn exacerbates vacuolization in pancreatic follicles. The decrease in LAMP-2 levels suggests a decrease in lysosomal degradation, which may be associated with reduced internal hydrolase activity. These findings underscore the critical role of autophagy in the pathogenesis of SAP and highlight its potential as a target for the prevention and treatment of SAP. Early intervention with PHI improved autophagic flux in SAP rats by decreasing p62 levels and increasing LAMP-2 levels. This suggests that PHI may alleviate the fusion blockade of autophagic vesicles with lysosomes and enhance the lysosomal degradation function of pancreatic cells during early-stage SAP treatment in rats, preventing exacerbation of pancreatic injury. The absence of statistically significant changes in LC3-II levels suggests that the enhanced autophagy may have simultaneously increased flux recovery and consumption. Transmission electron microscopy revealed that SAP did not prevent the formation of autophagic vesicles, but rather increased their formation and accumulation. The autophagy completion pathway was restored after PHI intervention, resulting in enhanced degradation and a relative decrease in autophagic vesicles.

Autophagy is an important mechanism in the development of SAP. The role of the mTOR pathway in the development of SAP has received considerable attention. The mTOR-related targets are central to the initiation of autophagy and are regulated by various signaling pathways, including PI3K/Akt, MAPK and NF-κB.^32–35 The PI3K/Akt/mTOR signaling pathway is crucial for the regulation of autophagy. Autophagic activity is suppressed by phosphorylation of the activated PI3K/Akt/mTOR signalling cascade.^36–38 Studies have shown that xanthohumol can restore autophagic flux in experimental SAP mice by inhibiting the AKT/mTOR pathway. ³⁹ Thus, monitoring the expression levels of proteins associated with the PI3K/Akt/mTOR pathway not only helps to elucidate autophagic mechanisms, but also to find the therapeutic approaches for SAP by targeting autophagic pathways. This research found that PHI can rescue the autophagic flux in SAP rats by inhibiting the PI3K/Akt/mToR signaling pathway. This intervention can prevent the progression and worsening of SAP. The SAP group showed higher expression levels of LC3-II and p62, lower levels of LAMP-2 and increased expressions of p-PI3K, p-Akt and p-mTOR proteins compared to the CON group. The results indicate that rats with SAP exhibited impairment of the autophagy pathway, leading to accumulation of autophagic vesicles and abnormal activation of the PI3K/Akt/mTOR signaling pathway. In contrast, pancreatic tissues from the SAP+PHI group showed decreased levels of p62, p-PI3K, p-Akt, p-mTOR and increased LAMP-2 expression compared to the SAP group. The results suggest that PHI can effectively suppress the early activation of the PI3K/Akt/mTOR pathway, which facilitates the successful completion of autophagy in pancreatic tissue and prevents further pancreatic damage. Previous studies have shown that activation of the PI3K/Akt/mTOR pathway attenuates the transition from LC3-I to LC3-II, ⁴⁰ thereby inhibiting the formation of autophagic vesicles. This is consistent with the results of the current study, in which no statistically significant differences in LC3-II levels were observed. This is likely to be due to an increase in autophagic vesicle formation followed by depletion during smooth autophagic flux.

Progress in modern medicine depends on identifying and exploiting the intrinsic mechanisms of disease. ⁴¹ Autophagy modulators, PI3K-AKT-mTOR pathway inhibitors, AMPK agonists and lysosomal inhibitors have been used to treat tumours, neurodegenerative diseases and metabolic disorders. ¹³ Despite these advances, the clinical application of autophagy modulators is hampered by their poor selectivity and unclear mechanisms of action, posing significant challenges to researchers. Recent studies have highlighted PHI as a promising clinical drug, noting its lack of significant toxic side effects both in vivo and in vitro. ¹³ SAP remains a fatal disease with a mortality rate of up to 30% and, despite extensive basic research, effective drug targets are still lacking. In recent years, natural products have gained attention as potential treatments for SAP, with PHI emerging as a potentially active monomeric compound for multiple diseases. ⁴² Our current study is the first to demonstrate that PHI can mitigate autophagy damage induced by sodium taurocholate in SAP rats, revealing a novel functional application of this drug and providing new insights into the combination of clinical drugs in the treatment of SAP. Future research should focus on a comprehensive investigation of the signaling targets of PHI in relation to cellular autophagy. In addition, the integration of various research tools such as genomics, transcriptomics, metabolomics and proteomics could facilitate the screening of differentially expressed genes, metabolites and proteins. These approaches will elucidate the molecular mechanisms and targets responsible for the broad pharmacological effects of PHI, thereby advancing medical development.

The present study has several limitations that need to be considered. Firstly, the therapeutic effect of PHI was assessed by designing three experimental subgroups: the control group (sham operation group), the SAP group and the PHI group. Comparisons were made between the SAP and PHI groups using the control group as a baseline. Although the results were clear, they lacked specificity regarding the therapeutic effect. Future studies should explore alternative reagents to PHI or use gene silencing techniques to increase the robustness of the experimental results. Secondly, although PHI is known to have multiple pharmacological effects and to involve numerous signaling pathways, our study focused exclusively on its regulation of autophagy via the PI3K/Akt/mTOR pathway. It remains a challenging question whether other signaling components play a critical role in autophagy generation in the SAP model and whether PHI affects these components. Our future research will explore the mechanisms of PHI and autophagy using network pharmacology, enrichment experiments or knockout models to facilitate more comprehensive studies. Thirdly, no systematic and in-depth toxicological assessment of PHI has been performed in our study, highlighting the urgent need to elucidate its toxicological effects and mechanisms to ensure safety and efficacy. Understanding the interaction of PHI with other drugs is also crucial, as prolonged or excessive use may result in adverse effects. ³⁵ Fourth, the sample size in this study was chosen empirically. Finally, this study was limited to in vivo animal models and further research including cellular and clinical studies is needed.

Conclusion

In conclusion, this study is the first to demonstrate that early intervention with PHI can mitigate pancreatic pathological injury in a SAP rat model. The underlying mechanism involves promoting the resolution of abnormal autophagy in pancreatic tissue induced by SAP through inhibition of the PI3K/AKT/mTOR pathway. This process helps to rescue the impaired autophagic flux, thereby achieving the protective effect on the organism. The results of this study provide a theoretical basis for the clinical treatment and prevention of SAP in patients. However, the study has certain limitations. It is expected that future research by innovative clinicians will address these limitations and apply these findings to further advance human health.