Mst1 participates in the progression of severe acute pancreatitis-induced intestinal barrier dysfunction via inhibiting autophagy and enhancing apoptosis

Materials and animals

Mst1 knockout (Mst1^−/−, C57BL/6 background, generation N10) mice and their wild-type littermates (WT) were obtained from K&D gene technology (WuHan, China) and genotyped by Western blotting and real-time PCR. All mice were housed in a conventional facility with 12h dark-light cycles and allowed free access to adequate food and water. All animal experiments were strictly performed in accordance with the guidelines for the Care and Use of Laboratory Animals formulated by the Laboratory Animal Welfare and Ethics Committee of Northwest University (Xi’an), as well as the National Research Council’s Guide for the Care and Use of Laboratory Animals, and the ARRIVE guidelines. This study was formally approved by the Laboratory Animal Welfare and Ethics Committee of Northwest University (Xi’an) (Approval No.: NWU-LAEC-XA-2023-071). All procedures were designed to minimize animal suffering and discomfort, and every effort was made to reduce the number of experimental animals used, in strict adherence to the 3R principle.

Caerulein (#CM05482) were purchased from Proteintech Group, Inc. (Wuhan, China). LPS (#L4524), bafilomycin A1 (#88899-55-2) and TNF-α (Escherichia coli lipopolysaccharide, 055: B5, #L2880) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). α-Amylase assay kit (#C016-1-1), lipase assay kit (#A054-1-1), endotoxin assay kit (#E039-1-1), diamine oxidase (DAO) assay kit (#A088-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse TNF-α ELISA kit (#CSB-E04741m), interleukin 1β (IL-1β) ELISA Kit (#CSB-E08054m), and interleukin 6 (IL-6) ELISA Kit (#CSB-E04639m) were obtained from CUSABIO Biology, Inc. (Wuhan, China). TUNEL detection kit (#C1088) were obtained from Beyotime Biotechnology Inc. (Shanghai, China). Anti-cleaved caspase-3 (#ab214430), anti-bcl-2 (#ab16904), anti-bax (#ab182734), anti-Mst1 (#ab51134), anti-beclin-1 (#ab207612), anti-p62 (#ab240635), anti-ZO-1 (#ab307799), anti-Claudin 1 (#ab307692), and anti-Occludin (#ab216327) antibodies were obtained from Abcam (Cambridge, UK). Anti-p-Mst1 (#49332) and LC3A/B (#4108) were purchased from Cell signaling (Danvers, MA, USA).

Experimental SAP animal model and survival study

The SAP mouse model was established as previously reported.^14–16 Briefly, mice received intraperitoneal injections of caerulein (50 μg/kg per hour) for 10 times, with LPS (10 mg/kg) injected intraperitoneally at the time of the last caerulein injection. Control mice received equivalent volumes of saline. SAP mice were sacrificed by intracardiac puncture to collect blood samples for biochemical and pathological assessments at 48 hours after the first caerulein injection. For survival study, the living condition of SAP mice was closely monitored, and the mortality was recorded twice daily for 5 days after the first caerulein injection. Littermate mice were used in the survival study. Each group contained 15 mice. The sample size (n=15 per group) was determined based on a power analysis with an expected effect size of 30% reduction in mortality, α=0.05 and considering a potential 20% attrition rate.

Serum lipase, amylase, diamine oxidase (DAO) and endotoxins detection

Blood was drawn from the heart and centrifuged at 4000 rpm for 10 minutes to separate serum. Serum levels of lipase, amylase, DAO activity and endotoxins were measured using commercial kits according to the manufacturers’ protocols.

HE staining and TUNEL staining

Pancreas and intestinal segments were collected, fixed in 4% paraformaldehyde overnight at room temperature, and then routinely embedded in paraffin, sectioned at 5 μm thickness, and stained with HE to observe histopathological changes under a light microscope. Pathological scores were calculated in a double-blind manner as previously described.^7,17,18 Pancreatic pathological scores were based on the degree of hemorrhage, edema, neutrophil infiltration, and necrosis. Intestinal damage severity was scored on a 0–5 scale (0, none; 1, mild; 2, moderate; 3, marked; 4, severe; 5, extremely severe).

For TUNEL staining, paraffin sections of intestinal segments were dewaxed, rehydrated, and incubated with proteinase K for 20 min at 37°C. After treatment with 3% H₂O₂ for 10 min at room temperature, sections were processed using a TUNEL assay kit following the manufacturer’s instructions. Cell nuclei were counterstained with DAPI (#ab285390, Abcam). TUNEL-positive cells were imaged using a fluorescence microscope (Nikon Eclipse NI-E; Nikon Corporation) and counted in three randomly selected fields per section to assess intestinal epithelial apoptosis.

Enzyme-linked immunosorbent assay (ELISA)

Serum concentrations of TNF-α, IL-1β, and IL-6 were determined using commercially available ELISA kits according to the manufacturers’ instructions.

Cell culture and transfection

MODE-K cells (the mouse intestinal epithelial cell line, #BFN608006456, Bluefbio. Inc., Shanghai, China) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. After serum starvation for 12 h, exponentially growing cells (≈80% confluence) were transfected with adenoviral vectors overexpressing Mst1 (Ad-Mst1, MOI=100, customized by Hanbio Biotechnology, Shanghai, China) for 24 h. Cells transduced with control adenoviral vectors (Ad-Control, MOI=100) served as controls. All cells were then stimulated with 10 ng/mL TNF-α or equivalent saline in the presence or absence of bafilomycin A1 for an additional 24 h. At the end of the experiment, cells were collected for Western blot analysis.

Western blot analysis

Total protein was extracted from intestinal tissues or MODE-K cells, and protein concentrations were determined using the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibodies, followed by HRP-conjugated secondary antibodies, and signals were detected using enhanced chemiluminescence. Immunoblots were analyzed using a gel imaging system (Bio-Rad, CA, USA). Expression levels of ZO-1, claudin-1, occludin, Mst1, p-Mst1, beclin-1, p62, LC3, cleaved caspase-3, Bcl-2, and Bax were measured. GAPDH was used as a loading control.

Statistical analysis

All data are expressed as mean ± standard deviation. Statistical differences were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons. Survival data were analyzed by the Kaplan Meier method with the log-rank test for multiple comparisons. A P value < 0.05 was considered statistically significant.

Mst1 knockout improved the accumulative mortality and relieved intestinal barrier dysfunction of SAP mice

Mst1 knockout greatly reduced the mortality of SAP mice. As shown in Figure 1(a), the 5-day cumulative mortality in WT+SAP mice was approximately 75%, whereas that in Mst1^-/-+SAP mice was about 55%, representing a marked reduction. WT+SAP mice exhibited severe pancreatic injury and intestinal barrier dysfunction, as evidenced by increased serum levels of lipase (Figure 1(b)), amylase (Figure 1(c)), DAO (Figure 1(d)), endotoxin (Figure 1(e)), and the inflammatory cytokines TNF-α, IL-1β, and IL-6 (Figure 1(i)–(k)). Histopathological examination of the pancreas revealed extensive widening of interlobular spaces, inflammatory cell infiltration, acinar cell necrosis, and hemorrhage (Figure 1(f)), leading to a significantly higher pancreatic pathological score in WT+SAP mice (Figure 1(g)). Similarly, ileocolic tissues from WT+SAP mice showed focal necrosis, epithelial cell detachment, basement membrane disruption, villous stroma broadening, edema, and congestion (Figure 1(f)), with a significantly increased intestinal pathological score (Figure 1(h)).OPEN IN VIEWER

Mst1 knockout did not reduce serum lipase or amylase levels (Figure 1(b) and (c)) nor improve pancreatic histopathology or pathological scores in Mst1^-/-+SAP mice (Figure 1(f) and (g)). However, Mst1 knockout significantly reduced serum DAO (Figure 1(d)) and endotoxin (Figure 1(e)) levels, ameliorated ileocolic histopathological changes (Figure 1(f) and (h)), and decreased serum levels of TNF-α, IL-1β, and IL-6 (Figure 1(i)–(k)). These results indicate that intestinal barrier dysfunction is a critical factor in the progression of SAP, and that Mst1 knockout significantly improves intestinal barrier function, thereby alleviating systemic symptoms and improving prognosis.

Mst1 knockout up-regulated tight junction proteins and alleviated apoptosis in the ileocolic mucosa tissue of SAP mice

We next examined the expression of the tight junction proteins ZO-1, claudin-1, and occludin in ileocolic mucosal tissue. As shown in Figure 2(a)–(d), the expression of all three proteins was markedly reduced in WT+SAP mice. In contrast, Mst1 knockout significantly upregulated these proteins in Mst1^-/-+SAP mice. TUNEL staining revealed a significant increase in TUNEL-positive cells in the ileocolic mucosa of WT+SAP mice, whereas Mst1 knockout markedly reduced the number of apoptotic cells (Figure 2(e)). These findings suggest that reduced tight junction protein expression and increased apoptosis contribute to intestinal barrier dysfunction in WT+SAP mice, and that Mst1 knockout alleviates this dysfunction by restoring tight junction proteins and reducing apoptosis.OPEN IN VIEWER

Mst1 knockout enhanced autophagy in the ileocolic mucosa of SAP mice

We further analyzed the expression of Mst1, p-Mst1, and autophagy-related proteins in ileocolic mucosal tissue. Mst1 and p-Mst1 levels were significantly elevated in WT+SAP mice (Figure 3(a)–(d)), accompanied by decreased beclin-1 and LC3-II expression and increased p62 expression (Figure 3(a), Figure 3(e)–(g)), indicating impaired autophagy. As expected, Mst1 knockout resulted in undetectable Mst1 and p-Mst1 expression in Mst1 ^-/- mice and Mst1 ^-/- +SAP mice (Figure 3(a)–(d)). Notably, Mst1 knockout markedly enhanced autophagy, as evidenced by increased beclin-1 and LC3-II expression and decreased p62 expression (Figure 3(a), Figure 3(e)–(g)). These results suggest that phosphorylation-dependent activation of Mst1 contributes to impaired autophagy in WT+SAP mice, and that Mst1 knockout protects intestinal barrier function by promoting autophagy.OPEN IN VIEWER

Mst1 overexpression inhibited autophagy and promoted apoptosis in MODE-K cells subjected to TNF-α stimulation

MODE-K cells transfected with Ad-Mst1 and stimulated with TNF-α (10 ng/mL) showed significantly increased Mst1, p-Mst1, and p62 expression, and decreased beclin-1 and LC3-II expression (Figure 4(a)–(f)). Furthermore, autophagic flux, assessed using the lysosomal inhibitor bafilomycin A1, was inhibited following Ad-Mst1 transfection, as shown by decreased LC3-II and increased p62 accumulation (Figure 4(g)–(i)). These data indicate that Mst1 overexpression blocks autophagic flux. Additionally, Ad-Mst1 transfection increased the expression of cleaved caspase-3, Bcl-2, and Bax compared with the TNF-α alone group (Figure 5(a)–(d)). Collectively, these results demonstrate that Mst1 overexpression inhibits autophagic flux and promotes apoptosis in TNF-α-stimulated MODE-K cells.OPEN IN VIEWER

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Figure 5.

Mst1 overexpression promoted apoptosis in MODE-K cells subjected to TNF-α stimulation. The expression of apoptosis related molecules in MODE-K cells transfected with Ad-Mst1 or Ad-Control following stimulation with 10 ng/ml of TNF-α were investigated. (A) The representative immunoblots of Cleaved caspase-3, bcl-2, and Bax in MODE-K cells. GAPDH was used as a loading control. The levels of Cleaved caspase-3 (B), bcl-2 (C), and Bax (D) were analyzed. Data are expressed as mean ± S.D., n=3 independent experiments, **P < 0.01 vs. Con group, ^##P < 0.01 vs. TNF-α group.