Sulfiredoxin-1 attenuates injury and inflammation in acute pancreatitis through the ROS/ER stress/Cathepsin B axis

Abstract

Acinar cell injury and the inflammatory response are critical bioprocesses of acute pancreatitis (AP). We investigated the role and underlying mechanism of sulfiredoxin-1 (Srxn1) in AP. Mild AP was induced by intraperitoneal injection of cerulein and severe AP was induced by partial duct ligation with cerulein stimulation or intraperitoneal injection of L-arginine in mice. Acinar cells, neutrophils, and macrophages were isolated. The pancreas was analyzed by histology, immunochemistry staining, and TUNEL assays, and the expression of certain proteins and RNAs, cytokine levels, trypsin activity, and reactive oxygen species (ROS) levels were determined. Srxn1 was inhibited by J14 or silenced by siRNA, and overexpression was introduced by a lentiviral vector. Transcriptomic analysis was used to explore the mechanism of Srxn1-mediated effects. We also evaluated the effect of adeno-associated virus (AAV)-mediated overexpression of Srxn1 by intraductal administration and the protection of AP. We found that Srxn1 expression was upregulated in mild AP but decreased in severe AP. Inhibition of Srxn1 increased ROS, histological score, the release of trypsin, and inflammatory responses in mice. Inhibition of Srxn1 expression promoted the production of ROS and induced apoptosis, while overexpression of Srxn1 led to the opposite results in acinar cells. Furthermore, inhibition of Srxn1 expression promoted the inflammatory response by accumulating and activating M1 phenotype macrophages and neutrophils in AP. Mechanistically, ROS-induced ER stress and activation of Cathepsin B, which converts trypsinogen to trypsin, were responsible for the Srxn1 inhibition-mediated effects on AP. Importantly, we demonstrated that AAV-mediated overexpression of Srxn1 attenuated AP in mice. Taken together, these results showed that Srxn1 is a protective target for AP by attenuating acinar injury and inflammation through the ROS/ER stress/Cathepsin B axis.

Fig. 1. SRXN1 expression is upregulated in mild AP but reduced in severe AP.

A In total, 22 genes commonly altered in both datasets by a cutoff of p < 0.05 and log₂ fold change >0.5 or <−0.5. B H&E staining of pancreas in the control and the groups with cerulein-induced AP (4-, 8-, and 12-hourly injections), partial pancreatic duct ligation followed by injection of cerulein (50 µg/kg body weight) 2 days after surgery, and 3-hourly intraperitoneal injections of 3.3 g/kg L-arginine. C The expression of Srxn1 RNA in acini, ducts, and islets in the cerulein-induced AP and control mice. D, E The expression of Srxn1 RNA and protein in the cerulein-induced AP and the two SAP groups. F The histological score of the SAP groups was higher than that of the cerulein-induced AP group. G, H The trypsin activity of SAP was higher than that of the cerulein-induced AP group. I The MDA of pancreatic tissue in SAP was higher than that of the cerulein-induced AP group. J The GSH/GSSH ratio of pancreatic tissue in SAP was lower than that of the cerulein-induced AP groups. Con control; SAP severe acute pancreatitis; 4 T, 4-hourly injections; 8 T, 8-hourly injections; 12 T, 12-hourly injections; n = 8; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. Inhibition of SRNX1 worsens AP in vitro and in vivo.

A Schematic of the in vitro assay using isolated acini. B Cerulein (10 nM) for 10 min significantly promoted acini’s edema, and pretreatment with J14 (5 μM) for 6 h further promoted it. C, D Pretreatment with J14 (5 μM) for 6 h significantly increased trypsin activity induced by cerulein (100 nM, 1 h) in acini. E siRNA1 exhibited the highest efficiency and was used in the subsequent assays. F, G The trypsin activity of the siRNA+cerulein group was significantly higher than that of the cerulein group in acini. H J14 (in 10% DMAC + 10% Tween 80 + 80% saline) was intraperitoneally administered at a dose of 50 mg/kg body weight once a day for 2 weeks, and cerulein (50 µg/kg body weight) was administered by 8-hourly injection on the 13th day. I, J J14 strongly increased the histological score of cerulein-induced AP. K, L J14 significantly increased the trypsin activity of cerulein-induced AP. M Srxn1 siRNA was chemically modified and suitable for in vivo application. siRNA was administered by caudal vein injection at a dose of 20 nmol/mouse two times per week for a total of 4 weeks, and cerulein was injected 2 days after the last siRNA injection. N siRNA knockdown of the RNA and protein expression of Srxn1 in pancreatic tissue. P, Q Srxn1 siRNA increased the histology score of the pancreas induced by cerulein. Con control; NC negative control; N. S no significance, *p < 0.05, **p < 0.01, ***p < 0.001; data represent five or more experiments for in vitro assays and eight or more for in vivo assays.

Fig. 3. SRXN1 attenuates AP by inducing oxidative stress and apoptosis.

A J14 (5 μM) significantly increased the DCF signal induced by cerulein (100 nM) in isolated acini. B J14 increased the MDA concentration and decreased SOD activity upon cerulein treatment in mice. C The cleaved caspase 3 level was substantially induced by cerulein (100 nM), and cotreatment with J14 further augmented it in acinar cells. D, E The cleaved caspase 3 was induced by cerulein at a dose of 50 µg/kg body weight eight times, and injection of J14 further promoted it in mice; TUNEL assay showed the same trend. F Acinar cells were isolated and cultured for 3 days, and Srxn1 siRNA was transfected for another 3 days and then cerulein (100 nM) was added. Amylase served as an indicator of acinar cells. G, H Lentivirus vector (mCherry) was transfected to overexpress Srxn1; IF staining of cleaved caspase 3 showed that Srxn1 overexpression prevented apoptosis induced by cerulein in acini. N. S no significance; Con control; NC negative control; OE overexpression; *p < 0.05, **p < 0.01, ***p < 0.001. Data represent three or more experiments for in vitro assays and eight for in vivo assays.

Fig. 4. Inhibition of Srxn1 results in accumulation and activation of M1 macrophages and neutrophils.

A, B Infiltrating M1 phenotype macrophages and neutrophils were stained with CD68 and Ly6g, respectively, in pancreatic tissues. C Flow cytometry analysis of blood showed an increase of neutrophils (CD45⁺/CD11b⁺/Ly6G⁺) in J14 + cerulein group. D Flow cytometry analysis of pancreatic tissues showed an increase of infiltrating neutrophils (CD45⁺/CD11b⁺/Ly6C⁺/Ly6G⁺) and macrophages (CD45⁺/CD11b⁺/F4/80⁺) in J14 + cerulein group. E BMDMs were isolated from marrow and stimulated for 7 days with MCSF (20 ng/mL) to differentiate into M1 phenotype macrophages and then cocultured with acinar cells. F Srxn1 siRNA largely increased the protein expression of IL6 and TNFα upon stimulation with cerulein compared with the control. LPS (500 ng/mL) served as a positive reference. G IF staining of CD68 and amylase showed that macrophages phagocytized more trypsin released from Srxn1-knockdown acini than normal acini. The white circle indicates acinar cells. H Schematic of coculture between acinar cells and neutrophils. I Neutrophils cocultured with acini transfected with Srxn1 siRNA showed significantly elevated RNA expression of Tnfα, Il6, and Cxcl10 compared with those cocultured with normal acini. LPS (500 ng/mL) served as a positive reference. Con control; *p < 0.05, **p < 0.01, ***p < 0.001. Data are three or more experiments for in vitro assays and eight for in vivo assays.

Fig. 5. ER stress-mediated activation of cathepsin B is responsible for the Srxn1 inhibition-mediated effects.

A Administration of cerulein via 8-hourly or 12-hourly injections led to significantly increased expression of p-PERK, XBP1s, and ATF4 in pancreatic tissues. B The transcriptomic profiles of acinar cells with Srxn1 knockdown or control stimulated by cerulein (50 nM) for 6 h. C GSEA showed that siRNA led to the enrichment of apoptosis, TNF, and protein-processing signaling pathways in acinar cells. D siRNA led to markedly increased expression of ER stress markers in acinar cells. E J14 significantly induced ER stress upon stimulation with cerulein compared to cerulein alone in mice. F Sal003 induces ATF4 expression, and GSK2606414 inhibits p-PERK and ATF4 expression. G Activation of ER stress by Sal003 facilitated apoptosis, while inhibition of ER stress by GSK2606414 inhibited apoptosis in acinar cells. In the presence of J14, inhibition of ER stress alleviated apoptosis nearly to the level of the cerulein group. H, I Sal003 increased trypsin activity, while GSK2606414 decreased trypsin activity in acinar cells. J Cathepsin B RNA expression was decreased by cerulein and Srxn1 siRNA. K Cerulein activated CTSB, which could be further enhanced by J14. L Inhibition of ER stress alleviated the activation of CTSB, while inducing ER stress markedly promoted the activation of CSTB in acinar cells. Con control; *p < 0.05, **p < 0.01, ***p < 0.001. Data represent three or more experiments for in vitro assays and eight for in vivo assays.

Fig. 6. AAV-mediated overexpression of Srxn1 protects against AP.

A Schematic of the microinfusion system for intraductal administration of AAV. B Fluorescence imaging of pancreas (upper) and pancreatic sections (lower) after AAV administration for 14 days. C SRXN1 RNA and protein was upregulated by AAV-Srxn1 in pancreatic tissues. D AAV-Srxn1 markedly reduced amylase and lipase activity in plasma compared with control. F Cleaved caspase 3 was decreased in AAV-Srxn1 group. G IHC and IF staining of CD68 and Ly6G showed a reduced infiltration of M1 macrophages and neutrophils by AAV-Srxn1 in pancreas. H Flow cytometry analysis of blood showed a decrease of neutrophils (CD45⁺/CD11b⁺/Ly6G⁺) in AAV-Srxn1 group. I Flow cytometry analysis of pancreatic tissues showed a decrease of infiltrating neutrophils (CD45⁺/CD11b⁺/Ly6C⁺/Ly6G⁺) and macrophages (CD45⁺/CD11b⁺/F4/80⁺) in AAV-Srxn1 group. J AAV-Srxn1 decreased the expression of p-PERK, XBP1s, and the active form of CSTB in the pancreas. **p < 0.01, ***p < 0.001. n = 5.