3.1 DCQD or 3-MA could inhibit serum Amylase, TNF-α, Beclin1 and ROS levels in AP rats.
Amylase serum level can be used as an important indicator for judging the occurrence and development of acute pancreatitis. TNF-α is one of the earliest and key endogenous pro-inflammatory cytokines released in the process of inflammatory response. Beclin1 and ROS are the key factors to measure the level of autophagy. Therefore, we detected the expression of serum Amylase, TNF-α, Beclin1 and ROS in AP group at different time points, autophagy inhibitors and after administration of DCQD. As Fig. 1 shows, compared with the Ctrl group, the expression of serum Amylase and TNF-a, Beclin1 and ROS in AP group increased significantly and further developed over time. After administration of autophagy inhibitors and DCQD, the concentration of serum Amylase (Fig. 1A) and TNF-a (Fig. 1B), Beclin1 (Fig. 1C) and ROS (Fig. 1D) decreased significantly at each time point. Linear regression analysis of serum Beclin1 concentration and ROS concentration indicated that there was a significant correlation between serum Beclin1 and ROS concentration in rats (Fig. 1E). Linear regression analysis of serum Beclin1 concentration and TNF-α concentration indicated that serum Beclin1 concentration was significantly correlated with TNF-α concentration in rats (Fig. 1F).
3.2 DCQD or 3-MA could improve pathological damage of pancreatic tissue in AP rats, and the therapeutic effect of DCQD or 3-MA at different time is different in AP rats.
The pathological changes of the pancreas were observed in four groups of rats. In the Ctr group, there were no obvious changes in the pancreas, and no obvious ascites in the abdominal cavity. Pancreatic swelling was observed in AP group at 3h. In AP group, the pancreas was obviously swollen at 6h, and bleeding spots were observed on some surfaces, accompanied by a small amount of ascites. In the AP group, there were pale yellow to dark brown ascites in the abdominal cavity at 24 hours, with varying amounts of ascites, obvious gastrointestinal dilatation, pancreatic spot and flake bleeding, blackening, saponified spots on the omentum and the surface of the pancreas (Fig. 2A).
The pathological changes of the pancreas were observed in four groups of rats at different treatment times. In the Ctr group, the pancreatic alveolar structure was intact, the interlobular interval was clear, and there was no obvious inflammatory cell infiltration; in the AP group, the pancreatic alveoli and interstitial edema were obvious, the interlobular interval was blurred, and a small amount of inflammatory cell infiltration was visible, occasionally erythrocyte infiltration was seen; in the AP rats, the pancreatic tissue structure was slightly disorganized at 6h, the pancreatic alveoli were pitting necrosis, inflammatory cell infiltration was visible, and some interstitial congestion and hemorrhage were seen; in the AP rats At 24h, the pancreatic alveolar structure was disrupted, with varying degrees of necrosis and peripheral hemorrhage, and large sheets of necrosis were seen, accompanied by a large number of inflammatory cell infiltrates; the AP + DCQT and AP + 3-MA groups had significantly improved pancreatic pathological changes at each time point compared with the AP group.
The pathological results showed that L-arginine model was successfully established in rats. Both DCQD and autophagy inhibitors could improve pancreatic pathology and maintain pancreatic function (Fig. 2B). Serum Beclin1 concentration was correlated with pancreatic pathological score by linear regression analysis, suggesting that rat serum Beclin1 was significantly correlated with pancreatic pathological score (Fig. 2C).
3.3 DCQD or 3-MA could cover the cellular structure of pancreatic cells of AP rats.
Therapeutic effect of DCQD or 3-MA on glandular vesicle cells by transmission electron microscopy. Six hours after modeling, the Ctr group showed uniform distribution of intracellular zymogen particles, high density of distribution, normal size of nucleus, intact nuclear membrane structure, regular arrangement of endoplasmic reticulum, normal size of mitochondria, dense, clear internal cristae, no obvious autophagic vesicles or autophagic vacuole formation; in the AP group, the normal structure of glandular vesicle cells disappeared, calcium soap spots appeared, the number of autophagic vacuoles increased significantly, microscopically the pancreatic lobular structure was destroyed, and capillary normal structure was seen. In the AP + DCQD group, the capillary duct wall was loosely structured, but the erythrocytes were regularly shaped, the interstitial contents of the lobules were increased, the inflammatory cells were scattered, and the number of autophagic vacuoles was reduced compared with the AP group; in the AP + 3-MA group, the capillary wall was intact, the erythrocyte structure in the lumen was intact, and a small number of autophagic vesicles were seen, which were reduced compared with the AP group. (Fig. 3)
3.4 DCQD or 3-MA could inhibit autophagy related protein Beclin1 in different time points
Detection of Beclin1 protein expression levels in pancreatic tissues of four groups of rats at different time periods by western blot. Figure 4A shows that Beclin1 protein expression was significantly increased in the pancreatic tissue of AP group. Beclin1 was significantly higher in the AP group at 3 hours (Fig. 4B), 6 hours (Fig. 4C) and 24 hours (Fig. 4D) compared to the Ctr group. Beclin1 expression was significantly lower after DCDQ and 3-MA treatment compared with the AP group. Immunohistochemical results of pancreatic tissue showed that Beclin1 positivity in pancreatic tissue was mainly in the cytoplasm of pancreatic alveolar cells. In Ctr group, Beclin1 expression in pancreatic alveolar cells was low, and there was no significant difference at each time point; Beclin1 positive cells were significantly more expressed in the AP group at each time point compared with the Ctr group. And more positive cells were found in the AP group at 6h and 24h, with darker cell cytoplasm coloring and stronger expression; Beclin1 positive cells were reduced in the AP + DCQT and AP + 3-MA groups compared with the AP group at each time point, and cell cytoplasm coloring was significantly lighter and expression was weakened (Fig. 4E).
3.5 DCQD or 3-MA could inhibit autophagy related protein LC3-II in different time points
The intensity of microtubule-associated protein LC3 expression is closely related to autophagic activity. LC3 is subdivided into two isoforms. When autophagy is low level, it exists in the form of cytoplasmic soluble LC3 I; when autophagy is activated, LC3 I undergoes ubiquitination to form type II LC3, the amount of which is proportional to the number of autophagic vesicles(Kabeya et al.,2000;Zhou et al.,2022). Detection of LC3-II protein expression levels in pancreatic tissues of four groups of rats at different time periods by western blot. LC3-II expression was significantly higher in the AP group at 3h, 6h and 24h, and was positively correlated with time (Fig. 5A). Compared with the Ctr group, LC3-II was significantly higher in the AP group at 3h (Fig. 5B), 6h (Fig. 5C) and 24h (Fig. 5D), and after DCQD and 3-MA treatment, LC3-II was significantly reduced compared with the AP group. The positive expression of LC3-II in pancreatic tissue was found in the cytoplasm of pancreatic alveolar cells, but not in the nucleus. Immunohistochemical (Fig. 5-E) results showed that the expression of LC3-II in pancreatic alveolar cells was low in the Ctr group, and there was no significant difference at each time point; the expression of LC3-II in the AP group was significantly higher than that in the Ctr group at each time point, and there were more positive cells in the AP group at 6h and 24h, with darker cytoplasm coloration and enhanced expression; the expression of LC3-II in the AP + DCQT and AP + 3-MA groups was significantly lower than that in the AP group at each time point. DCQT and AP + 3-MA groups had fewer LC3-II positive cells, lighter cytoplasmic coloration and weaker expression at each time point compared with the AP group.