The Effects of Da-Chai-Hu Decoction Alleviating Pancreatic Exocrine Dysfunction by Inhibiting Endoplasmic Reticulum Stress of Acinar Cell in Mice with Chronic Pancreatitis

Figure 1. Continued.

Figure 1. DCHD Alleviates Pancreatic Damage and Exocrine Dysfunction in CP. (A) Representative figures of pancreatic tissues in different groups; (B) Pancreas-to-body weight ratio; (C) H&E staining and Sirius Red staining of pancreatic tissues; (D) Pathological scores based on H&E staining; (E) Representative immunohistochemical staining of amylase in pancreatic tissues (400×); (F) RT-qPCR analysis of Cpa-1 gene expression; (G) Western blot analysis of amylase protein expression levels. The region delineated by the blue dashed box in the upper image of each group is magnified in the corresponding lower image, thus enabling detailed observation of pancreatic tissue alterations. Data are mean ± SD. n=6. *P<0.05, **P<0.01, ***P<0.001.

Figure 2. DCHD reduces pancreatic inflammation and fibrosis in CP mice. (A) Representative immunohistochemical staining of α-SMA, Collagen I and F4/80 in pancreatic tissues (400×); (B) Western blot analysis of α-SMA and IL-6 protein expression levels; (C and D) RT-qPCR analysis of fibrosis-related genes (C) and inflammation-related genes (D) in CP mice. Data are mean ± SD. n=6. *P<0.05, **P<0.01, ***P<0.001.

Figure 3. Continued.

Figure 3. Transcriptomic Analysis of Pancreatic Tissue. (A) PCA analysis; (B) Volcanic plot shows differences in gene expression; (C–E) Heatmaps display the expression of Inflammation (C), Fibrosis (D) and ERS (E) related differentially expressed genes in each group; (F–I) GO and KEGG enrichment analysis.

Figure 4. Network Pharmacology Analysis of DCHD in Regulating ERS in CP. (A) Clustering of the overall samples; (B) Differential metabolite K-means diagram; (C) Tic diagram of sub class3 metabolites detected by mass spectrometry; (D) Venn diagram showing the overlap between CP, DCHD and ERS related targets; (E and F) GO (E) and KEGG pathway (F) enrichment analysis of DCHD-CP-ERS targets, the area marked by the red dashed box in the figure represents the PI3K/AKT signaling pathway.

Figure 5. DCHD Inhibits Pancreatic ERS in vivo and the Mechanism. (A) Western blot analysis of ERS related proteins ATF6, DDIT3, and GRP78; (B) RT-PCR analysis of ERS related gene; (C) Immunofluorescence double staining showing DDIT3 and Amylase expression; (D) Western blot analysis the protein expression of P-PI3K, PI3K, P-AKT, and AKT; (E) Immunofluorescence double staining of Amylase and P-PI3K, Amylase and P-AKT in pancreatic tissues. Data are mean ± SD. n=6. *P<0.05, **P<0.01, ***P<0.001.

Figure 6. Continued.

Figure 6. Mechanisms of medicated serum of DCHD in regulating ERS in Pancreatic Acinar Cells. (A and B) Western blot analysis of ATF6, GRP78, DDIT3 (A), P-PI3K, P-AKT, PI3K, AKT (B) expression in 266–6 cell treated with or without DCHD-containing serum; (C) Western blot analysis of GRP78, DDIT3, P-PI3K, PI3K expression in 266–6 cell treated with or without PI3K inhibitors (LY294002); (D) RT-PCR analysis the mRNA expression of Bid and Apaf1 in CP and DCHD treated mice pancreatic tissue; (E) TUNEL staining to detect cell apoptosis treated with or without DCHD-containing serum (400×); (F) RT-PCR analysis the expression of Cpa1 and Ptf-1αin 266–6 acinar cell treated with or without DCHD-containing serum; (G) Immunofluorescence staining for amylase in 266–6 cells treated with or without DCHD-containing serum (600×). Data are mean ± SD. n=6. *P<0.05, **P<0.01, ***P<0.001.

Figure 7. Mechanism diagram of DCHD inhibiting ERS and restoring exocrine pancreatic function in CP.