Chaihu Guizhi Ganjiang Decoction Ameliorates Pancreatic Fibrosis via JNK/mTOR Signaling Pathway

Abstract

Pancreatic fibrosis is a pathological characteristic of chronic pancreatitis (CP) and pancreatic cancer. Chaihu Guizhi Ganjiang Decoction (CGGD) is a traditional Chinese medicine, which is widely used in the clinical treatment of digestive diseases. However, the potential anti-fibrosis mechanism of CGGD in treating CP remains unclear. Here, we conducted a series of experiments to examine the effect of CGGD on the CP rat model and primary isolated pancreatic stellate cells (PSCs). The results revealed that CGGD attenuated pancreatic damage, decreased collagen deposition, and inhibited PSC activation in the pancreas of CP rats. However, compared with the CP group, CGGD had no effect on body weight and serum amylase and lipase. In addition, CGGD suppressed autophagy by downregulating Atg5, Beclin-1, and LC3B and facilitated phosphorylation of mTOR and JNK in pancreatic tissues and PSCs. Moreover, the CGGD-containing serum also decreased LC3B or collagen I expression after rapamycin (mTOR inhibitor) or SP600125 (JNK inhibitor) treatment in PSCs. In conclusion, CGGD attenuated pancreatic fibrosis and PSC activation, possibly by suppressing autophagy of PSCs through the JNK/mTOR signaling pathway.

Keywords: Chaihu Guizhi Ganjiang Decoction; JNK/mTOR; autophagy; chronic pancreatitis; pancreatic stellate cells.

FIGURE 1

Typical chromatograms of mixed standard compounds (A) and CGGD (B). (1) liquiritin apioside, (2) liquiritin, (3) isoliquiritin apioside, (4) baicalin, (5) cinnamic acid, (6) wogonoside, (7) glycyrrhizic acid, and (8) wogonin.

FIGURE 2

Effects of CGGD on the body weight, serum amylase and lipase, and histological changes in CP rats induced by DBTC. (A) Body weight at different time points. (B) Serum amylase and lipase detected by ELISA. (C) Pancreatic morphological changes observed by H&E staining and scored by a pathologist (original magnification, × 200). n = 7–10, ^* p < 0.05 compared with the control group; ^# p < 0.05 compared with the CP group.

FIGURE 3

Effects of CGGD on collagen deposition and PSC activation in the pancreas. (A) Sirius red staining for collagen deposition in the pancreas (original magnification, ×200). (B) Immunohistochemistry staining of α-SMA (brown staining) in pancreatic tissues. Hematoxylin was used to counterstain nuclei (original magnification, ×200). (C) qRT-PCR analysis of α-SMA, COL I, FN, MMP2, and TIMP2. (D) Western blot analysis of α-SMA, COL I, and FN. Data are expressed as mean ± SD (n = 3). ^* p < 0.05 compared with the control group; ^# p < 0.05 compared with the CP group.

FIGURE 4

Influence of CGGD on autophagy and the mTOR pathway in the pancreas. (A) Immunohistochemistry staining of LC3B (brown staining) in pancreatic tissues. Hematoxylin was used to counterstain nuclei (original magnification, ×200). (B) mRNA levels of Beclin-1, Atg5, and LC3B in the pancreas detected by qRT-PCR. (C) Western blot analysis of Beclin-1, Atg5, and LC3B-I/II in the pancreas. (D) Western blot analysis of p-mTOR and mTOR in the pancreas. GAPDH was used as the loading control. Data are expressed as mean ± SD (n = 3). *p < 0.05 vs. control; #p < 0.05 vs. CP.

FIGURE 5

CGGD-containing serum (CGGDs) inhibited PSC activation and autophagy. PSCs were treated with 20% or 50% control serum (CONs) or CGGDs, respectively, for 24 h. (A) RT-PCR analysis of α-SMA, COL I, and FN. (B) Western blot analysis of α-SMA, COL I, and FN. (C) Densitometry analysis of the western blot bands of (B). (D) mRNA levels of Atg5, Beclin-1, and LC3B detected by RT-PCR. (E) Western blot analysis of Beclin-1, Atg5, P62, and LC3B. The data are expressed as fold changes over the value of the 20% CONs group. The data are expressed as the mean ± SD of three independent experiments. *p < 0.05 vs. the 20% CONs group; **p < 0.05 vs. the 50% CONs group.

FIGURE 6

Effect of CGGDs on PSC autophagy and activation through the JNK/mTOR pathway. (A) Western blot analysis of p-ERK1/2, p-P38, p-JNK, p-AKT, p-AMPK, and p-mTOR after PSCs were treated with 20% or 50% CONs or CGGDs, respectively, for 24 h. (B) Protein levels of p-mTOR, LC3B-I/II, and COL I in PSCs detected after pretreatment with rapamycin (Rapa, an mTOR inhibitor) for 1 h followed by treatment with 50% CONs or 50% CGGDs for 24 h. (C) Expression levels of p-JNK, LC3B-I/II, and COL I in PSCs measured by western blot after treatment with SP600125 (a JNK inhibitor) for 1 h and 50% CONs or 50% CGGDs for 24 h. (D) Immunofluorescence staining used to detect the expression levels of α-SMA (green) and LC3B (red) after PSCs were pretreated with Rapa or SP600125 for 1 h followed by treatment with 50% CONs or 50% CGGDs for 24 h. The nuclei were stained with DAPI (blue) (original magnification: ×200). The data are expressed as the mean ± SD of three independent experiments. *p < 0.05 vs. the 20% CONs group, **p < 0.05 vs. the 50% CONs group, △p < 0.05 vs. the CONs group, #p < 0.05 vs. the CGGDs group, p < 0.05 vs. the CONs + Rapa group, and $p < 0.05 vs. the CONs + SP600125 group.

FIGURE 7

Graphical abstract of CGGD-ameliorated pancreatic fibrosis through deactivation of PSC by inhibiting autophagy via the JNK/mTOR pathway.