Tibetan Medicine Shi-Wei-Gan-Ning-San Alleviates Carbon Tetrachloride-Induced Chronic Liver Injury by Inhibiting TGF- β 1 in Wistar Rats

Abstract

Background: Shi-Wei-Gan-Ning-San (SWGNS) is a classic Tibetan prescription, which has obvious clinical effects in the treatment of viral hepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, and other liver injuries. However, animal studies and mechanism studies are still lacking. This study aimed to investigate its hepatoprotective efficacy and pharmacological mechanism in animal experiments.

Methods: Chronic liver injury was induced by oral administration of carbon tetrachloride (CCl₄) in Wistar rats for 13 weeks. SWGNS was administered orally to rats at doses of 235, 705, and 1410 mg/kg for 13 weeks. Blood samples were collected for biochemical, ELISA, and radioimmunoassay. Livers were harvested for H&E and immunohistochemical staining. The major constituents of SWGNS were analyzed by HPLC. In vitro experiments were used to explore the protective effect of Crocin on BRL-3A in the environment of H₂O₂.

Results: SWGNS reversed weight loss is induced by CCl₄. Serum assays showed that SWGNS reduced CCl₄-induced alanine aminotransferase, aspartate aminotransferase, total bilirubin, and γ-glutamyltransferase levels and increased the total protein and albumin levels. Histopathological evaluation showed that SWGNS alleviated hepatic steatosis, fibrosis, and inflammation. Furthermore, SWNGS reduced CCl₄-induced elevations of TGF-β1, hyaluronic acid, laminin, and collagen IV in serum and reduced the high expression of α-SMA in tissues. Moreover, Crocin I and II are the main components of SWGNS. Crocin attenuated the damaging effects of H₂O₂ on BRL-3A.

Conclusions: In conclusion, SWGNS alleviated CCl₄-induced chronic liver injury by inhibiting the TGF-β1 pathway. This plays an important role in promoting traditional Tibetan medicine in clinical practice.

Figure 1

SWGNS formula recovered the bodyweight of the CCl₄-treated rats. ^#P < 0.05. ^##P < 0.01. The CCl₄-induced model group versus the normal control group.^∗P < 0.05. ^∗∗P < 0.01. SWGNS at 705 mg/kg treated rats versus the CCl₄-induced model group (n = 10–13 each group).

Figure 2

SWGNS improved enzymatic activity of ALT, AST, and γ-GGT and the levels of TP, ALB, and TBIL in the CCl₄-induced hepatic fibrosis. ^#P < 0.05 and ^##P < 0.01 versus the normal control group; ^∗P < 0.05 and ^∗∗P < 0.01 versus the CCl₄-induced model group (n = 10–13 each group).

Figure 3

SWGNS alleviated CCl₄-induced liver pathological changes. (a) Representative photos of H&E staining. 200X magnification.(a) normal control; (b) CCl₄ model control; (c) CCl₄+ bifendate at 200 mg/kg; (d) CCl₄+ SWGNS at 235 mg/kg; (e) CCl₄+ SWGNS at 705 mg/kg; (f) CCl₄+ SWGNS at 1410 mg/kg. (b) Histopathology scores of each group. ^##P < 0.01 versus the normal control group, ^∗P < 0.05. ^∗∗P < 0.01 versus the CCl₄ model group (n = 13 each group).

Figure 4

SWGNS protects against CCl₄-induced liver injury by mediating TGF-β1. (a) Serum TGF-β1 concentration; (b) serum HA concentration; (c) serum LN concentration; (d) serum CIV concentration. ^∗P < 0.05 and ^∗∗P < 0.01, compared to the CCl4 model group. n = 10–13. (e) Representative photos of α-SMA in the liver section. (a) Blank control; (b) CCl4 model group; (c) CCl4+ bifendate at 200 mg/kg; (d) CCl4+ SWGNS at 235 mg/kg; (e) CCl4 + SWGNS at 705 mg/kg; (f) CCl₄+ SWGNS at 1410 mg/kg. 100X magnification.

Figure 5

The HPLC chromatogram of standard Crocin I and II sample (a) and SWGNS sample (b). 1, Crocin I; 2, Crocin II.

Figure 6

The role of Crocin in protecting BRL-3A cells from a peroxidative environment. (a) Cell viability. (b) ALT. (c) AST. (d) MDA.(e) Total SOD. ^#P < 0.05, compared to the control group.^∗P < 0.05, compared to the H₂O₂ group. n = 5.