Ameliorative effects of Qingganjiuwei powder, a traditional Mongolian medicine, against CCl₄-induced liver fibrosis in rats

Abstract

Ethnopharmacological relevance

Qingganjiuwei powder (QGJWS) is a well-known traditional drug containing nine kinds of medicinal materials. This drug is commonly used in the Inner Mongolia region and exerts remarkable clinical effects on hepatic protection.

Aim of the study

To investigate whether QGJWS inhibits liver fibrosis in rats and to reveal its potential mechanisms.

Methods

Liver fibrosis model was induced by CCl₄ for 8 weeks in SD rats. Next, rats were intragastrically administered quantum satis doses of QGJWS (0.525, 1.575, 4.725 g/kg per day) or Silymarin (SIL; 120 mg/kg per day) for 8 weeks. Afterwards, the rats were sacrificed, and serum aminotransferase (ALT and AST) levels, histopathological changes as well as the mRNA and protein expression of matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase1 (TIMP1), collagen type Ⅰ(COL1), α-smooth muscle actin (α-SMA), combined with phosphorylation levels of extracellular signal-regulated kinase (ERK), C-Jun amino-terminal kinases (JNKs) and stress-activated protein kinase-2 (p38) protein in liver tissues were measured in each groups, respectively.

Results

The symptoms and signs of the model rats were consistent with the diagnostic criteria of liver fibrosis. By contrast, treatment with QGJWS clearly improved the general condition of rats. Also, the morphology and structure of liver can be ameliorated, there are fewer hepatocyte necrosis and lymphocytic infiltration and pseudolobuli in QGJWS treatment groups as demonstrated by histopathological analysis, thus helping bring about lower METAVIR scores. QGJWS administration also dramatically decreased serum ALT and AST levels. Further immunohistochemistry, western blotting and Real-Time PCR analysis revealed that QGJWS significantly enhanced the mRNA and protein expression of MMP2, MMP9, and downregulated the expression levels of COL1, TIMP1 and α-SMA. Furthermore, QGJWS reduced the activities of mitogen-activated protein kinases (MAPKs) pathway in liver by inhibited the phosphorylation of ERK, JNKs and p38 proteins.

Conclusions

QGJWS offers notable protection against CCl₄-induced liver fibrosis in rats, which may be due to its ability to inhibited the MAPKs signaling pathway.

Introduction

As a common but challenging clinical disease, liver fibrosis involves a pathological spectrum ranging from isolated deposition of hepatic extracellular matrix (ECM) to cirrhosis. Even worse, liver fibrosis provides fertile ground for hepatoma formation, which is one of the main causes of cancer death (Bray et al., 2018; Kwon et al., 2015; Wallace and Friedman, 2014).

Chronic liver injury, induced by viral hepatitis type B infection, aflatoxin exposure or alcoholism, may contribute to liver fibrosis. During this period, the hepatic stellate cells (HSCs) are activated due to continuous pathological stimulus and secrete large amounts of ECM. After a relatively long period of time, the deposited ECM changes the structure of the liver and accelerates the progression of fibrosis. Eventually, the disease progresses to the formation of cirrhosis. Studies have shown that liver fibrosis is a reversible disease by early prevention or treatment. However, the existing treatment fails to meet necessary requirements due to its complex pathogenesis (Friedman et al., 2018). Currently, researchers aim to identify drugs with precise curative effects to slow the process of liver fibrosis or even reverse it (Friedman et al., 2013; Trautwein et al., 2015).

In this studies, rat model involved an artificial liver injury induced by CCl₄, a selective hepatotoxic drug, which can trigger inflammation and fibrosis. This model simulated clinical liver fibrosis in morphology and pathophysiology (Yanguas et al., 2016).

SIL was used as a positive control due to its positive effects on liver fibrosis via retardation of the proliferation of HSCs and inhibited inflammatory response. Furthermore, SIL decreased the activation of the MAPK signaling pathways, which was consistent with previous studies (Ezhilarasan et al., 2016; Saber et al., 2018).

QGJWS, one of the Mongolian drugs, is commonly used in the Inner Mongolia region to treat patients with chronic hepatic disease (Bai, 2015; Wang et al., 2017). This formula is comprised of the following 9 natural materials: Calculus bovis factitius (specimen No.: 00024812), Dianthus superbusL. (specimen No.: 00014132), Faeces Trogopterpri (specimen No.: 00122715), Swertia bimaculata (Siebold & Zucc) Hook. f. & Thomson ex C.B. Clarke. (specimen No.: 0037480), Scabiosa comosaFisch. ex Roem. & Schult. (specimen No.: 00122741), Radix aucklandiae P.e. (specimen No.: 00077818), Momordica cochinchinensis (Lour.) Spreng. (specimen No.: 000633746), Clematis armandiiFranch. (specimen no.: 00046861), Crocus sativus L. (specimen No.: 00016576) in a ratio of 20:20:20:15:10:10:10:2:1 (Table 1) (Chinese Pharmacopoeia Committee, 1998), the voucher specimens of herbs were deposited at Mongolian medicine library (IMEM) of Inner Mongolia University for the Nationalities, Tongliao, China.

According to the Mongolian medicine theory, each herb has its own role in formula. Researches over last decade in vitro and vivo has demonstrated that in QGJWS, Calculus bovis factitius showed hepatoprotective effects on NAFLD by correcting lipid and glucose metabolic disorders (He et al., 2017); Both Crocus sativus and its main constituents here are kaempferol and safflomin A has immunomodulatory, anti-inflammatory and antioxidant capacities (Li, 2017; Pan et al., 2016); Protocatechuic acid, the main metabolite of Faeces trogopterpri exerted an anti-fibrotic effect in liver by reduced the activation of hepatic stellate cells (Jiang et al., 2017); Dianthus superbus was found to possess the cytotoxic activities against cancer cells, and also ranked very high in antioxidant properties in previous study (C.L. Ding et al., 2013); Momordica cochinchinensis has anti-tumor effects, besides its promoted cholesterol excretion and has a suppressive effects on fatty liver (Satsukawa et al., 2016; Tien et al., 2005); The main chemical constituents in Swertia bimaculata include swertiamain and sweroside, the former showed antidiabetic and antihyperlipidaemic activities, and alleviated NAFLD in mice, while the latter exerted marked anti-fibrosis effects on CCl₄-induced mouse model, resulted in inhibition of COL1 and TIMP1, of which the underlying mechanism was related to farnesoid X receptor-miR-29a regulation (Gong et al., 2020; Yang et al., 2019); Scabiosa comosa is a traditional Mongolian medicine in treating liver fibrosis, and showed good effects by selectively inhibiting Smad 3 phosphorylation (Ma et al., 2018); Costunolide, the main active ingredients of Radix Aucklandiae, has the activity against cancers; Costunolide, the main active ingredients of Radix Aucklandiae, as well as Clematis armandii both induced anti-tumor and hepatoprotective activities, the former is mediated by STAT3 and Notch3/HES1 pathway inhibition, and the latter through modulation of mitochondria and EGFR signaling pathway (Ge et al., 2020; Gong et al., 2020; Pan et al., 2016; Yan et al., 2019).

QGJWS is often used as an anti-fibrosis drug in clinic, our previous studies demonstrated that QGJWS (at a dose of 0.08 g/kg) had a curative effect on patients with alcoholic liver fibrosis by decreasing the serum transaminase levels and liver Doppler ultrasound parameters (Ge et al., 2017). However, the mechanisms of QGJWS against liver fibrosis remain poorly understood. This study aimed to further explore the effects of QGJWS in treating liver fibrosis.