Carbon tetrachloride (CCl₄) was purchased from Aladdin (part no.: C112043, China). Commercial kits for SOD, GSH-Px, hydroxyproli, MDA, AST/GOT, and ALT/GPT were purchased from Jiancheng Co. (Nanjing, China). 85% Baicalin was purchased from Fusion Biology Co. (Part No. 21967-41-9, China).

Experiments were carried out on 17 ICR male rats (Jestier, China) of 18–22 g body weight. They were housed at 22–24°C and exposed to alternate cycles of 12 h light and dark. They were given free access to a standard pellet diet and tap water. The animal study was approved by the Laboratory Animal’s Ethic Committee of the Hospital. It was carried out throughout the experiment following the international laboratory animal use and care guideline.

We first prepared a 2% CCl₄ solution by mixing CCl₄ and corn oil thoroughly mixed for 2 min. Then the CCl₄-corn oil solution was administered subcutaneously at a dose of 0.1 mL/kg of body weight, twice per week and continued for 6 weeks, in order to induce the liver fibrosis.

The animals were randomly divided into 5 groups: (1) Control group (normal rats), in which 3 rats were injected subcutaneously with corn oil with no CCl₄. (2) CCl₄ control group, in which 5 rats were administered 2% CCl₄ without adding baicalin. (3) CCl₄ + low dose baicalin group, in which 3 rats were administered with 2% CCl₄ and baicalin (100 mg/kg). (4) CCl₄ + intermediate dose baicalin group, in which 3 rats were administered with 2% CCl₄ and baicalin mixture (200 mg/kg) (5) CCl₄ + high dose baicalin group, in which 3 rats were administered with 2% CCl₄ and baicalin (400 mg/kg).

At the end of six weeks, animals were scarified. The blood samples were obtained from orbital veins. The serum samples were collected by centrifugation in a Sorvall RC centrifuge (Thermo Scientific, Germany) of the whole blood at 4000 rpm for 10 min. The samples were stored frozen at −80°C until further use. The livers were immediately removed and were fixed in 10% formalin for histological analysis.

Relative weights of liver, kidney and spleen were represented as the percentage of the total body weight in gram.

The activities of ALT and AST were measured by spectrophotometry using commercial kits, according to the manufacturer’s instructions. The levels of LN, HA, PC III were obtained by radio-immunoassay kits, according to the manufacturer’s instructions. TGF-beta1 was determined by the rat ELISA kit.

The tissue homogenates of livers were used to determine SOD, MDS, and GDH-PX levels, according to the manufacturer’s instructions.

Liver collage concentration was determined by measuring the level of hydroxyproline, according to the method published previously (Sun et al., 2007).

Livers were isolated from rats and the tissues were fixed using 10% formalin for 24 h. The fixed tissues were then dehydrated and immersed in paraffin solution, followed by cutting into small sections for staining. The hematoxylin-eosin staining was used to observe liver injury. The tissues were stained with Masson staining reagents to detect the deposition of collagen and stained with Sirius red staining reagents to measure the type and content of collagen. The pathological observation was evaluated by a Nikon microscope with a digital camera and polarization microscope.

Western immunoblotting analysis of liver tissue was undertaken using the following monoclonal antibodies: β-actin was purchased from Zsbio (Beijing, China). α-SMA was purchased from Abcam Inc. (Shanghai, China). JAK2, phosphor-JAK2 (or p-JAK2), phosphor-ErK (or p-ErK), Erk, Akt, phosphor-Akt (or p-Akt) and cleaved Caspase 3 were purchased from CST (Beverly, MA). Rock1, P-53, Bcl-2, and Bax were purchased from Santa Cruz biotechnology (Santa Cruz, CA). These antibodies were used to identify proteins expressed. The extracted proteins were subjected to electrophoresis on a 10% SDS-PAGE after normalization for protein content. After resolution, the protein samples were electrotransferred onto PVDF.

The membrane was blocked for 1 h in 0.1% nonfat dry milk in PBS. Membranes were incubated overnight at room temperature with the primary antibody in TBS. Membranes were washed twice for 5 min in PBS before the addition of the secondary antibody in TBS containing 0.1% nonfat dry milk for 1 h. The membranes were then washed in TBS for 5 min followed by water for 5 min. Reactive bands were identified using enhanced chemiluminescence and autoradiography according to the manufacturer’s instructions.

Quantitative data was demonstrated as the mean ± standard deviation (SD). The significance of the difference vs. the CCl₄ group was analyzed by a Student’s t-test. A Mann–Whitney rank sum test was used to measure the degree of histopathological liver fibrosis. The P-value less than 5% (P < 0.05) was considered to be statistically significant.

The roles of baicalin on the rats’ body weight, and the index of liver, spleen and kidney were evaluated, as shown in Fig. 1. The liver, kidney and spleen index were measured as the percentage of the total body weight. The results in Figs. 1B and 1C show that the body weight, the liver, kidney, and spleen index do not exhibit any statistically difference between normal and liver-fibrosis rats. The baicalin treatment does not have a significant impact on these factors.

The effect of baicalin on the liver function was evaluated by measuring the therapeutic serum regimens, hyaluronic acid (HA), Procollagen type III (HPCIII), Aspartate aminotransferase (ALT), and Alanine aminotransferase (AST), on the hepatic fibrosis in CCl₄ rat model (Hu et al., 2010; Ozer et al., 2010). The results are summarized in Fig. 2. The significantly higher levels of HA, HPCIII, AST and ALT in the rats’ serum in the CCl₄ group were observed than that of the control group. However, HA and HPCIII levels (Figs. 2A and 2B) were effectively reduced at a higher dose of baicalin (above 100 mg/kg). For example, by treating with 400 mg/kg baicalin, the HA and HPCII levels were reduced significantly by approximately 80% and 40%, respectively. Additionally, baicalin also significantly suppressed CCl₄-induced increase in AST level at relatively lower baicalin dose (Fig. 2D), while ALT level does not show a notably change upon treating baicalin (Fig. 2C). This result indicated that baicalin has a protective effect for the CCl₄-induced liver injuries.

Next, we investigated the effect of baicalin on the hepatic anti-fibrotic and anti-oxygenation capability by evaluating the hydroxyproline (HyP), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in liver. HyP is an amino acid, and it exists almost exclusively in collagens (Alcock et al., 2019; Katayama et al., 1997; Kim et al., 2009). Measurement of HyP in fibrotic tissues has been used as a reliable method to study fibrosis and to investigate the potentially anti-fibrotic agent (Kim et al., 2009). As shown in Fig. 3A, the content of HyP in the livers after treating CCl₄ were significantly higher than those in the normal livers, suggesting the liver fibrosis has been formed in the CCl₄ treated rats. After treating different doses of baicalin, the hepatic HyP levels were markedly decreased. This finding suggests that baicalin is an effective anti-fibrotic agent in CCl₄ injury rats.

Moreover, MDA, SOD, and GSH-Px are commonly used as biomarkers to evaluate the oxidative stress and the antioxidant status in liver injuries (Katayama et al., 1997; Romani et al., 1988; Sampey et al., 2003). As can be seen in Figs. 3C and 3D, GSH-Px and SOD levels are reduced in CCl₄-groups, suggesting that the end-product of lipid peroxidation due to the oxygenation process is higher in CCl₄ group than in the control group. As increasing the dose of baicalin treatment (Figs. 3C and 3D), the SOD and GSH-Px levels effectively increase. In contract, MDA level does not display a statistical difference among the group studied (Fig. 3B).

Liver fibrosis is often investigated by the accumulation of proteins in extracellular matrix, which occurs in the most types of chronic liver diseases (Chen et al., 2015; Li et al., 2019). To determine whether the protective effect of baicalin on CCl₄-induced liver injury is mediated by cell apoptosis, the protein levels of AKT, phosphorylate (p-) AKT, ERK, and phosphorylate (p-) ERK in cell were detected. The results indicated that CCl₄ injection enhanced the levels of AKT, p-AKT, p-JAK2, ERK, p-ERK and ROCK1, compared with the levels in control group (Figs. 4 and 5). However, as shown in Fig. 5, treating with a small dose of baicalin does not significantly affect the p-JAK2 and ERK level in CCl₄-injuried rats. These levels were only suppressed significantly at higher doses of baicalin. On the other hand, a small dose of baicalin (100 mg/kg) can significantly suppress AKT, P-AKT, and ROCK1 level in the CCl₄-injuried rats. It has been shown that high responsiveness of cell apoptosis is related to the increased expression of AKT and ROCK1 levels in liver cells (Liu et al., 2015b). Our findings indicate that baicalin treatment may suppress the AKT and ROCK1 signaling pathway in CCl₄ induced liver injury, suggesting the potential capability of baicalin to suppress cell apoptosis.

B-cell lymphoma-2 (Bcl-2) is considered to be an anti-apoptotic protein, and Bcl-2 associated X protein (Bax), mammalian target of rapamycin (mTOR), p53 and caspase-3 are known to be the biomarker of apoptosis (Cao et al., 2013; Guo et al., 2002). To better understand the role of baicalin on the cell apoptosis in liver injury, the western blot analysis was performed to detect the expression of apoptosis markers at mRNA and protein levels. As indicated in Figs. 6 and 7, the relative expression of Bax, Bcl-2, mTOR, p53, and cleaved caspase-3 was significantly enhanced after CCl₄-treatment, compared with the control. However, this enhancement was markedly attenuated by treatment of baicalin. Several studies have shown that high level of Bax, Bcl-2, mTOR, and p53 associates with cell apoptosis (Gao et al., 2002, 2003; Liu et al., 2019). Thus, these results suggest that baicalin may attenuate hepatocyte apoptosis in liver injury in the dose dependence matter.

Histological characteristics of the liver in the control group showed a normal lobular architecture and structure (Fig. 8A). However, after treating with CCl₄, the extensive hepatocellular damages were observed, as evidenced by the presence of hepatocellular degeneration and infiltration inflammatory cells (Fig. 8B). The treatment of baicalin in various dosages can attenuate the degree of injury changes (Figs. 8C–8E). These results confirmed that baicalin has the anti-fibrotic effects in CCl₄–liver injury model, and the effect is dose dependent.

The effect of baicalin on the changes of liver cells was presented in Fig. 9. The VG staining results indicated that CCl₄ treated liver showed extensive changes in microstructures, including marked enlarged domains of portal inflammation, hepatic necrosis, and fibrotic septa (Fig. 9B). No abnormalities were observed in the control group (Fig. 9A). Baicalin treatment could significantly alleviate the degree of liver injury in the cellular level, and their cell morphologies are close to that of normal cells.