Protective Effect of Mongolian Medicine Youning Bawei Powder on Liver Injury Induced by CCl4 in Mice

Hada WU, LIMA A

Inner Mongolia International Mongolian Hospital, Hohhot 010065, China

Abstract [Objectives] To study the protective effect of Mongolian medicine Youning Bawei Powder on liver injury induced by carbon tetrachloride (CCl4) in mice. [Methods] The experimental mice were divided into 5 groups: normal group, model group, positive control group, low-dose group and high-dose group of Mongolian medicine Youning Bawei Powder. The model group was induced by CCl4, the positive control group was treated with liver-protecting tablet, and the Mongolian medicine group was treated with Youning Bawei Powder for one month. The liver tissue injury of mice in each group was observed by HE staining, and the levels of serum ALT, AST, SOD and MDA were detected. [Results] Mongolian medicine Youning Bawei Powder significantly improved the pathological liver injury induced by CCl4, significantly decreased the content of ALT, AST and MDA in serum of mice with CCl4 liver injury, and significantly increased the activity of serum SOD. [Conclusions] Youning Bawei Powder, a Mongolian medicine, has a protective effect on liver injury induced by CCl4 in mice.

Key words Mongolian medicine Youning Bawei Powder, Liver injury, Protective effect

1 Introduction

Youning Bawei Powder, a Mongolian medicine, is a classic prescription derived from the famous work of Yi Fa Zhi Hai, and it has the functions of clearing liver heat, detoxification and so on. It mainly treats hepatobiliary diseases of Zisuhira type in clinic. Clinical observation shows that Mongolian medicine Youning Bawei Powder has a significant effect on the improvement of main symptoms, signs and liver function of chronic hepatitis B and C[1-2]. In this study, the liver injury induced by CCl4in mice was used as a model to study the preventive and therapeutic effect of Mongolian medicine Youning Bawei Powder on liver injury in mice.

2 Materials and methods

2.1 Experimental materialsThe experimental animals were 40 Kunming mice, 20-26 g, half male and half female (provided by Beijing Weitong Lihua Company). The experiment was conducted in the Animal Laboratory of Inner Mongolia International Mongolian Hospital. Mongolian medicine Youning Bawei Powder is composed of turquoise, gypsum, safflower,D.moldavica,Meconopsis,D.superbus,FaecesTrogopterori,etc., provided by the pharmacy of Inner Mongolia International Mongolian Hospital. According to the pharmaceutical standard of the Ministry of Health (Mongolian Medicine Volume) ZZ-8425, it was prepared into 22.5 and 45 mg/mL suspension before use. The liver-protecting tablet (OTC Z20003336) was provided by Heilongjiang Sunflower Pharmaceutical Group Co., Ltd., and it was prepared into a suspension with a mass concentration of 32 mg/mL before use. The ELISA kits of MDA and SOD were purchased from R&D Systems. ALT and AST were purchased from Wuhan Boster Biological Engineering Co., Ltd. The feed was purchased from Sino Biological Preparation Co., Ltd.

2.2 Main instrumentsAutomatic biochemical analyzer (Beckman DXC600), PGEN-ERAL visible spectrophotometer, tissue homogenizer (Changzhou Guohua FS-1), TG16-WS high-speed desktop centrifuge, VANOXAHB-LB universal microscope, Mot-ic6.0 digital medical image analysis system, gel image analysis system,etc.

2.3 Experimental methods

2.3.1Modeling and sample collection. There were 40 Kunming mice, 20-26 g, half male and half female. After 5 d of laboratory adaptive feeding, the mice were randomly divided into 5 groups (n=8): normal group, model group, positive control group (intragastric dose of liver-protecting tablet: 630 mg/kg/d), high and low dose groups of Mongolian medicine Youning Bawei Powder (900 and 450 mg/kg/d, respectively). The normal group and the model group were given the corresponding volume of water according to body weight, once a day for 21 d. On the 21std, 3 h after intragastric administration, all the other five groups except the normal group were intraperitoneally injected with 0.1 mg/mL CCl4peanut oil solution (5 mg/kg), and the normal group was intraperitoneally injected with normal saline (5 mg/kg). The blood obtained was centrifuged at 3500 r/min for 10 min, and the content of ALT, AST, SOD and MDA in serum was measured. After the liver tissue was fixed with 10% formalin, HE staining was used for histopathological examination.

2.3.2Observation indexes and detection methods. The serum ALT and AST were detected by automatic biochemical analyzer. The content of MDA and SOD in serum was determined by ELISA kit and it was operated strictly according to the instructions. The liver tissue fixed with formaldehyde was stained with HE, and the pathological changes of liver tissue were observed under light microscope.

3 Results

3.1 Liver histopathological changesIn the CCl4liver injury model group, the hepatocytes were zonal necrotic and the lobular structure was partially destroyed. The necrotic area of hepatocytes spread to a lobular area or connected to the area between the central vein and the portal area, and the hepatocytes showed diffuse degeneration. The positive control group and Mongolian medicine Youning Bawei Powder group could reduce cell steatosis, and cause inflammatory cell infiltration in part of confluence area and cytoplasmic loosening of some hepatocytes. In the CCl4injury model group, there were extensive turbid swelling, vacuoles, steatosis and focal necrosis of hepatocytes, accompanied by swelling and necrosis of bile duct cells. In the treatment group, the structure of hepatic lobule was clear and complete, and the hepatocytes with vacuolar degeneration were rare.

3.2 Changes in liver functionThe content of serum ALT and AST in the model group was significantly higher than that in the blank group. The content of serum ALT and AST in the positive control group and the low and high dose groups of Mongolian medicine Youning Bawei Powder was significantly lower than that in the model group, and the difference was significant (Table 1).

Table 1 Comparison of the content of ALT and AST in serum of mice in each group (U/L, n=8)

3.3 Changes in serum MDA and SOD contentThe content of serum MDA in the model group was significantly higher than that in the normal group (P<0.05). The content of serum MDA in the positive control group and the low and high dose groups of Mongolian medicine Youning Bawei powder was significantly lower than that in the model group (P<0.05). The content of serum SOD in the model group was significantly lower than that in the normal group (P<0.05). The content of serum SOD in the positive control group and the low and high dose groups of Mongolian medicine Youning Bawei Powder was significantly higher than that in the model group, and the difference was significant (P<0.05), as shown in Table 2.

Table 2 Comparison of serum MDA and SOD content of mice in each group n=8)

4 Discussion

Experimental liver injury induced by CCl4is a pathological model commonly used to study the mechanism of hepatoprotective drugs. Liver injury is a complex pathological process involving many factors. A variety of enzymes, free radicals and lipid peroxidation have changed greatly in the process of liver injury. Some studies have shown that free radicals and lipid peroxidation play a very important role in liver damage and liver disease. Free radicals are produced when CCl4is activated by liver microsomal P450. Free radicals can covalently bind with macromolecules in hepatocytes and attack submembrane unsaturated lipids, resulting in lipid peroxidation, damage to hepatocytes, destruction of cell membrane structure, and entry of cytoplasmic ALT and AST into the blood. As a result, the activity of serum ALT and AST is increased. The animal experimental model of liver injury induced by carbon tetrachloride showed that Youning Bawei Powder, a Mongolian medicine with the effect of clearing liver heat and detoxification, could increase the level of SOD and decrease the level of MDA in serum of experimental animals, suggesting that its effect of protecting liver and lowering enzymes was related to scavenging active oxygen free radicals and anti-lipid peroxidation[5-8]. The results showed that Youning Bawei Powder could significantly reduce the increased ALT and AST induced by CCl4in mice. Histopathological observation showed that Mongolian medicine Youning Bawei Powder could significantly alleviate the liver pathological changes of mice induced by CCl4, and there was no significant change in the basic structure of liver tissue in mice treated with high and low doses of Mongolian medicine Youning Bawei Powder. The steatosis of hepatocytes and the infiltration of inflammatory cells in the liver tissue in the model group were significantly lower than those in the model group, and the effect of the high dose of Mongolian medicine Youning Bawei powder was the most obvious, and the effect was better than that of the positive treatment group. This showed that Youning Bawei Powder had a certain protective effect on carbon tetrachloride-induced liver injury, and its effect was related to the applied dose. However, the exact mechanism of Mongolian medicine Youning Bawei Powder protecting hepatocytes remains to be further studied.