Viral hepatitis has various routes of infection, and the population suffering from viral hepatitis is widespread. China has the largest population of hepatitis B patients worldwide and the morbidity associated with hepatitis C is increasing annually [1]. Persistent infection and repeated viral attacks are important factors in liver cirrhosis and primary hepatic carcinoma (PHC) [2].

Currently, Western medicine usually treats viral hepatitis by inhibiting virus replication, protecting hepatocytes, reducing transaminases, and improving immune function. Drugs such as nucleosides and interferon have good short-term curative effects, but their long-term effects are unsatisfactory. Moreover, the high medical expenses, the severe toxic side effects of drugs, and viral drug resistance, are problematic [3,4]. It would be very useful to identify a drug that inhibits acute liver immunoreaction and lipid peroxidation, removes free radicals, reduces the levels of cytotoxic factors, and modulates liver metabolism [5,6].

Schemes based on traditional Chinese medicine have long been used to treat liver disease, and valuable experience has been accumulated. Recently, the pharmacological mechanisms and the clinical medicinal value of some Chinese herbal medicines have been clarified, and their lower costs, fewer toxic effects, and reliable long-term curative effects make them worthy of study [7].

Qizhuyigan capsule contains seven traditional Chinese medicinal materials, including the root of red-rooted salvia, felwort, white atractylodes rhizome, Astragalus membranaceus, and radix isatidis. The preparation can eliminate damp-heat, and regulates the liver and spleen. The capsule is used clinically to treat various types of chronic hepatitis, and has good curative effects, but its mechanism of action has not been determined [8].

The study mainly explored the mechanism of action of Qizhuyigan on liver function in a mouse model of immunological liver injury to provide a theoretical foundation for the treatment of immunological liver injury and offer an experimental basis for further development of the Qizhuyigan capsules.

Drugs and reagents

Qizhuyigan capsules formulated from 15 g of the root of red-rooted salvia, 15 g rough gentian, 15 g Rhizoma Atractylodis Macrocephalae, 15 g Astragalus membranaceus, 12 g Radix isatidis, 10 g Semen brassicae, and 6 g Curcuma zedoary were obtained from the Binzhou People’s Hospital. Bifendate pills (batch number: 2014122081) were purchased from Guangzhou Baiyun Moutain Xingqun Pharmacy Co., Ltd. (China), malondialdehyde (MDA), superoxide dismutase (SOD), allophanamide (Shanghai Hongshun Biotech. Co., Ltd., China), tumour necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA), and interleukin 6 (IL-6) kits (Beijing Biolab Science and Technology Ltd., China) were also purchased.

Animals and instruments

One hundred and twenty male mice (body weight range 18 – 22 g) were obtained from the Experimental Animal Centre of Binzhou Medical College, Shandong, China, and were acclimated for 7 d in the Binzhou People’s Hospital, Shandong, China. Instruments used included an AU5800 automatic biochemical analyser (Beckman Coulter, Inc., USA), a TG18G-II high-speed centrifuge (Shanghai Yanqi Biotechnology Co., Ltd., China), an RX-MBY enzyme-labelling instrument (Beijing Ruixi Science and Technology Ltd., China), a UV-765 ultraviolet and visible spectrophotometer (Shanghai Junke Biotech. Co., Ltd., China), and an AUW120D analytical balance (Shimadzu Inc., Japan).

Grouping and modelling

The 120 mice were divided randomly into control, test, bifendate, and Qizhuyigan groups. The model used was reported previously [9]. Specifically, except for the control group, the other groups were gavaged with tap water at 2.5 g/kg. Mice in the bifendate group were gavaged with 12 mg/kg of the drug, and mice in the Qizhuyigan group were gavaged with water containing the contents of a Qizhuyigan capsule (1,000 mg/kg), for 10 days. On the last day of the experiment, all mice except those in the blank control group were injected intravenously with lipopolysaccharides (0.4 mg/kg) through the tail vein after the last dosing. Furthermore, each mouse was fixed with a fixator, with its tail outside the fixators. The tail was repeatedly wiped with of 75 % (v/v) ethyl alcohol. The tail vein was punctured with a syringe at an angle of 30° and a white line was seen along the vein. The absence of resistance during drug injection indicated that the tip of the needle had penetrated the vein, and residual drug was then injected continuously. Then, the needle was removed and the injection site was pressed with a cotton swab to stop the bleeding. Mice were provided with water, but no food, after the injection. They were assessed 12 h after the injection.

This study was approved by the Medical Ethics Committee of Binzhou People’s Hospital (approval no. ZXQ20160115BZ) and followed the protocols set out in the Declaration of Helsinki [10].

Determination of biochemical parameters

After the mice were anaesthetised with diethyl ether, the eyeballs were removed with tweezers. Next, 1.5 mL of blood was collected using a sterile blood collection tube and centrifuged at (5,000 revolutions/min at a centrifugal radius of 6 cm for 10 min, and serum was recovered. An automatic biochemical analyser was used to measure levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver tissues were collected, washed with normal saline at 4°C, and dried with filter paper. Samples (0.2 g) of liver tissues were taken and 10 % (w/v) homogenates were prepared, and centrifuged at 4,500 revolutions/min at a centrifugal radius of 6 cm for 10 min. The supernatants were removed and the levels of MDA, SOD, TNF-α, and IL-6 measured following standard operating instructions provided with the kits.

Statistical analysis

Analyses were conducted using SPSS version 20.0. Data are expressed as means ± standard deviation (SD). Enumerated data were assessed using χ² tests, whereas categorical data were processed using t-tests. P < 0.05 was taken to indicate statistical significance.

Serum ALT and AST levels

The serum ALT and AST levels of the test group were significantly higher than those of the control group (both p < 0.05), indicating that the animal model had been established successfully. The serum ALT and AST levels of the bifendate and Qizhuyigan groups were much lower than those of the test group (both p < 0.05). The ALT and AST levels were lowest in the Qizhuyigan group ().

MDA and SOD contents of liver homogenates

Compared with the control, SOD levels in the hepatic tissue homogenate of the test group decreased, but MDA levels increased (p < 0.05), indicating peroxidation damage. Compared with the bifendate group, the Qizhuyigan group exhibited clearly reduced MDA levels in hepatic tissue homogenate (p < 0.05) and increased SOD levels (p < 0.05). MDA and SOD contents changed the most in the Qizhuyigan group, indicating that Qizhuyigan enhanced anti-lipid peroxidation ability ().

TNF-α and IL-6 contents of liver homogenates

Compared with the control group, the TNF-α and IL-6 levels in the liver homogenate of the test group were significantly increased (both p < 0.05), indicating more serious damage to liver tissues in the former group. Compared with the test group, the bifendate group and the Qizhuyigan group exhibited significantly reduced TNF-α and IL-6 levels (both p values < 0.05); this was especially true of the Qizhuyigan group, suggesting that the anti-hepatic injuryability of Qizhuyigan may be related to inhibition of the release of the cytotoxic factors TNF-α and IL-6 ().

Viral hepatitis is a relatively common infectious disease. The occurrence of immunoreactions in the liver is an important factor causing liver injuries in patients with viral hepatitis. Currently, effective drugs for treating chronic viral hepatitis are lacking, but good curative effect may be achieved if the treatment is focused on regulating immunity. The occurrence of liver injury in patients with chronic viral hepatitis appears to be significantly correlated with immune dysfunction and immunodeficiency [11]. The animal model of immunological liver injury used in this study was associated with extensive cytokine release (e.g., TNF-α and IL-6) and oxygen radicals, similar to the pathogenesis of liver injury induced by chronic viral hepatitis. Thus, the mouse model was suitable for the purpose [12].

Traditional Chinese medicine suggests that liver injury is caused by insufficient anti-pathogenic energy, damp-heat, and pestilent toxin. The basic therapies used to treat Chronic Hepatitis B (CHB) are based on toxin elimination, phlegm reduction, relief of fatigue, correction of deficiencies, and tonification. Traditional Chinese medicine can help treat various difficult diseases, and scholars are focusing on applying traditional Chinese medicine to develop drugs to treat liver diseases.

The Qizhuyigan capsule used in this study was developed based on clinical experience using traditional Chinese medicine to treat viral hepatitis, along with features of traditional Chinese medicine, such as diagnosis and treatment based on an overall analysis of the illness and the patient's condition, as well as modern pharmacology. The capsule consists of seven traditional Chinese medicine materials, including the root of red-rooted salvia, felwort, white atractylodes rhizome, Astragalus membranaceus, and radix isatidis. Astragalus membranaceus is frequently used in traditional Chinese medicine, with effects such as tonifying qi, lifting yang, strengthening the exterior, reducing sweat, inducing diuresis to alleviate oedema, and promoting fluid and blood production. Work using modern pharmacological techniques [13,14] has revealed that Astragalus membranaceus contains active ingredients, such as astragaloside, which can improve cardio-pulmonary function, enhance immunity and promote the body’s resistance to disease, specifically its ability to resist viruses.

ALT and AST levels can directly reflect the degree of liver injury. Previous research has found that damage to 1 % of hepatocytes can double the levels of ALT and AST in the liver [15]. Levels of ALT and AST in the test group were clearly increased compared with the blank control group, and those in the bifendate and Qizhuyigan groups were significantly decreased, indicating that Qizhuyigan had a protective effect in this mouse model of immunological liver injury.

Lipid peroxidation plays an important role in the occurrence and development of immunological liver injury. MDA is the end product of lipid peroxidation, the content of which is positively proportional to the degree of lipid peroxidation [16].

SOD is important in balancing oxidant and antioxidant content in the body, and can remove superoxide anion free radicals and protect cells from damage [17]. Thus, levels of SOD and MDA can reflect the degree of damage caused by peroxidation. Our results indicate that Qizhuyigan can significantly lower MDA and increase SOD in mice, indicating positive anti-lipid peroxidation effects.

TNF-α not only directly kills hepatocytes, but also promotes liver injury [18]. IL-6 has functions in regulating immunoresponses and promoting the growth of hepatocytes [19]. The results of this study suggest that Qizhuyigan may effectively protect the liver by significantly lowering TNF-α and IL-6 content and reducing the release of inflammatory factors.

Qizhuyigan protected mouse liver function in animals with induced immunological liver injury. The mechanism of action appeared to be related primarily to the inhibition of immune function and lipid peroxidation in the liver, thereby providing some theoretical pharmacological foundation for the clinical treatment of chronic viral hepatitis using Qizhuyigan capsules.