Experimental study on the efficacy of Fuganling granula on protecting against immunological hepatic injury

Abstract

To study the efficacy of Fuganling granula (FGL, 复肝灵颗粒) in treating mouse immunological hepatic injury that was caused by Bacille Calmette Guerin (BCG) and lipopolysaccharide (LPS), a total of 60 mice were adopted, among which, 50 mice were given intraperitoneal injection with BCG and LPS to establish an immunological liver injury model and then were randomly divided into 5 groups (10 mice/group): 4 groups received treatment of FGL orally at the doses of 100 mg/kg (high-dosage), 50 mg/kg (middle-dosage), 25 mg/kg (low-dosage) and bifendate orally at the dose of 80 mg/kg, respectively. One group was treated with distilled water orally. The remaining 10 mice were given distilled water intraperitoneally as the normal control group. The indices of thymus, liver and spleen, and the activities of the alanine aminotransferase (ALT), aspartate aminotransferase (AST) in the serum were detected. Compared to the normal rat, the model group’s thymus index decreased significantly. The liver index and spleen index increased significantly. The activities of serum ALT and AST increased significantly (all P< 0.01). Compared to the model control group, the group treated with FGL in high-dosage, middle-dosage or low-dosage can decrease the activities of ALT and AST and the group treated with FGL in high-dosage and middle-dosage can increase the thymus index significantly (P< 0.01). This experiment established the immunological liver injury model successfully and found that FGL has a remarkably protective effect on this kind of immunological hepatic injury.

Keywords

Fuganling granula / immunological liver injury / bacille calmette guerin / lipopolysaccharide / mice

Introduction

Fuganling granula (FGL, 复肝灵颗粒) is a prescription in the traditional Chinese medicine praeparatum which contains Huangxiangcao, Muxi [Melilotus officinalis (L.) Desr.], virgate wormwood herb, christina loosestrife and licorice root. According to the organization prescription theory of traditional Chinese medicine, it combines the secret family recipe with the organization prescription theory of traditional Chinese medicine which distinguishes from traditional Chinese drugs. FGL is new drug praeparatum which has gained clinical test documentation from the State Food and Drug Administration. The research confirmed [1] that FGL has noticeable curative effect for treating viral hepatitis and have remarkable effects on protecting hepatic cells from immunological injury caused by carbon tetrachloride. In our study, carbon tetrachloride was used to establish the immunological liver injury model in mouse and observe the changes in liver function and structure after treatment by BCG and LPS to explore the protecting mechanism in liver.

Materials and methods

Materials

The animals were purchased from the Animal Center of Shandong University, and animal experiments were carried out in accordance with the Prevention of Cruelty to Animals Act 1986 and the National Institute of Health guidelinesfor the care and use of laboratory animals for experimental procedure. Sixty Kunming mice were adopted, weighing 18-22 g, female and male half each. FGL was processed according to manufacture technology procedure in Hong Ji Yang drug factory and dissolved in normal saline before using. Bifendate was purchased from Beijing Xiehe Pharmaceutical Factory, Batch No. 20010216. Plasmogen injectable preparation of Bacille Calmette Guerin (BCG) was procured from the Beijing Institute of Biological and Specification, the specification is 80 mg/mL, Batch No. 20010216, prepared by double distilled water temporarily. GPT kit and GOT kit was purchased from the Institute of Biological Products of Shanghai. The ultraviolet-visible light-photometer of UV-756 is product of the Third Analytical Apparatus Factory of Shanghai. Pathological sections were cut in a cryostat (F81-HP; Julabo, Germany) and the microscope was from the Olympus Company (Japan).

Methods

Immunological liver injury model established in mice

Fifty mice were injected with BCG and LPS to induce immunological liver injury as reported previously [2], then injected with BCG (0.2 mL per mouse, 1∶5 dilution, 10⁶ bacteria per mL) via the vena caudalis to start an allergic response. Ten days later, LPS was injected (8.5 μg per mouse) via vena caudalis to establish an immunological mouse liver injury model.

Animal grouping and treatment

Sixty mice were randomly divided into 6 groups (10 mice each group), 4 groups were treated with FGL orally at the doses of 100 mg/kg, 50 mg/kg, 25 mg/kg, respectively, or treated with bifendate orally at the dose of 80 mg/kg, and 1 group was given distilled water once a day for 6 days via intragastric administration. The last 10 mice were given distilled water by intraperitoneal injection as the normal control group.

Biochemical indicator analysis

After treatment for 16 h, blood samples were collected to detect the glutamic pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST), then the mice were killed, the thymus, liver and spleen were collected, weighed and calculated. A part of the liver tissue was taken and washed with cold normal saline, then 0.5 g liver tissue was weighed and homogenated with cold normal saline.

Pathology examination

According to the routine method, hepatic tissue was excised at the same site and fixed with formalin, imbedded with paraffin, stained with hematoxylin and eosin (HE) and observed through a light microscope.

Statistical analysis

Data were expressed as \overline{x}\pm s. The statistical analysis was carried out using F test in group comparison, using q test in group comparison and using t test between pre-therapy and post-treatment comparison by SPSS 10.0 statistical software. Differences were considered statistically significant at P < 0.05.

Results

Effect of FGL on the index of thymus, liver and spleen

As Table 1 shows, compared with the normal control group, the weight of the liver and spleen increased significantly but the weight of thymus decreased significantly in the immunological liver injury model (P < 0.01). Compared with model control group, the weight of liver decreased in the Bifendate group and all of the FGL groups (P < 0.01). The weight of spleen decreased in the Bifendate group (P < 0.05). The weight of spleen decreased significantly in all of FGL group (P < 0.01). The weight of thymus increased significantly (P < 0.01). Compared with the Bifendate group, the weight of liver decreased significantly in three of the FGL groups (P < 0.01) and the weight of spleen decreased in the FGL middle dose group and large dose group (P < 0.05), while there was no significant difference in FGL low dose group.

Effect of FGL on activities of ALT and AST

As Table 2 shows, compared with normal control group, the activities of ALT and AST increased significantly in immunological liver injury model (P < 0.01), but decreased significantly in the FGL large-dosage group, middle-dosage group and low-dosage group compared with the model control group (P < 0.01), indicating that FGL has remarkably protective effects on this kind of immunological hepatic injury. Compared with Bifendate group, the activities of ALT and AST of serum also decreased in the Bifendate group (P < 0.01).

Effect of FGL on peroxidized production of malondialdehyde (MDA)

As Table 3 shows, compared with normal control group, MDA in mice hepatic tissue increased significantly in immunological liver injury model (P < 0.01), FGL of each dose group could decrease the MDA significantly and achieved or approached the level of the normal control group while it was increased by injection of BCG+ LPS (P < 0.01).

Effect of FGL on pathology of immunological liver injury model

Observed under light microscope, the liver cell cord of mice were in a regular arrangement in the normal control group. The structure of the hepatic lobules was clear. The nucleus of hepatic cell was larger and round. The nuclear membrane was clear, the cytoplasm was abundant and there was no-inflammatory cell infiltration in portal area and its structure is clear. Compared with the normal control group, it is clear that hepatic cells were swollen, and there was cytoplasm rarefaction, ballooning degeneration and focal necrosis developed in the model control group. Hepatic cell damage was relieved markedly in each FGL dose group. The structure of the hepatic lobule was distinct and most of the hepatic cell appearances were similar to those of the normal control group. There was no evident on pathological change (Fig. 1).

Discussion

The liver has a set of arterial systems, two sets of venous systems and a set of lymphatic system. Its oxygen consumption is only second to brain. Similar to hepatic cellular edema, degeneration caused by immunological liver injury [3-7] and hepatic tissue damage can result in a vicious cycle of hepatic cell hypoxia-edema-degeneration-necrosis. The immunological liver injury model is induced by BCG and LPS. Its pathogenetic mechanism is similar to the immunological functional disorder of human hepatitis, and it is an ideal model to screen and study regarding drugs for liver protection [8-11]. In our study, BCG was injected in mice in advance to induce polynucleation neutrophilic granulocyte or macrophage aggregation and a sensitization response in the liver. After LPS was injected, sensitized Kupffer cells stimulated these cells to release panimmunity effector molecule such as free radicals, NO, TNF2α, IL21 and so on, which have toxic actions on hepatic cells. Among theses, free radical has strong oxidization, and it attacks cell membrane and mitochondrial membrane through a peroxidatic reaction and reacts with unsaturated fatty acid and generates lipid peroxidation in the cell membrane, leading to damages of the mitochondria, DNA and RNA in tissue. The change of the MDA’s level reflected the change of oxygen free radical indirectly, so the level of MDA can be used to appraise the level of oxygen free radical and the degree of lipid peroxidation. In this experiment, after BCG and LPS were injected, ALT and AST were increased in the mouse serum, and the degeneration and necrosis of disseminated cells developed in liver tissue. MDA increased significantly in the cytoplasm of hepatic cells. All of these indicated that lipid peroxidation in the hepatic cell is one of the important hepatic injury mechanisms in this model. In vivo, the content of Huangxiangcao, Muxi in FGL could increase the quantity and activity of macrophages [12], strengthen the function of the anti-virus by stimulating the activity of lymphocytes to protect the liver and enhance immunization. FGL also has obvious therapeutic action on edema. The content of baical skullcap root in FGL has an obvious effect on the pharmacologic action such as anti-viral, restraining lipid peroxidation reaction and so on [12-14]. Further more, baical skullcap root could lower MDA levels and succinic dehydrogenase (SDH) activity. The content of licorice root has the effect of protecting liver [15-17].

In this experiment, it was shown that FGL at each dose in the groups could relieve pathological damage of the liver in model mice, relieve hepatic cell necrosis and inflammatory cell infiltrate, cause ALT and AST in the serum to return to normal levels and decrease the level of MDA in hepatic cytolymph. The pathological result displayed that FGL could relieve the degree of inflammation and necrosis, which is consistent with the previous reports [18-20]. Therefore, FGL has the functions such as anti-inflammation, adjusting immunization, decreasing enzyme, protecting hepatic cell membrane, improving hepatosis and so on. It also has significant effects on preventing and delaying experimental hepatic injury, preventing occurrence and development of hepatic fibrosis, enhancing phagocytosis of reticuloendothelial system in vivo and improving nonspecific immunity of organism. Therefore, this research may provide a theoretical proof for the clinical application of FGL.

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