| Background:The liver is one of the vital substantive organs in the human body, and it is an important part of the digestive system. The physiological function of the Liver is very complex. Bile secretion is a basic function for the live as a digestive organ, but it is only a little part of the physiological function of the liver. In addition, it is involved in the synthesis, conversion, storage and decomposition of carbohydrate, lipid, protein, vitamins and other nutrients. Furthermore, hormones, metabolites, drugs and other substances are metabolized or detoxified in the liver, so liver is the most active metabolic organ. Liver also has a certain immune function and even hematopoietic function in the embryonic period. Thus, the important role of the liver in the human body is quite obvious. Liver damage, structural or functional, caused by various factors, is bound to affect human health and even endanger life. Common factors affecting the function of the liver are drinking, infection of pathogens (viruses, parasites, etc.), autoimmunity, high-fat diet and so on. In developed countries, the main cause of liver disease and liver related death is alcoholic liver disease. China is a country with high incidence of liver disease. Viral hepatitis, especially hepatitis B, accounts for the main part of liver disease. Epidemiological survey in 2006 showed that the hepatitis B surface antigen carrying rate is 7.18% in Chinese population aged 1 to 59. According to this survey, about 93 million people in China have been infected with hepatitis B virus, and about 20 million people of them are chronic hepatitis B patients. HBV infection rate has dropped as vaccination program rolled out. But how to treat the people with chronic hepatitis B is still a medical problem. Many Drugs have been developed to treat viral hepatitis, such as the liver protecting drugs and antiviral drugs. These drugs were widely used in clinic to improve clinical symptoms, clear the virus and prevent the development of the complications. But side effects, virus resistance, high cost, long treatment cycle and less therapeutic effect are limiting factors in many cases. So we still need to develop more efficient, economical and safe drugs to treat viral hepatitis.Liver injury can have a variety of causes. Different etiology of liver injury makes different pathogenesis, and a single etiologic factor may have multiple pathogenic mechanisms. Therefore, the pathogenic mechanism of liver injury is very intricate and is not fully understood yet. Known liver damage mechanism involves damage of membrane integrity, mitochondrial dysfunction, damage of endoplasmic reticulum, oxidative stress, hepatocyte calcium overload, immune disorders, release of inflammatory cytokines, necrosis and apoptosis. No matter what the liver injury was caused by, liver function indices or (and) the histopathology of liver change can be detected to evaluate the damage.Liver injury is a modern medical term. Chinese medicine has no record about the exact word "liver injury", but we can find many similar records in traditional Chinese medicine which is similar to the clinical symptoms and signs of liver injury patients. These diseases and syndromes were associated with liver injury. TCM therapy is based on syndrome differentiation which means TCM physicians pay much more attention to the summarization of the pathological change of a disease at a certain stage in its course of development. Disease differentiation combined with syndrome differentiation is also quite important. They include Biao and Ben, that is, the principle of treating a disease by analyzing both its root cause and symptoms. It is significant to distinguish what is primary and what is secondary. Besides, factors such as climatic and seasonal conditions, geographic localities, and the patient’s personal conditions must be considered in treatment. Chinese Medicine Therapy has good effect on liver diseases proved by clinic and experiments. The therapy for chronic liver disease with TCM is one of the specific and preponderant disciplines in our country. Chinese medicine treatment of liver injury or liver disease both experimental research and clinical practice, where suitably applied, all achieved good results.Zhi-Zi-Da-Huang decoction is a preponderant recorded in Treatise on Febrile Diseases and Miscellaneous Diseases by Zhang Zhongjing. Studies have shown that Zhi-Zi-Da-Huang decoction has a protective effect on alcoholic liver injury. The mechanism may be related to reduce alcohol induced peroxidation. The treatment of other types of liver injury has not been reported in literature. The mechanism of the treatment on liver injury has not been further studied yet.Objectives:This study aims to establish a mouse model of CC14 induced acute liver injury. To investigate the protective effect of Zhi-Zi-Da-Huang decoction against CC14-induced acute liver injury. The serum ALT and AST changes and liver tissue histopathology will be detected to evaluate the mouse model and the therapeutic effect on liver injury. The expression of proteins related to apoptosis and the level of MDA and SOD in liver tissue will be detected to explore its protective effect through antioxidant and anti-apoptosis. To provide the experimental evidence for the clinical use of Zhi-Zi-Da-Huang decoction.Method:1. Preparation of ZZDHD:According to the original composition of ZZDHD recorded in Treatise on Febrile Diseases and Miscellaneous Diseases, Gardenia jasminoides Ellis (10 g), Rheum palmatum L. (5 g), Citrus aurantium L. (12 g) and Sojae Semen Praeparatum (10 g) were weighed separately. Then the herbs were immersed in 370 ml distilled water (1/10, w/v) for 30 min and then reflux-extracted for 1 h. The extracted solution was filtered through five layer gauzes and the procedure was repeated once again by adding 370 ml water. The two water extracts were combined and concentrated into low concentrations (0.3 g/ml), concentration (0.6 g/ml) and high concentration (1.2 g/ml) liquid by rotary evaporator.2. Animal groups and establishment of mouse model:72 male KM mice were randomly divided into 6 groups:control group, model group, bifendate (200 mg mg/kg) group, ZZDHD low dose group (6 g/kg), ZZDHD medium dose group (12 g/kg) and ZZDHD high dose group (24 g/kg),12 mice in each group. Mice in each group were intragastric administrated with the corresponding concentration of the drug or normal saline (control group and model group) once a day for 5 consecutive days.1 hour after the last intragastirc administration, mice in control group were intraperitoneal injected with peanut oil, while mice in all other groups were intraperitoneal injected with 0.12%(v/v) CCl4, at dose of 10ml/kg body weight. All mice were fasted but can drink water freely. At 16 h after injection, all the mice were sacrificed under anesthesia. Blood samples were collected and centrifuged at 3000r/min for 10 min at room temperature 1 h after collection to collect the serum. Liver tissue was isolated from each mouse. Serum samples and liver tissue samples were stored at-80℃ for further experiments.3 Blood and tissue analysis3.1 assessment of liver function Biochemical parameters of serum ALT and AST in mice were determined using the corresponding diagnostic kits in accordance with the manufacturer’s instructions.3.2 Histological Examination of Liver Tissue For the histological investigations, liver tissues were removed from a portion of the left lobe, and parafin section were cut at 4μm thickness. After hematoxylin and eosin (H & E) staining, the slides were observed for conventional morphological evaluation under a light microscope and photographed.3.3 Assay of hepatic MDA and SOD Each liver tissue sample was homogenized in nine volumes of ice cold 0.86% NaCl saline and centrifuged at 3000 rpm for 10 min at 4℃. Supernatant was used to determine the MDA, SOD and total protein concentrations by using the commercially available diagnostic kits. The levels of MDA and SOD were normalized with the total protein content.3.4 Western blot assay The liver tissues were harvested and homogenized in RIPA lysis buffer to prepare of whole-protein extracts. The lysates were centrifuged at 12,000 rpm at 4℃ for 20 min. Protein concentrations were determined with the BCA protein assay kit and 150μg of protein were loaded per well on SDS-PAGE. Subsequently, proteins were transferred to PVDF membranes. Membranes were blocked at room temperature for 2 h with blocking solution (5% skimmed milk in Tris-buffered solution plus Tween-20 (TBST):50 mM Tris-HCl,150 mM NaCl, pH = 7.5,0.1% v/v Tween 20). Membranes were then incubated overnight at 4℃ with primary antibodies for Bax, Bcl-2, cleaved caspase-3 and β-actin in blocking solution. After three 5 min washing in TBST, membranes were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody in blocking solution. The proteins were visualized using an enhanced chemiluminescence Western blot detection kit. The relative expression of target proteins was quantified by the Image-Analysis system.4 Statistical analysis All data were reported as mean±SD and analyzed using the SPSS 13.0 statistical software. Statistical analysis was conducted by one-way analysis of variance (ANOVA). If the variances were homogeneity, LSD was used to the multiple comparisons. If the variances were heteroscedastic, Dunnett’s T3 was used to the multiple comparisons. Significant difference at p< 0.05 was accepted for all the tests.Result:1. ZZDHD protects against CCl4-induced Hepatic dysfunction Serum activities of ALT and AST are biochemical markers of hepatic damage. Compared with the control group, serum ALT and AST levels in model group were significantly increased with significant statistical difference (P< 0.05), which indicated that CC14 induced acute liver injury model was established successfully. Positive control drug bifendate significantly reduced ALT level (P< 0.05), ZZDHD at three different dose can prevent the CCl4-induced increase of ALT (P< 0.05), medium and high dose can reduce the level of AST (P< 0.05). There is a significant difference among AST and ALT in three doses (P< 0.05), which presented a dose-dependent manner. The level of ALT in ZZDHD high dose group has no significant difference (P> 0.05) compared with that of bifendate group, but AST level was significantly lower (P< 0.05). ZZDHD can decrease CCl4-induced serum ALT and AST levels in mice.2. ZZDHD alleviated CC14-induced histopathological changes in the liver The liver section was observed under a light microscope after hematoxylin and eosin (HE) staining. The normal liver lobule structure was integrity with hepatic cords in order. The structure of hepatocytes was clear and the central vein and portal area were clear. In model group, hepatocyte showed that many focal necrosis around the central vein. Steatosis, ballooning degeneration and lymphocytic infiltration were observed in the model group. Compared with the model group, the liver tissue necrosis area decreased, and cell swelling, degeneration and other pathological changes were reduced to varying degrees in the positive control group and the drug administration group. The high dose of ZZDHD had the best effect.3. ZZDHD alleviated CCl4-induced Oxidative Liver Injury The hepatic level of MDA was assessed as an indicator of lipid peroxidation in oxidative liver damage. Compared with the control group, the level of hepatic MDA in model group increased significantly (P< 0.05), and hepatic SOD activity significantly reduced (P< 0.05). Compared with the model group, positive control drug bifendate and three doses of ZZDHD groups could significantly decrease the content of MDA and increase SOD activity in liver tissue (P< 0.05). There was significant difference among three different does of ZZDHD on the levels of MDA and SOD (P<0.05). High dose group showed the best effect. The levels of MDA and SOD had no significant difference between bifendate group and ZZDHD high dose group (P>0.05), which showed that the same effect. The results showed that ZZDHD has an antioxidant effect by decreasing MDA and increasing SOD activity.4. ZZDHD Decreases CCl4-Induced Apoptosis of Hepatocytes Compared with the control group, the expression level of Bax and cleaved caspase-3 in model group increased (P<0.05), and the expression level of Bcl-2 decreased (P<0.05), indicating that CC14 induced apoptosis in mouse liver cells. Compared with model group, the expressions of Bax and cleaved caspase-3 were down-regulated and bcl-2 was up-regulated in bifendate group and ZZDH three different dose groups (P< 0.05). The expressions of cleaved caspase-3 in ZZDHD medium and high dose group were less than that in bifendate group(P<0.05).Conclusion:(1) The acute liver injury model was successfully reproduced by intraperitoneal injection of 0.12% CC14 in KM mice.16 hours after injection was the appropriate time point to collect the blood samples and liver tissues tto measure the changes of serum ALT, AST and hepatic pathological changes.(2) ZZDHD has protective effect on against CC14-induced acute liver injury.(3) The hepatic protective effect of ZZDHD might be related with alleviation of oxidative stress and effect of anti-apoptosis. |
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