The Relationship And Medication Between Apoptosis-regulated Proteins Bcl-2,Bax And Nonalcoholic Steatohepatitis

Objective:NAFLD is a clinical syndrome, and has a spectrum ranging from that of fatty liver alone to nonalcoholic steatohepatitis (NASH), which may progress to liver cirrhosis and can progress to liver cell necrosis, fibrosis and cirrhosis of the liver. At present the mechanisms underlying NAFLD are focus on "two hits" theory posed by Day. With the study of apoptosis, more and more researches demonstrate that apoptosis is the key steps when fatty liver alone progress to nonalcoholic steatohepatitis (NASH), and closely related to inflammation,fibrosis. In the two of apoptosis signal transduction pathways, Bcl-2 family members' ratio is the key factor, especially the most representative members Bcl-2 and Bax, which have the simily structure but the opposite function, the ratio is directly determines the fate of liver cells when they accept the death message, the cell towards survival or death. In this study, we established rat NAFLD model by high-fat and high-cholesterol diet to try to explore the following vievpoint:(1)To observe the expression of apoptosis-regulated proteins Bcl-2,Bax in liver of rats in every stage of NAFLD model; (2)To investigate whether Jiangzhi Yigan granules can treat and prevent NAFLD through regulating the expression of apoptosis-regulated proteins Bcl-2,Bax or not. This investigation may provide new clues for the pathogenesis of NAFLD and instruct clinical medication.Methods:1. Animal Model and Specimen Collection:70 male Wistar rats fed a normal diet after one week, were randomly divided into normal control group (N group):normal diet; the model group (M group), drug treatment group (D group):given high fat diet (85% normal diet, 13% lard and 2% cholesterol). The experimental set up 4,8,12 weeks time point,10 rats for each time point, groups corresponding to the N1, N2, N3, M1, M2, M3, D. Drug treatment group was given Jiangzhi Yigan Chongji (1ml/100g rat body weight, its concentration is 0.8g/ml, twice every day) by intragastric administration separately after the end of 8th week, meanwhile the normal and model group were separately given life drinking water by the same means. All the rats were sacrificed after 4,8,12 weeks. Since the abdominal aortic blood, quickly remove the liver, according to conventional preparation of serum, liver homogenate, and to retain a small amount of liver tissue cryopreservation in-70℃.2. Detect indicators:measured body weight, body length, wet weight of liver, and calculate the liver index (liver wet weight/body weight×100%), Lee's index (0.33 weight/body length×1000) and other physical indicators. Automatic biochemical analyzer determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood lipids content, light microscope to assess steatosis, inflammatory activity and fibrosis; The levels of Bcl-2 and Bax expression were determined by Western Blot.Results:1. The liver lobules of normal group rats structural integrity, liver cord around the central venous actinomorphous distribution, cells were polygonal, various biochemical indices were within the normal range; the rates of Bcl-2 and Bax keep the balance.2. After 4 weeks, the model rats exhibited mild steatosis, no inflammatory cell infiltrationand and no fibrosis; increase of aminotransferase; compared with normal group, the expression of Bax protein was more pronounced, while the expression of Bcl-2 was decreased, Bcl-2/Bax was also decreased significantly (P<0.01).3. After 8 weeks, control with the N2 group, model group M2 rats, simple fatty liver were observed, no inflammatory cell infiltration and fibrosis S1; serum transaminase levels were significantly higher than normal, TC, TG, ALT, AST; liver Bax expression than the control group increased significantly, while Bcl-2, Bcl-2/Bax decreased (P<0.01).4. After 12 weeks, severe steatohepatitis with fibrosis S2 of M3 rats; compared with normal group, the level of aminotransferase and blood fat were increased in model group; liver Bax expression than the control group increased significantly, while Bcl-2, Bcl-2/Bax decreased; compared with M3 group, the treated group every biochemical manifestation shew improvement significantly, the expression of Bax in liver was decreased, Bcl-2 and Bcl-2/Bax increased; but still lower than normal group.Conclusion:1. High-fat and high-cholesterol diet can successfully copy the NAFLD rat model; lipid metabolism disorders, inflammatory injury and apoptosis are closely related to the occurrence of NAFLD.2. Bcl-2 and Bax can be expressed in normal rat liver, the expression of Bax protein was more pronounced from 4 week and continued to rise until 8 week and 12 week in the model group, compared with the normal group the expression of Bcl-2 was decreased, Bcl-2/Bax in the model group, particularly at 12 week decreased in a time-dependent manner.3. Jiangzhi Yigan granule has a good anti-therapeutic effects for combat NAFLD. Its molecular mechanism may be partly through up-regulating the expression of Bcl-2 of the live, down-regulating Bax and keeping the balance of Bcl-2 and Bax.