Study On The Mechanism Of Buyang Jiedu Huayu Granules In Improving Microcirculation Disorder In Chronic Liver Failure Rats

Objective: Established the animal model of chronic liver failure(CLF),to observe the effect of Buyang Jiedu Huayu granule(BYJDHY)on liver microcirculation,and to study the expression of endothelin-1(ET-1)and nitric oxide(NO)in chronic liver failure rats.Objective to explore the effect of BYJDHY on chronic liver failure and the relationship between the change of protein gene content and its relationship,and to reveal its possible mechanism of improving liver regeneration microenvironment and preventing chronic liver failure.Methods: Seventy SPF grade male SD rats,weighing 180-250 g,were fed adaptively for one week.The experimental animals were randomly divided into 4 groups: control group(Control),model group(Model),Buyang Jiedu Huayu granule treatment group(BYJDHY),eNOS signal pathway inhibitor + Buyang Jiedu Huayu granule(L-NAME+BYJDHY)intervention group,10 rats in control group,and the other 20 rats in each group.Except the control group,all rats were injected intraperitoneally with 50% carbon tetrachloride vegetable oil solution(CCl4: Olive oil = 1:1)2.0ml/kg for 8 weeks,and then injected intraperitoneally with 1.5ml/kg until the 10 th week.After modeling,BYJDHY group and L-NAME+BYJDHY group were given BYJDHY 18.9g /(kg · d)by gavage for 2 weeks,and L-NAME+BYJDHY group were given L-NAME 8mg /(kg · d)by intraperitoneal injection for 1 week.At the same time,1.5ml/kg CCl4 vegetable oil solution was injected intraperitoneally every three days to maintain the timeliness of chronic liver failure rats.The Control group was given the same amount of distilled water instead.During the modeling and intervention,the general situation of rats in each group was observed,and the changes of body weight and abdominal circumference were monitored.48 hours after the end of treatment,blood was taken and liver was dissected.After HE and Masson staining,the pathological changes of liver tissue were observed under light microscope.The levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by ELISA,RT-PCR was used to detect the mRNA expression of Gab1,Akt and eNOS in liver tissue.Western Blot was used to detect the protein expression of Gab1,Akt and eNOS in liver tissue.Results:(1)General situation: there was no significant difference in the control group.With the extension of medication time,compared with the control group,the hair color of the model group,the BYJDHY group and the inhibitor group was dark yellow,the glossiness decreased,and part of the hair fell off.The results showed that the body weight was decreased,the reaction to external stimulation was slow,the rats liked to curl up,the body was thin,the abdomen was swollen,the ascites was obvious,the spirit was depressed in varying degrees,the food intake was reduced,the dyspepsia,loose stools,yellow urine were observed.Some rats had symptoms of hematuria,skin disease,abscess,etc.these symptoms also confirmed the serious metabolic disorder in the experimental animals Chaos.Among them,the diet,activity and spirit of rats in BYJDHY group were better than those in model group.(2)Weight change: there was no significant difference in the initial weight before the experiment among the control group,model group,BYJDHY group and L-NAME+BYJDHY group(P>0.05);after 12 weeks of experiment,the weight of the control group increased significantly,compared with the model group,BYJDHY group and L-NAME+BYJDHY group(P < 0.05).The difference was statistically significant;compared with BYJDHY group,the weight increase of the model group was small(P < 0.05).After the fifth week,the weight of rats in the control group increased significantly compared with the other groups.The weight of rats in the other three groups increased,but the increase was significantly lower than that of the control group.After 10 weeks,with the intervention of traditional Chinese medicine,the growth rate of rats in the model group was increased compared with that in the same period.(3)Abdominal circumference change: there was no significant difference in the initial abdominal circumference before the experiment,control group,model group,BYJDHY group and L-NAME+BYJDHY group(P > 0.05); after 12 weeks of experiment,the abdominal circumference of rats in each group increased in varying degrees.Compared with the model group,the BYJDHY group and L-NAME+BYJDHY group,the abdominal circumference of the control group was smaller(P < 0.05),compared with the BYJDHY group and L-NAME+BYJDHY group,the abdominal circumference of the model group was larger(P < 0.05).(4)HE and Masson staining of liver tissue were observed under light microscope: in the control group,the structure of hepatic lobules was normal,no obvious necrosis or cholestasis of hepatocytes,no obvious injury or fibrosis of central vein,no obvious infiltration of inflammatory cells in hepatic sinuses,no lymphocyte infiltration in portal area,no obvious injury of bile duct cells,no obvious hyperplasia of fibrous tissue in liver tissue,no atypia or mitosis of hepatocytes The pathological grade was 0.In the long-term CCl4 treated rat liver tissue sections,we observed significant changes in the structure of typical cirrhotic organs.In the model group,the hepatic lobules were significantly fibrous tissue hyperplasia,the normal hepatic lobules were reduced,most of them were replaced by the typical pseudo lobules,the inflammatory cells in the pseudo lobules were diffusely infiltrated,the hepatic sinuses and small hepatic vessels were dilated and hyperemia,obvious edema,the hepatocytes were diffusely scattered in steatosis,disordered arrangement,and punctate necrosis,which were the obvious manifestations of liver cirrhosis,and the pathological changes were graded as grade 6.Compared with the model group,the structure of hepatic lobules in the inhibitor group was improved.The liver cells were turbid and swollen in varying degrees,with a small amount of balloon like changes.There were punctate necrosis,central venous fibrosis,fibrous tissue hyperplasia in liver tissue,obvious pseudo lobule formation,and pathological changes were graded as grade 5.In the BYJDHY group,the structural disorder of hepatic lobule was improved to some extent,and the liver fibrosis,liver degeneration and liver necrosis were improved to varying degrees.The pathological changes were graded as grade 4.(5)Serum biochemical results: compared with the control group,levels of ALT,AST,TBIL in the model group,BYJDHY group and L-NAME+BYJDHY group were significantly increased,ALB was significantly decreased(P < 0.05);compared with the model group,levels of ALT,AST,TBIL in the BYJDHY and L-NAME+BYJDHY group were significantly lower,ALB was significantly higher(P<0.05),TBIL of BYJDHY group was lower than that of inhibitor group(P<0.05);ALB of BYJDHY group was higher than that of L-NAME+BYJDHY group(P < 0.05).(6)NO / ET-1 results: Compared with the control group,the contents of NO and ET-1 in the liver tissue of the model group,the BYJDHY group and L-NAME+BYJDHY group were significantly increased(P < 0.05);compared with the model group,the levels of NO in BYJDHY group and L-NAME+BYJDHY group were lower than those in the model group(P < 0.05);the level of NO in BYJDHY group was higher than that in L-NAME+BYJDHY group(P < 0.05).(7)RT-PCR: Compared with control group,the expressions of eNOS,Gab1 and Akt mRNA in liver tissue of model group,BYJDHY group and L-NAME+BYJDHY group were significantly decreased(P < 0.05);compared with the model group,the expressions of eNOS mRNA in liver tissue of BYJDHY group were significantly higher than those of model group(P<0.05);the expressions of Gab1,Akt and eNOS mRNA in liver tissue of BYJDHY group were significantly higher than those of model group(P < 0.05).The mRNA expression level in BYJDHY group was significantly higher than that in L-NAME+BYJDHY group(P < 0.05).(8)WB: Compared with control group,the expressions of eNOS and Gab1 in liver tissue of model group,BYJDHY group and L-NAME+BYJDHY group were significantly higher compared with the model group,the protein contents of Gab1,Akt and eNOS in BYJDHY group and L-NAME+BYJDHY group were significantly higher than those in the model group(P < 0.05);the protein contents of BYJDHY group and L-NAME+BYJDHY group were significantly higher than those in the model group(P < 0.05)The protein contents of Gab1,Akt and eNOS in liver tissue of BYJDHY group were significantly higher than those in L-NAME+BYJDHY group(P < 0.05).Conclusions:(1)CCl4 can successfully construct the model of chronic liver failure rats.(2)Buyang Jiedu Huayu granule can regulate the concentration of vasoactive substances no and ET-1 in rats with chronic liver failure,improve liver microcirculation and alleviate liver hypoxia.The mechanism of improving microcirculation in rats with chronic liver failure may be through regulating Gab1-Akt-eNOS signaling pathway,reducing hepatic pathological angiogenesis,maintaining effective blood flow and improving hepatic microcirculation.