Screening And Evaluation Of Anti-fibrosis Effect Of Drugs Used By Wa Nationality People In Liver Diseases

Objective: This research project intends to take the rat hepatic stellate cells HSC-T6 and liver fibrosis model rats as the research objects,Leverage Yunnan's abundant Wa medicine resources to screen out medicines that may have the role of preventing and treating liver fibrosis through investigation.The Wa medicines screened by the investigation acted on HSC-T6 in vitro,and the effects of drugs on HSC-T6 cell proliferation and cell viability were analyzed and evaluated by Celigo Cytomter full-field cell scanning analyzer,Active medicinal materials that promote its apoptosis.Then,through the selection of a medicinal material with the best screening experiment effect in vivo,an experimental study was conducted on a rat model of liver fibrosis.Method:Cell experiment:HSC-T6 cell line was purchased from the Cell Bank of Kunming Institute of Zoology,Chinese Academy of Sciences,Rat hepatic stellate cells HSC-T6 cells were cultured in RPMI-1640 culture medium(containing 10% fetal bovine serum,100 U / mL penicillin,100 U / mL streptomycin)in an incubator at 37°C and 5% CO2 saturated humidity,Take HSC-T6 cells in logarithmic growth phase,inoculate each group of cells with 1×104 cells / well in a 96-well plate,add the corresponding concentration of experimental herbs(filtered through 0.2um filter),The96-well plates were taken out 24 h,48h and 72 h after the medicinal materials were stimulated respectively,and the cell proliferation and cell viability were detected with Celigo Cytomter full-field cell scanning analyzer?Animal experiment:Select 85 SD rats,male,weighing 200±20g,randomly divided into 6 groups,10 blank groups,and 15 in each group.They are the blank group,the model group,the compound Biejia Ruangan tablet positive group,and thehigh,medium,and low dose groups of Artemisia halodendron.Except for the blank group,all the other groups were induced by compound factors to establish an animal model of liver fibrosis--40% oil CCL4 subcutaneous injection,the first injection was5.0ml / kg,and every 3 days thereafter,3.0ml / kg Subcutaneous injection of 40% oil CCL4,continuous injection for 4 weeks,at the same time feeding with high-fat feed and 10% ethanol at a dose of 2ml / kg,once every other day.Calculate the daily gavage dose for the week based on the weight weighed each week.In the positive group,compound Biejia Ruangan tablets were given by intragastric administration(concentration 0.027 g / ml,prepared with physiological saline after being ground into powder),Artemisia chinensis high(concentration0.5g/ml),medium(concentration0.25 g / ml)and low(concentration 0.125 g / ml)groups were fed with 20 ml / kg of water extract of Artemisia halodendron,the blank group and model group were given20 ml / kg of drinking water.Observe and record the general situation during the experiment.After 4 weeks of experiment,fast for 12 hours,weigh the last weight and record it,electronic scale weighs the liver wet weight and calculate the liver organ coefficient,automatic analysis method detection Rat liver function indexes(TBIL,DBIL,AST,ALT,GGT,HDL,LDL,TG);Four indexes of liver fiber(HA,LN,PC?,IV-C)were detected by enzyme-linked immunosorbent assay;liver pathological tissue sections were made by Masson staining method to observe the degree of liver fibrosis.Result:1.Cell experiment research shows:At 24h:Compared with the model group,when the concentration is 200,400,and 1000,P<0.01 is a significant difference,and when the concentration is 800,P<0.05 is a significant difference;the number 08 is at a concentration of 200.P<0.05 means that the difference is significant.Compared with the model group,at the concentration of1000 in the positive group,P<0.01 is a significant difference,and at the concentrations of 200 and 400,P <0.05 is a significant difference.Compared with the model group,when the concentration is No.09,13,17 at 200,P<0.01 is a significant difference;at the concentration of 200 in the positive group and No.11,P<0.05 is a significant difference.In the positive group and numbers 09,13,and 17 at a concentration of 400,P<0.05 indicated a significant difference.No.09,11,13,17,20 at a concentration of 800,P<0.01 is a significant difference.Nos.13,17,and 30,when the concentration is 1000,P<0.01 is a significant difference;when the positive group is 1000,P<0.05 is a significant difference.At 48h:Compared with the model group,when the number 23 is 400,800,1000,P<0.01 is a significant difference;when the number 22 is 1000,P<0.05 is a significant difference;compared with the model group In the positive group,when the concentration was 400 or 1000,P<0.05 indicated that the difference was significant.Compared with the model group,when the concentration is No.05 at 200,P<0.01 means that the difference is very significant.When the concentration of No.13 is 400,P<0.01 is a significant difference;when the concentration of the positive group and No.05 is 400,P<0.05 is a significant difference.Nos.05 and 13 have a significant difference when the concentration is 800,P<0.01 is the difference.Nos.13 and 30 had a significant difference at a concentration of 1000,P<0.01;at a concentration of 1000 in the positive group,P<0.05 indicated a significant difference.At 72h:Compared with the model group,No.23 at the concentration of 400,800,1000,P<0.01 is a significant difference;No.22 at the concentration of 1000,P<0.05 is a significant difference;No.08 at the concentration When it is 400,P<0.05 means that the difference is significant,and when the concentration is 800,P<0.01 means that the difference is very significant.Compared with the model group,P<0.05 for positive drugs at 400 and 1000 was considered significant.Compared with the model group,when the concentration is No.05 at 200,P<0.01 means that the difference is very significant.When the concentration of No.13 is 400,P<0.01 is a significant difference;when the concentration of the positive group and No.05 is 400,P<0.05 is a significant difference.Nos.05 and 20 have a significant difference at a concentration of 800,P<0.01;Nos.09,11,and 13 have a significant difference at a concentration of 800.No.05,13,17,30 at the concentration of 1000,P<0.01 is a significant difference;at the concentration of 1000 in the positive group and No.20,P<0.05 is significant.2.Animal experiments show that:2.1 General situation:According to the general morphological observation,the normal group of rats has bright and smooth coat color and normal drinking water intake.Compared with the normal group,the model group rats have significantly reduced water and food intake,dry and disordered coat color and lack of luster,andthe weight gain has even decreased,Mental atrophy,reduced activity,etc.Compared with the observation of the model group,the above symptoms of rats in each dose group and positive drug group of "Artemisia halodendron" have improved.2.2 Weight:Compared with the blank group,the weight value of the model group decreased,and P<0.01,the difference was very significant.Compared with the model group,the weight value of the compound Biejia Ruangan tablet positive group was207.7±27.71,P>0.05,and the difference was not statistically significant.Compared with the model group,the body weight values of Artemisia halodendron high,middle and low dose groups were 213.6±19.12 g,206.6±16.66 g,212.4±24.57 g,P> 0.05,the difference was not statistically significant.2.3 Coefficient of liver organs:Compared with the blank group,the liver organ coefficients of the model group and each dose group were significantly increased;compared with the model group,the liver organ coefficients of the positive group and the high and medium dose administration groups were reduced.The decrease in the high-dose group was most obvious,and the increase in the low-dose administration group was not statistically significant.2.4 Detection of four indicators of liver fiber:Compared with the blank group,the value of each group in the model group decreased,suggesting that liver fibrosis occurred in the model group.Compared with the model group,PC-? decreased and PC increased in the compound Biejia Ruangan tablet positive group,P<0.05,the difference was significant.Compared with the model group,PC-? decreased,HA increased,and Col-IV increased in the low-dose group,P<0.05,the difference was significant;compared with the model group,LN,HA increased,P<0.01,the difference has very significant significance.Compared with the compound Biejia Ruangan tablet positive group,LN increased in the low-dose group of Artemisia halodendron,P<0.05,the difference was significant;HA increased in the low-dose group of Artemisia halodendron,P<0.01,the difference was very significant.2.5 Serological testing:Compared with the blank group,the value of each group of the model group increased,P<0.01,the difference was very significant.Compared with the model group,the AST and TG of the compound Biejia Ruangan tablet-positive group decreased and the TG of the low-dose group of Artemisiachinensis increased,P<0.05.Compared with the compound Biejia Ruangan tablet positive group,the TG and LDL-C of Artemisia halodendron middle-dose group all increased,P<0.05,the difference was significant.2.6 Observation of liver pathology:Liver tissue pathological sections can directly observe the formation and progress of liver fibrosis,and are also a common method for testing liver fibrosis.In this experiment,the results of Masson staining of rat liver tissues intervened by the CCL4 compound method under light microscope showed that in the blank group: the rat liver tissues showed clear and complete hepatic lobule structure,neat arrangement of liver cords,no fibrous tissue hyperplasia,and normal liver tissues;Model group: Rat liver tissues are dim in color,hepatic lobular structure is destroyed and fibrous tissues are proliferated,central veins deviate or disappear,hepatic cords are disordered or even ruptured,liver plates are dissociated,hepatic sinuses become narrowed and blocked,and there is a large number of expansion of the manifold Infiltration of inflammatory cells,extensive hepatocyte balloon-like degeneration,steatosis,and necrosis of the hepatocytes in the central venous area and the peripheral area of the liver lobule,and the hepatocyte volume increased.The above pathological changes are basically consistent with the pathological changes of liver fibrosis,so it can be seen that the model meets the standards of liver fibrosis model.Compared with the model group,the liver in the positive group was reddish in color,the color quality improved,liver cell degeneration and necrosis were reduced,and fibrous tissue hyperplasia was relieved.Compared with the model group,the high-dose and medium-dose groups of Wa medicine Boontail Artemisia halodendron have delayed liver fibrosis,hepatocyte necrosis,steatosis,and inflammatory cell infiltration are significantly reduced.Only mild hyperplasia of collagen can be seen in the manifold Fibre and reticulum fibers and fibrous tissues remain after fiber absorption,and fibrous tissue hyperplasia is not obvious,but the low-dose group has no significant improvement in liver fibrosis,liver cell necrosis,steatosis,and fibrous tissue hyperplasia are not significantly relieved,suggesting Wa medicine cattle tail Artemisia high-dose and middle-dose groups can effectively reverse the liver fibrosis process in rats.Conclusion:1.Wafers of different concentrations,at different time points of action,can have varying degrees of effect on inhibiting the growth of hepatic stellate cell HSC-T6 cell lines.2.Artemisia halodendron can inhibit and reduce the level of liver fibrosis induced by CCL4 compound method,reduce liver index,liver function index and liver fibrosis index,and has the effect of protecting and improving the liver.