| Purpose:The formula of Zeming Hongshan Tea is the Classical prescription which summarized the treatment of non-alcoholic steatohepatitis(NASH)by Professor Lu Bingjiu.At first,this research was to observe the clinical therapeutic effect of the formula of Zeming Hongshan Tea.Then,through the establishment of the model of non-alcoholic steatohepatitis rats and fatty degeneration of liver cell LO2,The effects of Caspase-3,Cyclin D1 mRNA and protein expression observed invivo and invitro,the study on the formula of Zeming Hongshan Tea on anti-apoptosis mechanism of non-alcoholic steatohepatitis,so as to explore the efficacy and mechanism of the formula of Zeming Hongshan Tea.Material and method:In the clinical study:112 patients with Non-alcoholic steatohepatitis were randomly divided into three groups,including the combination of Traditional Chinese Medicine(TCM)and Western medicine group,TCM group and Western medicine control group.Besides of the basic treatment,the combination of TCM and Western medicine group received the formula of Zeming Hongshan Tea and Silibinin,TCM group received the formula of Zeming Hongshan Tea and western medicine control group just received Silibinin.At last,compaired with before effects,three groups effects and indexes results including the TCM symptom integral meter,BMI,transaminase,serum lipid level and liver B-ultrasonic examination of the patients,and to evaluate the safety of the formula.Researches inVivo 1:We divided 62 healthy male Wistar rats into two groups in random,including a normal control group which had 11 rats,and the rest 51 rats belong to the the high-fat model group.In the first eight weeks,we fed the rats in normal control group with the standard feed,and we gave the another group a high-fat diet in the high-fat model group.Then,we set up a model by randomly choosing one rats in these two groups and executing them and taking the livers to do a biospy for histological examination.The experimental model is successful.After that,we divided the rats in high-fat model group into three different doses formula of Zeming Hongshan Tea treated groups and western medicine control group in random,and each group contains 10 rats.And then,we gave the rats in normal control group and model group gastric perfusion with 0.9%saline,and the silibinin for the rats in western medicine control group,and traditional Chinese medicine for the rest for six weeks.By the end of the fourteenth week,we executed the rats,and then tooled the serum and liver,weighing the liver and measuring the liver index.We observed the pathological changes in liver by HE staining and the degree of lipid change.We tested the level of ALT,AST,TG,TC,LDL,HDL in serum,and the level of Cyclin D1,Caspases-3 mRNA and protein in rats' cells by RT-PCR and Western-blot methods.Finally,we figured out the mechanism and efficacy of anti-apoptosis that the formula of Zeming Hongshan Tea do to rats with non-alcoholic steatohepatitis invivo.Researches inVitro 2:At first,we built up the invitro model that free fatty acids induce L02 liver cells with steatosis.We cultured the human L02 liver cells in RPMI1640 medium that contained 10%fetal bovine serum,after the cells were gave in even distribution,we divided them into six groups,including normal control group,model group,high dose,medium dose,and low dose formula of Zeming Hongshan Tea treated group and western medicine control group,six vials per groups.We used the 0.5mmol/L free fatty acids to induce five groups L02 non-alcoholic fatty liver cells except for control group,after 24 hours,we randomly chose one bottle to observe the lipid droplets in L02 cells by oil red O staining.After the successful induction had been proved,we added 10%normal rat serum to normal control group and model group,and we added the 10%rats serum that contains correspondent medicine in low dose,medium dose,and high dose formula of Zeming Hongshan Tea treated group and silibinin group.After 24 hours culture,we collected the cells and observed the level of lipid in cells,in the meantime,we tested the activity of the ALT,AST and observed the apoptosis of cells by TUNEL staining.Then,we tested the level of Cyclin D1,Caspases-3 mRNA and protein in cells by RT-PCR and Western-blot methods.Finally,we figured out the mechanism of anti-apoptosis that the formula of Zeming Hongshan Tea do to L02 liver cells with steatosis invitro.Results:Experiment No.1 The clinical experiment of the formula of Zeming Hongshan Tea on NASH patients.1.The TCM symptom integral meterThe total effective rate of the combination of TCM and western medicine group was 85.71%.The total effective rate of the TCM group was 77.77%,And the total effective rate of the western medicine control group was 67.64%.The difference among three groups was compared significant difference(P<0.05).2.The BMI,transaminase,serum lipid levelAfter the treatment of patients of three groups,the BM1,transaminase(ALT,AST),serum lipid level(TG,TC)decreased significantly(P<0.05),HDL increased significantly(P<0.05).The comparison of transaminase:The result of the combination of TCM and western medicine group was the best of all(P<0.05),and the TCM group was better than the western medicine control group.The comparison of serum lipid level:The result of the compare of the TG?TC?LDL on the combination of TCM and western medicine group and the TCM group were decreased significantly,the result of compare of HDL on the combination of TCM and western medicine group and the TCM group were increased significantly.The difference was statistically significant among three groups(P<0.05).3.Liver B-ultrasonic examinationThe total effective rate of the combination of TCM and western medicine group was 77.14%,the TCM group was 66.67%and the western medicine control group was 61.76%.There was statistic different among the three groups(P>0.05).It meant that in the improvment of liver morphology,the combination of TCM and western medicine group was superior to the TCM group and the western medicine control group.4.Comprehensive efficacy evaluationThe total effective rate of the combination of TCM and western medicine group was 74.28%,the TCM group was 61.11%and the western medicine control group was 58.82%.The difference was statistically significant among the three groups(P<0.05).The result of total effective in the combination of TCM and western medicine group was the best of all(P<0.05),the TCM group was better than the western medicine control group(P<0.05).5.Safety AnalysisThere were no side effects and adverse events occurring during the process.Experiment No.2 The experimental study of the effect of the expression of Cyclin Dl,Caspase-3 mRNA and protein of the formula of Zeming Hongshan Tea on NASH model.1.The general conditions and pathological morphologyThe experiment had been lasted for 14 weeks.In this experiment,the rats in the control group remained actively and stayed normally in following aspects,such as growing,feeding,drinking,etc.They all have clean fur,bright luster and weight up,moreover,there is a zero death rate.During the set up of model,the rats in model group all had lags in response,dim fur,obvious weight up and there was a zero death rate,too.At the end of the 8th week,we randomly chose a rat in normal control group and model group respectively,and then we took the liver out to do a biospy for histological examination.If cellular swelling and lipid change occur,fat vacuoles were found in cytoplasm,and a dense inflammatory infiltrate present,then we had set up a successful NASH model.After 6 weeks medicine treating,the rats in high dose,middle dose,and low dose Zeming Hongshan Tea formula group and the western medicine group showed some recovery,among them,the rats in the high dose Zeming Hongshan Tea formula treated group had fastest recovery,they had a normal diet,bright fur and obvious weight down,and the rats in the rest of groups all had various degrees of improvement.Analysis of body mass and liver wet weight:Compared with the normal control group,the body mass and liver wet weight of rats in model group had a great increase,the difference is significant(P<0.01).Compared with the model group,the body mass and liver wet weight of rats in Zeming Hongshan Tea formula treated groups and western medicine control group had a great decrease,the difference was significant(P<0.05).Compared with the western medicine control group,the difference of the body mass and liver wet weight of rats in middle dose,high dose Zeming Hongshan Tea formula treated group was not statistically significant(P>0.05).2.Liver function and serum lipid levelCompared with the normal control group,The result of serum ALT,AST,TG,TC,LDL of the rats in model group significantly increased and HDL decreased remarkably while the indexes of ALT,AST,TG,TC,LDL significantly decreased and HDL increased remarkably in all the intervention groups(P<0.05 or P<0.01),in which the middle and high dose Zeming Hongshan Tea group improved the liver function and blood lipids was not statistically significant(P>0.05).3.The expression of Cyclin D1,Caspase-3 mRNA and protein in all groupsCompared with the normal control group,the level of Cyclin D1,Caspase-3 mRNA and protein had a obvious increase in model group which was significant difference(P<0.01);Compared with the model group,the level of Cyclin D1,Caspase-3 mRNA and protein had a obvious decrease in middle and high doses of Zeming Hongshan Tea formula groups and western medicine control group(P<0.01);Compared with the western medicine control group,the level of Cyclin D1,Caspase-3 mRNA in high dose of Zeming Hongshan Tea formula group had no obvious difference(P>0.05).Experiment No.3 The experimental study on steatosis of L02 cells Zeming Hongshan Tea anti-apoptosis mechanism1.Observe the lipid in liver cells by oil red O stainingCulturing the human L02 liver cells in 10%fetal bovine serum contained 1640 medium,then we set up the steatotic L02 liver cells model through the induction by free fatty acids.We observed the lipid content of liver cells in each group by optical microscope:In the normal control group,the cell edge was clear,cytoplasm was abundant and nuclear membrane integrated and no red lipid droplets were found in the cells.In the model group serious steatosis occurs,massive red and orange vesicular lipid droplets were found in cytoplasm and cells maintain a great activity and shape.Most of the cells were polygon,some of them became round and intercellular adhesion less closed.we call it a successful model of steatosis in liver cells.Cells eage were clear and the number of lipid droplets in cytoplasm decreased significantly in each dose group of Zeming Hongshan Tea formula and the western medicine control group,they all explained that Zeming Hongshan Tea formula and Silibinin had certain inhibitory action on intracellular fat accumulation.2.Test the activity of ALT,ASTALT:Compared with the normal control group,the level of ALT in the model group increased obviously.There was significant difference between two groups(P<0.01);Compared with the model group,the level of ALT in each dose group of Zeming Hongshan Tea formula and the western medicine control group decreased obviously.There was significant difference between two groups(P<0.01);Compared with the western medicine control group,the difference of each dose group of Zeming Hongshan Tea formula was not statistically significant(P>0.05).AST:Compared with the normal control group,the level of ALT in the model group increased obviously.The difference was statistically significant(P<0.01);Compared with the model group,the level of ALT in the middle and high dose group of Zeming Hongshan Tea formula and the western medicine control group decreased obviously.The difference was statistically significant(P<0.01 ? P<0.05);Compared with the western medicine control group,the difference of each dose group of Zeming Hongshan Tea formula was not statistically significant(P>0.05).3.Observe the apoptosis of cells by TUNEL stainingCompared with the control group,we find the rate of apoptotic liver cells in model group apparently increased.The difference was statistically significant(P<0.05);After drugs intervention,the rate of apoptotic liver cells in three doses groups of Zeming Hongshan Tea formula and the western medicine control group significantly decreased(P<0.01);The difference was statistically(P<0.01);Compared with western medicine control group,high dose group had no significant difference(P>0.05).4.The expression of Cyclin D1,Caspase-3 mRNA and protein in all groupsCompared with the normal control group,the level of Cyclin D1,Caspase-3 mRNA and protein had a slight increase in model group.The difference was statistically significant(P<0.01);Compared with the model group,the level of Cyclin D1,Caspase-3 mRNA and protein had a obvious decrease in middle and high doses of Zeming Hongshan Tea formula groups and western medicine control group(P<0.01);Compared with the western medicine control group,the level of Cyclin D1,Caspase-3 mRNA in high dose of Zeming Hongshan Tea formula group had no obvious increase(P>0.05).Conclusion:1.The formula of Zeming Hongshan Tea can treat NASH through improve BMI index,liver function,blood fat level,hepatic morphology and other aspects.There were no side effects and adverse events occurring during the process.According to the reduction of visceral fat accumulation to achieve the purpose of treating NASH.The effect was better than it was used combined with western medicine silibinin.2.The formula of Zeming Hongshan Tea can improve the adipose degeneration of NASH by protecting liver function,regulating blood lipid,improving the pathological aspects of liver tissue damage of rats.3.Pathological apoptosis of liver cells promotes the development of NAFLD during the conversion process of adipose degeneration of liver tissue,apoptosis of hepatocytes,inflammation and necrosis of liver tissue and hepatic fibrosis.4.The formula of Zeming Hongshan Tea can protect the liver cells,inhibit the inflammation and prevent or delay the progression of fatty liver.The formula of Zeming Hongshan Tea has some effects of anti-apoptosis,which may be associated with the decrease of the expression of Cyclin D1 and Caspase-3 in liver.5.According to the comparisons of results,the effect of high dose group of Zeming Hongshan Tea formula was the best. |
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