Experimental Study On Discussing Syndrome-treatment Characteristic And Evolution Axiom Of Traditional Chinese Medicine Of NAFLD By Using Treatment Analyze Syndrome

Objectives:With the standard of living enhancement,more and more people are suffering Nonalcoholic Fatty Liver Disease(NAFLD).NAFLD includes three pathology types of Nonalcoholic fatty liver,Nonalcoholic steatohepatitis(NASH),Adipose hepatic cirrhosis.At now,there has no ideal medicine to cure NAFLD;the Chinese traditional medicine has certain superiority.In different stage of NAFLD morbidity process,the pathogenesis of traditional Chinese medicine is different.My tutor, professor Sheng Guo-guang discovered in the clinical work that the basic pathogenesis of NAFLD were phlegm stagnation,obstruction of collaterals by blood stasis.But in different stage of morbidity process,the pathogenesis of NAFLD had the difference slightly:The early period pathogenesis was accompanied with Liver strongly fragrant spleen weakness, and then the later period pathogenesis was accompanied with damp depression heat-transmission and hepatorenal insufficiency.According to the clinical experience,professor Sheng Guo-guang used different treatments in view of NAFLD pathogenesis difference,had obtained the good curative effect on clinical.At the foundation of effective therapy in earlier period research,our topic adopts the rat model of NAFLD recognized by domestic and foreign.We observe the effect of Chinese medicine various therapies interfering in changed index of Blood serum biochemistry,Fatty acid metabolism,Inflammatory factor,Lipin peroxidation,Liver pathology,Mitochondrial COX gene expression in rat model of NAFLD;approach the pathogenesis of NAFLD and syndrome-treatment characteristic and evolution axiom of Traditional Chinese Medicine.Methods:The experiment was divided into three parts.The primary part:The establishment of rat models NAFLD and different traditional Chinese medicine treatments affect blood serum biochemistry target,liver organization pathology and so on of rat in the different time of NAFLD.(Experiment one to two)Eighty SPF grade Wistar rats,were randomly divided into 5 groups,each group of 16:Normal group,Model group,Comparison medicine treatment group(effectiveness of activating blood circulation to dissipate blood stasis and dissipating phlegm to decrease wet),Medicine A treatment group (effectiveness of dispersing the depressed liver-energy to invigorate the spleen,activating blood circulation to dissipate blood stasis and dissipating phlegm to decrease wet),Medicine B treatment group(effectiveness of dissipating phlegm to dissipate blood stasis, clearing away heat evil to decrease wet and benefiting hepatorenal function),To adopt the high fat diet(ordinary feedstuff to add 15%lards, 2%cholesterol,5%yolk powder)to duplicate the rat model of NAFLD.When the experiment were in 8,12 weeks,each group took 8 rats at random,took the blood serum and the liver of rat to detect index.Used Entire automated biochemical analysis instrument to detect the content of rat serum Triglyceride(TG),Total cholesterol(TC),Low density lipoprotein(LDL), Alanine aminotransferase(ALT),Aspartate aminotransferase(AST); Emission immunization detected Tumor necrosis factor-α(TNF-α), Computed liver index,to detect the content of superoxide dismutase(SOD), Mononuclear phagocyte system(MDA),Reduced glutathione hormone(GSH), Free fatty acids(FFA)of rat's hepatic tissue according to the instructions of kits.Hepatic tissue were stained with HE dyeing,the degree of hepatic steatosis and inflammation activity were evaluated under the optical microscope.The second part:Discussed syndrome-treatment characteristic of Traditional Chinese Medicine of NAFLD by using treatment analyze syndrome and effect of different treatments on the indexes of liver lipid metabolism,liver function,Insulin resistance,hepatic tissue pathology of NAFLD of rat.(Experiment three to five) Seventy two SPF grade Wistar rats,females and males were half and half, were divided into 9 groups at random,each group of eight:Normal group, Model group,Comparison medicine treatment group,Medicine A treatment group,Medicine B treatment group,from 4 to 8 weeks given Medicine A and from 8 to 12 weeks given Medicine B group,From 4 to 8 weeks given Medicine A and from 8 to 12 weeks given Medicine B group,From 4 to 8 weeks given Medicine B and from 8 to 12 weeks given Medicine A group,First blank and then from 8 to 12 weeks given Medicine A group,First blank and then from 8 to 12 weeks given Medicine B group.To adopt the high fat diet to duplicate the rat model of NAFLD,Used Entire automated biochemical analysis instrument to detect the content of rat serum Triglyceride(TG), Total cholesterol(TC),Low density lipoprotein(LDL),Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),Alkaline phosphatase(ALP),Total Bilirubin(TBil),Insulinum(INS),Insulin resistance(IR);Emission immunization detected Tumor necrosis factorα(TNF-α),Computed liver index,To detect the content of superoxide dismutase(SOD),Mononuclear phagocyte system(MDA),Reduced glutathione hormone(GSH),Free fatty acids(FFA)of rat' s hepatic tissue according to the instructions of kits.Hepatic tissue were stained with HE dyeing, the degree of hepatic steatosis and inflammation activity were evaluated under the optical microscope.The third part:the effect of different traditional Chinese medicine treatments on pathology and genetic expression of hepatic mitochondria of NAFLD.(Experiment six to seven) Forty SPF grade Wistar rats,were randomly divided into 5 groups,each group of 8:Normal group,Model group,Comparison medicine treatment group, Medicine A treatment group,Medicine B treatment group,to adopt the high fat diet to duplicate the rat model of NAFLD,to detect rat hepatic tissue pathology variance through electronic microscope,adopt the RT-PCR method to detect the expression of COXⅡ,Ⅲof rat hepatic mitochondria,adopt the Western-blot technique to detect the variance of COXIgene protein expression of rat hepatic mitochondria.Results:The primary part:The establishment of rat models NAFLD and different traditional Chinese medicine treatments affect blood serum biochemistry target,liver organization pathology and so on of rat in the different time of NAFLD.(Experiment one to two) The rat model of NAFLD was duplicated successfully.1.When the experiment were in 8 weeks,Medicine A treatment group had better curative effect than other treatment groups at reducing the blood serum value of AST,ALT, TG,TCH,FFA,LDL,TNF-α,the content of hepatic tissue MDA;raising the content of SOD and GSH-ST(P＜0.05 or P＜0.01).In pathology influence aspect,Medicine A treatment group had better curative effect than other treatment groups at relieving the degree of hepatic cell steatosis and the activity of pathology inflammation(P＜0.05 or P＜0.01).2.When the experiment were in 12 weeks,Medicine B treatment group had better curative effect than other treatment groups at reducing the blood serum value of AST,ALT,FFA,TNF-α,liver index,the content of hepatic tissue MDA;raising the content of SOD and GSH-ST(P＜0.05 or P＜0.01).In pathology influence aspect,Medicine B treatment group had better curative effect than other treatment groups at relieving the degree of hepatic cell steatosis and the activity of pathology inflammation(P＜0.05 or P＜0.01).The second part:Discussed syndrome-treatment characteristic of Traditional Chinese Medicine of NAFLD by using treatment analyze syndrome and effect of different treatments on the indexes of liver lipid metabolism,liver function,Insulin resistance,hepatic tissue pathology of NAFLD of rat.(Experiment three to five) When the experiment were in 12 weeks,First Medicine A then Medicine B treatment group had better curative effect than other treatment groups at reducing the blood serum value of TG,TCH,LDL,FFA,TNF-α,ALT,AST, ALP,TBIL,DBIL,INS,IR,liver index,the content of hepatic tissueMDA; raising the content of SOD and GSH-ST(P＜0.05 or P＜0.01).In pathology influence aspect,First Medicine A then Medicine B treatment group had better curative effect than other treatment groups at relieving the degree of hepatic cell steatosis and the activity of pathology inflammation (P＜0.05 or P＜0.01).The third part:the effect of different traditional Chinese medicine treatments on pathology and genetic expression of hepatic mitochondria of NAFLD.(Experiment six to seven)1.Compared with normal group,hepatic mitochondria had different degree pathology change in each model group.At the end of the 12 weeks,hepatic mitochondria duplicate and proliferate actively,had not the obvious shape and the structure change in Medicine B treatment group;Medicine B treatment group had better curative effect on protecting hepatic mitochondria than other treatment groups,such as Model group,Comparison medicine treatment group,Medicine A treatment group.2.Compared with normal group,hepatic mitochondria COXⅡ,ⅢmRNA,COX I protein had lower expression in each NAFLD model group.Medicine B treatment group had better curative effect than other treatment groups at raising expression of COXⅡ,ⅢmRNA,COXⅠprotein of hepatic mitochondria(P＜0.05 or P＜0.01).Conclusion:1.The animal model establishment To adopt the high fat diet to duplicate the rat model of NAFLD,at the experiment of 8th weeks,hepatic tissue pathology of rat Model group of NAFLD were consistent with pathological changes of Nonalcoholic fatty liver;at the 12th weeks,liver functional lesion,rising blood-fat and hepatic tissue pathological changes of rat Model group were consistent with pathological appearance of Nonalcoholic fatty liver.The rat model of NAFLD was duplicated successfully.2.Research on the hepatic pathomeehanism and the syndrome pattern of traditional Chinese medicine of NAFLD2.1The change of pathogenesis and the syndrome pattern of traditional Chinese medicine were in the different time of NAFLD. At the NAFLD experiment of 8th week,hepatic tissue pathological appearance of rat was hepatic steatosis of big soaks consistent with pathological changes of Nonalcoholic fatty liver;the result of multi-treatments of traditional Chinese medicine indicate that effectiveness of dispersing the depressed liver-energy to invigorate the spleen,activating blood circulation to dissipate blood stasis and dissipating phlegm to decrease wet treatments had better intervention therapeutic effect in the stage;At 12th week,hepatic tissue pathological changes of rat were mixing hepatic steatosis consistent with pathological appearances of Nonalcoholic steatohepatitis;the intervention result of traditional Chinese medicine indicated that effectiveness of dissipating phlegm to dissipate blood stasis,clearing away heat evil to decrease wet and benefiting hepatorenal function treatments had better therapeutic effect.Insulin resistance and fatty acid metabolism unbalanced were the main invasive factors in the period of Nonalcoholic fatty liver,Lipin peroxidation;oxidative stress and inflammatory reaction induced by kinds of cell factor were the main pathogenesis of NASH.Difference curative affects of traditional Chinese medicine different treatments in the NAFLD various periods indicated that the traditional Chinese medicine basic pathogenesis of NAFLD was phlegm stagnation,obstruction of collaterals by blood stasis.Traditional Chinese medicine basic pathogenesis was accompanied with Liver strongly fragrant spleen weakness in the period of Nonalcoholic fatty liver;Traditional Chinese medicine basic pathogenesis was accompanied with damp depression heat-transmission and hepatorenal insufficiency.2.2 Action mechanism of hepatic mitochondria in NAFLD Hepatic mitochondria had impaired function of fatty acid oxidation metabolism,decreased ability of eliminating free radical,which were the important factors of fatty acid metabolism unbalanced,Lipin peroxidation, oxidative stress,were the important reasons of NAFLD pathogenetic condition being advancement.3.Action mechanism of Chinese medicine differentiation of symptoms and signs treatment to the NAFLD3.1 Effect of traditional Chinese medicine different treatments interfering in the NAFLD various periods Traditional Chinese medicine divided the syndrome pattern of NAFLD into Liver strongly fragrant spleen weakness,phlegm stagnation,obstruction of collaterals by blood stasis,damp depression heat-transmission and hepatorenal insufficiency,according to the clinical manifestation in the morbidity process of NAFLD.According to different pathogenesis of NAFLD, we interfered in NAFLD by different treatments of effectiveness of dispersing the depressed liver-energy to invigorate the spleen, activating blood circulation to dissipate blood stasis and dissipating phlegm to decrease wet,clearing away heat evil to decrease wet and benefiting hepatorenal function.The experimental result indicated that treatments of dispersing the depressed liver-energy to invigorate the spleen,activating blood circulation to dissipate blood stasis and dissipating phlegm to decrease wet had better therapeutic effect in the early period,treatments of dissipating phlegm to dissipate blood stasis, clearing away heat evil to decrease wet and benefiting hepatorenal function treatments had better therapeutic effect in the later period. Curative effects of each traditional Chinese medicine treatments mainly displayed in improving liver function,liver index,the activity of pathology inflammation,reducing the inflammation factor level,free fatty acids,serum triglyceride,elevating ability of anti-oxygen free radical and so on.3.2 mechanism of Chinese medicine differentiation of symptoms and signs treatment interfered in the NAFLD Adopted treatments of dispersing the depressed liver-energy to invigorate the spleen,activating blood circulation to dissipate blood stasis and dissipating phlegm to decrease wet in the early period,and treatments of dissipating phlegm to dissipate blood stasis,clearing away heat evil to decrease wet and benefiting hepatorenal function treatments in the later period,which had better therapeutic effect in the process of preventing and curing NAFLD.Mechanism of treating NAFLD displayed in reducing serum triglyceride,low density lipoprotein,free fatty acids, inflammation factor level,relieving lipid peroxidation damage,liver inflammation activity,improving insulin resistance and so on.3.3 Pathology changes of hepatic mitochondria and mechanism of traditional Chinese medicine treatments interfered in NAFLD Cytochrome oxidase(COX)was the key enzyme,which judged oxidizing ability of mitochondria,lower expression of hepatic mitochondria COXⅡ,ⅢmRNA,COXⅠprotein indicated that Hepatic mitochondria had impaired function of fatty acid oxidation metabolism,decreased ability of eliminating free radical,which were the primary factors of fatty acid metabolism unbalanced,lipid peroxidation.The mechanism of traditional Chinese medicine intervened NAFLD was possibly adjusting the COX gene, the protein expression to prevent and to delay further development of NAFLD.