Efficacy And Mechanism Of Combination Of Kangxian Ruangan Recipe And Ursodeoxycholic Acid In Preventing Nonalcoholic Fatty Liver Fibrosis

Background and Objectives:Non-alcoholic fatty liver disease(NAFLD)refers to no history of excessive alcohol consumption,characterized by hepatic parenchymal fatty degeneration and fat accumulation in clinical pathological syndrome.The disease spectrum with the progression of the disease showed different manifestations including simple fatty liver,non-alcoholic steatohepatitis,non-alcoholic fatty liver fibrosis,and cirrhosis.Among them,the fibrosis stage is the stage with the highest mortality rate of liver-related diseases.Therefore,early diagnosis and treatments are crucial.Liver fibrosis from NAFLD in western medicine has the shortcoming of insignificant curative effect and large side effects.In recent years,basic experiments and clinical studies have found traditional Chinese medicine has a unique effect in preventing and treating liver fibrosis.Previous studies showed Kangxian Ruangan(KXRG)Fang which following the principles of formulating prescription could significantly improve the clinical symptoms and liver function in patients with chronic hepatitis B or post-hepatitis cirrhosis and reverse the liver fibrosis.However,the systemic clinical study on KXRG Fang treatment of hepatic fibrosis in NAFLD is still rare,the effect and related mechanisms are elusive.Therefore,this study has four purposes: firstly,to compare the efficacy and safety of KXRG Fang plus ursodeoxycholic acid versus ursodeoxycholic acid alone;secondly,comparing Anluo Huaxian Pills and ursodeoxycholic acid to investigate whether KXRG Fang combined ursodeoxycholic acid is much more effective;thirdly,the efficacy in different TCM syndromes of NAFLD related liver fibrosis;fourthly,the mechanisms of treatment of hepatic fibrosis in NAFLD.It provides a theoretical basis for the clinical screening of drugs for the prevention and treatment of NAFLD liver fibrosis.Methods:The study was divided into retrospective clinical studies and animal experiment studies.The former was performed on patients met the inclusion and exclusion criteria at the same time diagnosed with non-alcoholic fatty liver disease.According to the drugs taken are divided into three groups:group A: oral KXRG Fang combined with ursodeoxycholic acid;group B: oral Anluohuaxian pills combined with ursodeoxycholic acid,group C: ursodeoxycholic acid alone.To evaluate the liver function,four items of liver fibrosis,Child-Pugh classification,spleen ultrasound,TCM syndrome integral and A group TCM syndromes syndrome of the three groups of patients before and after treatments.Animal experiments were divided into two parts.Part 1:56 SPF Wistar rats were randomly divided into 7 groups with 8 rats in each group after a week of adaptive feeding:(1)control group(CTRL);(2)model group(MOL);(3)fuzheng huayu group;(4)colchicine group;5)normal dose kangxian ruangan(N-KXRG)group;(6)middle dose Kangxian ruangan(M-KXRG)group and(7)high dose Kangxian ruangan(H-KXRG)group.The rats in the control group were fed normal MCS diet,the rats in the model group and the five drug pretreatment groups were fed with the MCD diet.At the same time of normal feeding,the rats in each group were given the corresponding drug gavage:(FZHY group:15 g/kg/d;COL group: 0.1 mg/kg/d;N-KXRG group: 9.2 g/kg/d;M-KXRG group: 18.4 g/kg/d;H-KXRG group: 36.8 g/kg/d;the control group(CTRL)and model group: 2m L/100g/d saline).The whole process lasted 8 weeks.After 8 weeks,the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),?-glutamyl transpeptidase(GGT),total cholesterol(CHOL)and triglyceride(TG)in rat serum were detected according to commercial detection kit.The levels of serum HA,LN,IV-C,PC-III,TNF-?,and interleukin-10(IL-10)were detected by ELISA kit.HE and Masson staining were used to detect the pathological changes of liver tissues in each group.TUNEL staining was used to detect the apoptosis of hepatocytes in each group of rats.Western blotting was used to detect the expression of related proteins(caspases family and Bcl-2family)in the signal pathway of mitochondrial apoptosis.Part 2: 24 SPF Wistar rats were randomly divided into 3groups with 8 rats in each group after a week of adaptive feeding:(1)control group(CTRL);(2)model group(MOL);(3)kangxian ruangan(KXRG)group.The rats in the control group were fed normal MCS diet,while the rats in the model group and KXRG group were fed with the MCD diet.At the same time of normal feeding,the rats in each group were given the corresponding drug gavage:(KXRG group: 36.8 g/kg/d;the control group(CTRL)and model group: 2m L/100g/d saline).The whole process lasted 8 weeks.After 8 weeks,the serum levels of tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)in each group of rats were measured by ELISA.The hepatic histopathological changes in each group were detected by hematoxylin-eosin(HE)staining.Western blotting was used to detect the expression of related proteins of TGF-? signal pathways(TGF-?1,TGF-?2,Smad2,Smad3,and Smad7)and TLR4 signal pathways(TLR4,My D88,and NF-?B).Results:Retrospective clinical study results1.1 The comparison of clinical efficacy in the three groups:group A had a total efficiency of 90% and group B has 80%.The differences between group A and group C,group B,and group C were statistically significant(P<0.01).The total effective rate in group A was higher than that in group B,but the difference was no statistically significant(P>0.05).1.2 Biochemical indicators of hepatic function in the three groups: ALT and TBil: the difference among the three groups before and after treatment was statistically significant(P<0.01).There was a significant difference between group A and group B after treatment and group C after treatment(P<0.01).GGT: There was a significant difference between the three groups before and after treatment(P<0.01).There was no significant difference among the three groups after treatment(P>0.05). 1.3 The liver fibrosis indexes in the three groups were compared:HA and IV-C: There was a significant difference among the three groups before and after treatment(P<0.01).The comparison of decrease rate of the three groups after treatment was: A group>B group> C group,and the difference was statistically significant(P<0.01).Laminin(LN): There was a significant difference between group A and group B before and after treatment(P<0.01).The difference between the two groups before and after treatment was statistically significant(P<0.05).There was no significant difference between group A and group B after treatment(P>0.05).There was a significant difference between group A and group B after treatment and group C after treatment(P<0.01).1.4 The Child-Pugh scores of the three groups were compared:there was a significant difference among the three groups before and after treatment(P<0.01).There was a significant difference between group A and group C after treatment(P<0.01).There was no significant difference between group B and group C after treatment(P>0.05).1.5 The color Doppler index of the three groups was compared:there was no significant difference in the portal diameter among the groups before and after treatment(P>0.05).Spleen thickness: There was a statistically significant difference between group A and group B before and after treatment(P<0.01).The decrease rate of the three groups after treatment was: A group> B group> C group,and the difference was statistically significant(P<0.01).1.6 TCM comprehensive effect in the group A: five TCM syndrome total efficiency is as follows: phlegm and blood stasis syndrome> phlegm dampness internal resistance syndrome> liver qi stagnation syndrome> liver stagnation and spleen deficiency syndrome> damp heat accumulation syndrome,each The total effective rate was statistically significant(P<0.01).1.7 The syndrome scores of TCM syndromes in the group A: the difference among the five groups was statistically significant before and after treatment(P < 0.01).There was no significant difference after treatment(P > 0.05).2.Experiment one Biochemical tests revealed the liver function(ALT,AST,and GGT)and the levels of serum lipids(CHOL and TG)significantly decreased in all treatment groups compared with the MOL group(P < 0.01),especially in the H-KXRG group.The results of ELISA showed that compared with MOL group,the serum levels of fibrosis-related protein in each treatment group were significantly decreased(P < 0.01),the levels of proinflammatory cytokine TNF-? were significantly decreased(P < 0.01),while the levels of anti-inflammatory cytokines IL-10 levels were significantly increased(P <0.01)in all treatment groups,especially in the H-KXRG group.HE staining showed that compared with MOL group,hepatic lobule structure of rats in FZHY group and COL group were damaged lightly,inflammatory cell infiltration in hepatocytes and collagen fiber hyperplasia were alleviated.In different concentrations of KXRG-treated rats,the hepatic lobule structure was basically normal,the arrangement of hepatocytes was still neat,some of the hepatocytes degenerated around the central veins,the lesion area was smaller,the degeneration of hepatocytes was less,and the abnormal proliferation of fibrous tissue was not obvious,especially in H-KXRG treatment group.The results of Masson staining showed that compared with the MOL group,the collagen fibers in the liver of rats in each treatment group decreased to some extent,the degree of fibrosis was obviously improved,some of the thin blue fibers walked around the blood vessels,especially in H-KXRG treatment group.The results of TUNEL staining showed that compared with the MOL group,the apoptotic cells in the liver of rats in each treatment group were significantly decreased(P < 0.01),especially in H-KXRG group.Western blotting results showed that compared with the MOL group,the protein levels of Caspase-3,8 and 9 decreased in all treatment groups(P < 0.01),especially in the H-KXRG group.The expression level of pro-apoptotic protein-Bax was similar to that of Caspase 3,8,9,but the trend of anti-apoptotic protein(Bcl-2,Bcl-XL)and Bax showed the opposite direction.3.Experiment two The results of ELISA revealed the levels of TNF-? and IL-6 in KXRG treatment group were significantly lower than those in MOL group(P < 0.01).The results of HE staining showed that compared with MOL group,the hepatic lobule structure of KXRG treatment group was normal and the arrangement of hepatocytes was neat,some hepatocytes degenerated around the central vein,and the lesion area was smaller and the degree of degeneration of liver cells was lighter,abnormal proliferation of fibrous tissue is not obvious.Western blotting results showed that,on the one hand,the expression of TGF-?1,TGF-?2,Smad2 and Smad3 in KXRG treatment group was significantly lower than that in the MOL group(P < 0.01), while Smad7 protein was significantly increased(P < 0.01).On the other hand,compared with the MOL group,the expression of TLR4,My D88 and NF-?B p65 protein in KXRG-treated group was significantly lower than that in the MOL group(P < 0.01).Conclusions:1.Combination of Kangxian Ruangan Fang and ursodeoxycholic acid is much more effective than ursodeoxycholic acid alone in the treatment of non-alcoholic fatty liver fibrosis on improving liver function and spleen enlargement,and reducing Child-Pugh score and TCM syndrome integral.2.Compared with Anluo Huaxian Pills and ursodeoxycholic acid,combination of Kangxian Ruangan Fang and ursodeoxycholic acid has more advantages in anti-fibrosis,improving Child-Pugh score and secondary splenomegaly.3.Among different types of TCM syndromes,phlegm and blood stasis syndromes have the best efficacy.4.Kangxian Ruangan Fang can significantly improve liver function,blood lipid levels,hepatic pathological changes and liver cells apoptosis in NAFLD liver fibrosis rats,and the effect is more significant than the control drugs(Fuzhenghuayu and colchicine).5.Kangxian Ruangan Fang can significantly improve the levels of TNF-?,IL-6 in the rat model of NAFLD related liver fibrosis.6.Kangxian Ruangan Fang may play an inhibitory effect on hepatocyte apoptosis in the liver of NAFLD hepatic fibrosis rats by regulating the expressions of caspases family and Bcl-2family related proteins.7.Kangxian Ruangan Fang prevents and treats NAFLD rat liver fibrosis by regulating TGF-? signaling pathway and TLR4 signaling pathways.The scientific significance of this study: through the combination of clinical analysis and animal experiment,achieving the purpose of determination of the mechanism in anti-alcoholic fatty liver treatment of non-alcoholic fatty liver,providing a theoretical basis for clinical drug screening and treatment of NAFLD related liver fibrosis.