The Study On The Clinical Efficacy And The Mechanism Of Kangxian Ruangan Granule Against Hepatic Fibrosis Based On Hepatic Sinusoidal Capillarization

Objective:1.From the point of view of"phlegm and blood stasis"in traditional Chinese medicine,the clinical efficacy and relevant data of antifibrosis of Kangxianruangan Granule were evaluated scientifically,which provided the latest and reliable evidence-based medicine evidence for anti fibrosis of Kangxianruangan Granule.2.Based on the network pharmacology,the network data mining technology is used to screen the active ingredients of Kangxianruangan Granule in the treatment of liver fibrosis,and the potential targets and mechanisms of the ingredients are integrated and analyzed to explore the antifibrosis mechanism of Kangxianruangan Granule,so as to provide a scientific basis for antifibrosis and provide a certain basis for subsequent experimental research.3.Based on the capillarization of hepatic sinuses,the mechanism of Kangxianruangan Granule on liver fibrosis was studied by means of cell experiment and animal experiment.Methods:1.By means of the method of clinical control study,from the perspective of"Phlegm and Blood stasis",to study the clinical effect of Kangxianruangan Granule on the patients with liver fibrosis of phlegm and blood stasis type.Sixty patients with chronic hepatitis B?CHB?with liver fibrosis were divided into two groups:the treatment group treated with Kangxianruangan Granule combined with entecavir?ETV?,and the control group only treated with ETV.We observed the changes of TCM syndrome score,liver function index,liver fibrosis index and liver elasticity index before and after treated six months.2.With the help of bioinformatics analysis tool for molecular mechanism of traditional Chinese Medicine?Batman-TCM?combined with disenet,GENECARD,HPO,OMIM disease target database,through constructing the interaction target network of Kangxianruangan Granule active components and the related target network of liver fibrosis disease,mining the Kangxianruangan Granule antifibrotic target,using string for biological process and pathway enrichment analysis,to explore the antifibrotic target Research of Kangxianruangan Granule on the mechanism of action pathway.3.Cell experiment and animal experiment were used respectively?1?In the cell experiment,the serum containing medicine was preparated and extracted by the way of twice repeated administration on Wistar rats,then the primary hepatic sinusoid endothelial cell were respectively treated by the serum of Kangxianruangan Granule and the serum of control group.Western-blotting was used to detect the protein expression level of CD31 and vWF,and the effect of Kangxianruangan Granule on the molecular level of the endothelial cells in the hepatic sinuses was analyzed.Immunohistochemistry method was used to detect the expression level of CD31 and vWF in the cells.Scanning electron microscopy was used to detect the changes of fenestra of the endothelial cells in the hepatic sinuses.?2?In the animal experiment,Wistar male rats were given subcutaneous injection with CCl₄ olive oil mixed solution subcutaneously and fed with low protein-high fat food to induce hepatic fibrosis.With the method of decomposing prescriptions,Sorafenib was used as the control drug,to study the effects of Kangxianruangan Granule?MF?,Qubiejia?QJ?and Biejia?BJ?,respectively on the general situation and serum liver function of rats with hepatic fibrosis,as well as the MMP13 and TIMP-1 molecules in liver tissue and serum,liver histopathology,hepatic sinusoidal capillarization,even molecular level of hepatic sinusoidal capillarization.In addition,the molecules of NO,NOS,MDA,T-SOD and HYP in liver tissue were measured,and the effects of Kangxianruangan Granule group and its disassembled formula group on membrane lipid peroxidation and enzymatic defense system of liver tissue were observed.The levels of ALT,AST,ALB and IgG in serum,MMP-13,TIMP-1 and VEGF in liver tissue,CD31,vWF,collagen?and collagen?in liver tissue,H&E and Sirius red staining were used to detect the pathological changes of liver tissue,respectively.The expression level of MMP-13 and TIMP-1 in serum was detected by ELISA,and the indexes of NO,NOS,MDA,T-SOD and Hyp were detected by biochemical methods.Results:1.Clinical research results:In the clinical study of chronic hepatitis B?CHB?patients with liver fibrosis,there were 60 cases,29 males and 31 females,aged18-65 years,with an average of 52.5 years old,32 in the treatment group and28 in the control group.?1?There was no significant difference between the two groups before treatment?P>0.05?.?2?The results showed that:compared with the control group,the total effective rate?84.375%vs 60.71%?,the significant efficiency?53.125%vs28.57%?in the treatment group were significantly different?P<0.05?.?3?Before and after treatment,TCM syndrome score and total effective rate of single symptom were compared.The results showed that before treatment,TCM syndrome score of treatment group and control group was 23.40±4.14vs 22.80±3.85,t=0.5786,P>0.05;after treatment,TCM syndrome score of treatment group and control group was 8.75±3.28 vs 15.84±4.24,t=7.2916,P<0.01.The scores of TCM syndromes in the treatment group and the control group were significantly lower than that before treatment?t=15.6901,P<0.01?.Compared with the control group,the total effective rate of each single symptom in the treatment group was significantly higher,and the difference was statistically significant?t=11.406,P<0.01?.Compared with the control group,the treatment group of Kangxianruangan Granule combined with ETV can significantly reduce the clinical symptoms of patients:right flank pain,abdominal distension,fatigue,anorexia,loose stools,purple tongue,etc.?4?The results showed that ALT,AST,TBIL and GGT in the treatment group and the control group decreased after treatment,and there was significant difference between the two groups?P<0.05?.In addition,ALT,AST,TBIL and GGT were significantly different in the control group and the treatment group before and after treatment?P<0.01?.?5?The results showed that:compared with the control group,after giving the corresponding treatment,there were significant differences in HA,PC?,?-C and LN in the treatment group?P<0.01?;before and after the treatment,there were significant differences in HA,PC?,?-C and LN in the control group and the treatment group?P<0.01?.And compared with the control group,the index of the treatment group decreased more significantly after treatment.?6?The results showed that compared with the control group,the hardness index of liver in the treatment group decreased significantly?P<0.01?.In addition,there was no significant difference in the two groups before and after the treatment?P>0.05?,but there was significant difference between the two groups?P<0.01?.2.The results of network pharmacology analysis:seven kinds of drugs in Kangxianruangan Granule were input into the Batman TCM.One turtle,39Salvia miltiorrhiza,two skeletons,six curcumae,one seaweed,five oysters and 30 Hawthorn compounds were found respectively,totaling 84.Through Disgenet,GENECARD,OMIM and HPO,821 targets related to liver fibrosis diseases were searched and screened,and key targets of Kangxianruangan Granule and liver fibrosis intersection were integrated and screened.Using the software of Cytoscape 3.7.1 to construct the component target disease network,the core network was screened out with more than average degrees of freedom,and 95 related targets of Kangxianruangan Granule were obtained.2203biological processes,181 molecular functions and 162 KEGG pathways including MAPK,JAK-Stat,PI3K-Akt,Toll receptor,etc were obtained through the enrichment analysis of GO function and pathway/pathway of string.These pathways provide more powerful and reliable pharmacological evidence for the clinical use of Kangxianruangan Granule.3.Experimental research results:Experiment 1:Based on the capillarization of hepatic sinuses,the cellular experimental study on the anti fibrosis mechanism of kangxianruangan granules was carried out.?1?The effect of Kangxianruangan Granule on the morphology of sinusoidal endothelial cells:the sinusoidal endothelial cells were identified by immunofluorescence and fluorescence microscopy.Under inverted phase contrast microscope,compared with the control group,there was no significant change in the morphology of sinusoidal endothelial cells in the MF group.?2?The effect of Kangxianruangan Granule on the structure of the fenestra of the sinusoidal endothelial cells:the fenestra of the sinusoidal endothelial cells in the control group was narrow,and the fenestra structure was nearly occluded,and the number was small,while the fenestra structure of the sinusoidal endothelial cells in the Kangxianruangan Granule was clear,and the number of fenestra increased significantly.?3?The effects of Kangxianruangan Granule on CD31 and vWF cytokines in sinusoidal endothelial cells:the expression level of CD31 and vWF cytokines in primary sinusoidal endothelial cells in Kangxianruangan Granule group was significantly lower than that in the control group,The difference was statistically significant?CD31:0.0688±0.0150 vs 0.1151±0.0114,t=4.2565,P<0.05;VWF:0.0920±0.0089 vs 0.1319±0.0135,t=4.2740,P<0.05?.?4?The effect of Kangxianruangan Granule on the expression of CD31 and vWF protein in sinusoidal endothelial cells:compared with the control group,the expression level of CD31 and vWF protein in sinusoidal endothelial cells of primary generation in Kangxianruangan Granule group was significantly lower,the difference was statistically significant?CD31:0.3337±0.0290 vs0.4480±0.0128,t=6.2454,P<0.01;vWF:0.5603±0.0472vs0.7093±0.0448,t=3.9658,P<0.05?.Experiment 2:Based on the capillarization of hepatic sinuses,the mechanism of anti fibrosis effect of Kangxianruangan Granule was studied.?1?The effect of Kangxianruangan Granule on the general situation of rats:the weight growth of the model rats was inhibited,and the weight of the rats in other treatment groups was increased.Compared with the model group,the weight of the rats in the MF group increased significantly?P<0.05?.The mortality of liver fibrosis and the incidence of ascites in the MF group were significantly reduced?P<0.05?.There was no significant difference between liver index and spleen index?P>0.05?.?2?The effect of Kangxianruangan Granule on the liver function of rats:after the treatment of Kangxianruangan Granule group and its disassembled formula groups,the serum ALT,AST and IgG levels decreased significantly,while the serum ALB level increased significantly?P<0.05?.?3?The effect of Kangxianruangan Granule on the expression of MMP13 and TIMP-1 in liver tissue and serum of rats:qRT-PCR and ELISA showed that the expression of TIMP-1 mRNA in liver tissue and the level of TIMP-1 in serum were decreased in Kangxianruangan Granule group and its disassembled groups,while the expression of MMP13 mRNA and the level of MMP13 molecule in liver tissue and serum were increased significantly?P<0.01?.Compared with Sor group,the expression level of MMP13 and TIMP-1in liver tissue and serum was significantly regulated in the MF group?P<0.05?.?4?The effect of Kangxianruangan Granule on the histopathology of rat liver:in H&E staining,Kangxianruangan Granule group and its disassembled groups can significantly improve the pathological changes of rat liver tissue,the degree of degeneration and necrosis of hepatocytes,can also inhibit the proliferation of connective tissue in liver tissue,especially the MF group.Compared with Sor group,the improvement of liver fibrosis in MF group was more obvious.In Sirius red staining,compared with the model group,the content of fibrous tissue in the Kangxianruangan Granule group and its disassembled groups was significantly reduced,while that in the mother group was the most obvious.?5?The effect of Kangxianruangan Granule on the capillarization of hepatic sinuses in rats:compared with the model group,Kangxianruangan Granule and its disassembled groups,especially in the group of Kangxianruangan Granule,the structure of hepatocyte cords and hepatic sinuses are clear,the groove of hepatic sinuses can be seen,the degree of twist and stenosis of hepatic sinuses is obviously reduced,even disappeared,the basement membrane is thin or discontinuous,and some structures of hepatic sinuses are connected.Compared with the Sor group,the liver tissue of the MF group showed the most significant improvement in the degree of distortion and stenosis of the hepatic sinuses.?6?The effect of Kangxianruangan Granule on the level of capillarization molecules in hepatic sinuses of rats:the expression level of VEGF mRNA in Kangxianruangan Granule group and its disassembled groups can be significantly reduced.Compared with the Sor group,the difference between the Kangxianruangan Granule group and the Sor group is statistically significant.The protein levels of CD31,vWF,collagenan I and collagena IV were significantly reduced in the Kangxianruangan Granule group and its disassembled groups,especially in the Kangxianruangan Granule group.Compared with Sor group,the effect of Kangxianruangan Granule on the protein level of CD31,vWF,collagenan I and collagena IV was more significant?P<0.05?.?7?The effect of Kangxianruangan Granule on NO,NOS,MDA,T-SOD and HYP molecules in rat liver tissue:the expression level of MDA,NO,NOS and HYP in Kangxianruangan Granule group and its disassembled formula group was significantly reduced,while the expression level of T-SOD was increased,the difference was statistically significant.Compared with Sor group,the expression levels of NO,NOS,MDA,T-SOD and HYP were significantly regulated in the MF group?P<0.05?.Conclusion:1.Kangxianruangan Granule combined with ETV and ETV alone can significantly improve the clinical symptoms,liver function and liver fibrosis index of patients with chronic hepatitis B?CHB?.However,the treatment group of Kangxianruangan Granule combined with ETV has advantages in improving clinical symptoms,some liver functions and liver fibrosis indexes.Especially for the improvement of liver hardness,the combination of Kangxianruangan Granule and ETV has obvious advantages.So Kangxianruangan Granule can treat the type of liver fibrosis in"Phlegm and Blood stasis syndrome",and has the effect of softening liver and eliminating phlegm,promoting blood circulation and removing blood stasis.2.Through the network pharmacological prediction and analysis,it is found that the mechanism of Kangxianruangan Granule on liver fibrosis may be closely related to MAPK,PI3K-AKT,Jak-STAT,toll-like receptor,etc signal pathway.Kangxianruangan Granule have the pharmacological basis of antifibrosis.3.The results of cell and animal experiments show that Kangxianruangan Granule has an active therapeutic effect on the primary generation of sinusoidal endothelial cells in vitro and the capillarization of sinusoidal tissue in vivo.At the same time,in animal experiments,we found that the traditional Chinese medicine compound Kangxianruangan granule,QJ and BJ had obvious antifibrosis effects,especially the MF formula?Kangxian ruangan granule?had the best effect.The mechanism of Kangxianruangan Granule on hepatic fibrosis may be to prevent and treat hepatic fibrosis by inhibiting the capillarity of hepatic sinuses.