| Objective: To observe the clinical efficacy of tonifying kidney and removing blood stasis(Jisheng Shenqi Decoction,Sanqi Powder,Biie Jia)combined with tenofovir fumarate disofuroxil tablet in the treatment of hepatitis B cirrhosis with Yang deficiency and blood stasis syndrome,and to evaluate its safety.Furthermore,the molecular mechanism of the effect of kidney tonifying and stasis removing method combined with bone marrow mesenchymal stem cell transplantation on cirrhosis rats was further observed,and the correlation between the TCM theoretical basis of kidney tonifying and stasis removing method on promoting stem cell migration,homing,improving liver function and liver fibrosis and the modern molecular biological mechanism was discussed.Methods:(1)Clinical study: 62 patients with hepatitis B cirrhosis with Yang deficiency and blood stasis syndrome were randomly divided into observation group(n = 31)and control group(n = 31).The control group was treated with tenofovir fumarate diofuroxil tablets,and the observation group was combined with kidney tonifying and stasis removing method(Jisheng Shenqi Decoction plus Sanqi powder and Biie Jia)on the basis of the control group,with a course of 6 months.After the course of treatment,the clinical symptom score,liver function,liver fiber,HBV-DNA,liver chronological elastography,and B-ultrasound of liver and spleen in 2 groups before and after treatment were observed,and the clinical efficacy and safety of kidney-tonifying and stasis removing method in the treatment of hepatitis B cirrhosis with Yang deficiency and blood stasis syndrome were evaluated.(2)Animal study: 80 healthy SPF SD rats were randomly divided into 5rats for cell culture,10 rats as blank group,and the other rats were subcutaneously injected with 40%CCl4 olive oil solution(CCl4: olive oil was4:6)for 8 weeks to establish the rat model of liver cirrhosis.After successful modeling,the rats were divided into model group,high,middle and low dose of Bushen Huayu recipe + BMSCs transplantation group,BMSCs transplantation group and Bushen Huayu recipe(medium dose)group.In the BMSCs transplantation group,BMSCs;Bushen Huayu recipe was injected through the tail vein,and the high,middle and low dose of Bushen Huayu recipe plus BMSCs transplantation group were treated with high,middle and low dose Bushen Huayu recipe combined with BMSCs transplantation of caudal vein.All rats were intragastrically administered once a day,and BMSCs was transplanted once a week for 4 weeks from the end of the 8th week.At the end of the 12 th week,the rats were killed and blood samples were collected.The pathological morphology of liver was observed under high power microscope,and the liver function(ALT,AST,ALB,TBIL)and four items of liver fibrosis(HA,LN,PC-III,IV-C);ELISA)were measured by automatic biochemical analyzer.The levels of GCSF,IL-6,HGF and SCF in peripheral blood of rats in each group were detected by;Western blot method to detect the expression of SDF-l and CXCR4 protein in liver tissue of each group.Results:(1)Clinical research: After treatment,the scores of TCM syndromes,total scores,liver function(ALT,AST,ALB,TBIL),liver fiber(HA,LN,PC-III,IV-C)HBV-DNA,liver stiffness,portal vein diameter,and spleen thickness were significantly improved compared to before treatment,and the difference was statistically significant(P<0.05).Compared with the control group,the observation group improved the TCM syndrome score,total score,liver function(ALT,AST,ALB,TBIL),liver fiber(HA,LN,PC-III,IV-C),liver stiffness,The inner diameter of the portal vein and the thickness of the spleen were better than those of the control group,and the difference was statistically significant(P<0.05).However,there was no statistically significant difference in the HBV-DNA conversion rate between the two groups of patients after treatment(P>0.05).There were no obvious adverse reactions in the two groups of patients during the observation.(2)Animal studies: 1)General conditions of rats: rats in blank group had good mental state,activity and sensitivity,bright and lustrous hair color,normal food and water consumption,and normal defecation;During the modeling process,the rats in the model group gradually showed listlessness,retarded reaction,decreased activity,gradually coarse and dark yellow hair color,significantly reduced food intake and water intake,loose stools,and gradually decreased body weight.After 4W of intervention treatment,the daily activity,food intake,defecation,mental state,body hair brightness,body weight and other aspects of rats in BMSCs transplantation group,Bushen Huayu Formula group,Bushen Huayu Formula high-dose,medium-dose and low-dose + BMSCs transplantation group were improved to varying degrees compared with the model group.Among them,Bushen Huayu Decoction high-dose + BMSCs transplantation group had the best improvement.2)Changes of HE staining in liver tissue of rats in each group: in blank group,the structure of liver lobules was normal,the central vein was arranged radially,and there was no inflammatory necrosis or fibrous tissue hyperplasia;Model group: The structure was incomplete,with obvious infiltration of inflammatory cells,obvious proliferation of fibrous tissue,and formation of pseudoblobule.Compared with the model group,in the Bushen Huayu Decoction high-dose + BMSCs transplantation group,the pseudoblobule structure was significantly reduced and basically disappeared,with a small amount of fibrous tissue hyperplasia and liver cell regeneration.In the Bushen Huayu Decoction medium dose + BMSCs transplantation group,the degree of fibrosis was moderately alleviated,hepatoid cell regeneration was observed,and the inflammatory necrosis of liver tissue was reduced;Bushen Huayu Decoction low-dose + BMSCs transplantation group:the degree of fibrosis was slightly reduced,inflammatory infiltration was visible;BMSCs transplantation group: fibroplasia,pseudolobules and inflammatory infiltration were observed.Compared with the model group,the number of false lobules in Bushen Huayu Formula group was decreased,and the inflammatory infiltration was slightly reduced.3)Comparison of the four items of liver function and liver fiber in each group: Compared with the blank group,the liver function(ALT,AST,TBIL)and the four items of liver fiber(HA,PCIII,IV-C,LN)in the model group were significant ALB increased significantly,and ALB decreased significantly,both with significant differences(P<0.01);compared with the model group,liver function(ALT,AST,TBIL),liver fiber(HA,PCIII,IV-C,LN)levels decreased significantly,and ALB increased significantly(P<0.01);among the treatment groups,the treatment effect of Bushen Huayu Decoction high-dose + BMSCs transplantation group improved the most significantly(P<0.01);Bushen Huayu Decoction The medium dose + BMSCs transplantation group and the Bushen Huayu Decoction low-dose + BMSCs transplantation group have a more significant effect(P<0.01);the Bushen Huayu Decoction low-dose +BMSCs transplantation group has the same therapeutic effect as the BMSCs transplantation group(P> 0.05);Compared with the Bushen Huayu Decoction group,the treatment effect of BMSCs transplantation group was better(P<0.01).4)Comparison of G-CSF,HGF,SCF and IL-6 in each group:compared with blank group,the levels of G-CSF,HGF and SCF in model group were significantly decreased,while the level of IL-6 was significantly increased,with statistical significance(P<0.01);Compared with the model group,the levels of G-CSF,HGF and SCF in each treatment group were significantly increased,and IL-6 was significantly decreased,with statistically significant differences(P<0.01);After intervention,compared with each treatment group,Bushen Huayu Decoction high-dose + BMSCs transplantation group had the most obvious effect on the treatment of increased G-CSF,HGF,SCF and decreased IL-6(P<0.01);The effect of Bushen Huayu Decoction medium dose + BMSCs transplantation group was more significant than that of Bushen Huayu Decoction low dose + BMSCs transplantation group(P<0.01);The effect of Bushen Huayu Decoction low-dose + BMSCs transplantation group was similar to that of BMSCs transplantation group(P>0.05);Compared with Bushen Huayu Formula group,BMSCs transplantation group had better effects of increasing G-CSF,HGF,SCF and decreasing IL-6(P<0.01).5)Comparison of SDF-1 and CXCR4 protein expression in liver tissue: Compared with the blank group,SDF-1 and CXCR4 protein in liver tissue decreased significantly in the model group(P<0.01);Compared with model group,2 indexes of treatment group were significantly increased(P<0.01);The above indexes of Bushen Huayu Decoction high-dose + BMSCs transplantation group were significantly higher than those of the other two groups(P<0.01);Compared with the Bushen Huayu Decoction medium dose + BMSCs transplantation group,the above indexes increased more significantly(P<0.01 or P<0.05);There was no significant difference in therapeutic effect between Bushen Huayu Decoction low-dose + BMSCs transplantation group and BMSCs transplantation group(P>0.05);Compared with Bushen Huayu Formula group,BMSCs transplantation group had more obvious elevation of treatment(P<0.01).Conclusion(s): Kidney-tonifying and stasis removing method combined with tenofovir fumarate diofuroxil tablet in the treatment of hepatitis B cirrhosis patients with Yang deficiency and blood stasis syndrome can significantly improve the liver function,reduce the degree of liver fibrosis,portal hypertension and splenomegaly,and effectively relieve liver injury,with a clear efficacy and good safety.The pathologic characteristics of liver cirrhosis with Yang deficiency as the foundation and blood stasis as the standard have been preliminarily verified,and the treatment of liver cirrhosis with kidney tonifying and removing blood stasis has enriched the theoretical basis of treating liver cirrhosis with traditional Chinese medicine.Joint BMSCs transplantation of kidney and blood method can improve the liver cirrhosis in rats of hair,feeding,signs or symptoms such as spirit,mobility,and improve the liver function,fibrosis degree,reduce inflammation,reduce collagen deposition,and can promote the migration of BMSCs,homing,its mechanism may be through promoting the secretion of SCF,G-CSF,HGF,and based on SDF-l/CXCR4 axis. |
