Based On PI3K/AkT/mTOR Signaling Pathway To Explore Kidney Between Raw Pulp Method To Promote Bone Marrow Mesenchymal Stem Cell Transplantation For The Treatment Of Liver Cirrhosis Mechanism Research

Objective: The clinical part of this project retrospectively analyzes the clinical material of Jiawei Jishengshenqi Decoction combined with Entecavie in the treatment of patients with hepatitis B liver cirrhosis and spleen-kidney Yang deficiency syndrome,including clinical symptoms,liver function,HBV-DNA,liver stiffness,and abdominal ultrasound,etc.To explore the efficacy of the method of invigorating the kidney and cultivating marrow combined with antiviral drugs in the treatment of liver cirrhosis,and to provide new ideas for the clinical use of traditional Chinese medicines against liver fibrosis.In the animal experiment part,we further observed the effects of Jiawei Jishengshenqi Decoction combined with bone marrow-derived mesenchymal stem cells transplantation on the serology and histopathology of cirrhotic rats,for exploring the effect of Jishengshenqi Decoction ’ s therapeutic mechanism on promoting stem cell transplantation and treating liver cirrhosis.Methods:(1)Clinical experiments: Sixty patients with hepatitis B cirrhosis with spleen-kidney-yang deficiency syndrome were divided into western medicine group and chinese western medicine group,with 30 cases in each group.The western medicine group was treated with entecavir antiviral therapy.The chinese western medicine group was given Jiawei Jishengshenqi Decoction on the basis of the western medicine group.Jishengshenqi Decoction was coposed of 40 g Rehmannia glutinosa,20 g cornus officinalis,20 g Chinese yam,15 g Rhizoma alismatis,15 g Poria cocos,15 g paeonol,5g cinnamon,5g aconite(first fried and long fried),10 g plantain,10 g achyranthes,2g panax notoginseng,20 g vinegar turtle shell(fried first).The course of treatment is 6 months.The taking method is One dose a day,two warm doses in the morning and evening,200 ml per time.(2)Animal experiment: Fifty-five male SD rats were fed adaptively for one week.After weighed and numbered them,randomly separated 3 for separating BMSCs cultured and 8 as a black group,the remaining 44 rats were induced rat liver cirrhosis model by the way of intraperitoneal subcutaneous injection with carbon tetrachloride(CCL4).The intervention lasted for 8 weeks.At the end of 8 weeks,5 rats died during the modeling process,and the remaining 4 rats were killed at random to prove that the modeling was successful.The other 35 rats were randomly divided into:model group,BMSCs graft group,Jishengshenqi decoction group,BMSCs graft + Jishengshenqi decoction group,BMSCs graft + Jishengshenqi decoction + LY294002 inhibitor group.The BMSCs was injected from the tail vein at 9W,the BMSCs graft + Jishengshenqi decoction group was injected with BMSCs and the Jishengshenqi decoction suspension was gavaged simultaneously,and the BMSCs graft+ Jishengshenqi decoction + In the LY294002 inhibitor group,gavage of Jishengshenqi decoction suspension and inhibitor injection were performed.The blank group was gavage with the same volume of distilled water,and the model group received no intervention.BMSCs were transplanted once a week and gavage once a day for 4W.Taking materials after the intervention.Observing the liver function and histopathological changes of rats in each group after intervention;ELISA method to detect the levels of GCSF and EPO in peripheral blood.Western blot method was used to detect the expression of SDF-1,CXCR4,PI3 K,p-Akt and m TOR protein in liver tissue.Results:(1)Clinical retrospective analysis: A total of 60 effective cases were included in this study.The study found that the symptom score,clinical efficacy,liver function,HBV-DNA load,liver hardness,spleen length,spleen thickness and portal vein diameter of the two groups were significantly improved after treatment(P < 0.01 or P < 0.05),and the above indexes of the chinese western medicine group were better than those of the western medicine group(P < 0.01 or P < 0.05).(2)Animal experiment: Morphology of BMSCs: After culturing,irregular morphology,long-spun or long spindle-shaped cells can be seen.BMSCs graft group,After intervention,the rat mental state,nutritional status,activity sensitivity of the Jishengshenqi decoction group,BMSCs graft+Jishengshenqi decoction group,BMSCs graft+Jishengshenqi decoction+LY294002 inhibitor group have been varied degrees of improvement.Among them,the BMSCs graft + Jishengshenqi decoction group improved the most.HE staining situation of rat liver tissue in each group.The pathology of the liver tissue in the model group showed severe fibrosis,the formation of false lobules,hepatocyte degeneration and necrosis obviously,indicating that the model of liver cirrhosis was successful.After intervention,the degree of liver fibrosis,hepatocyte degeneration and necrosis of rats in each treatment group improved to different degree,among which the BMSCs graft+ Jishengshenqi decoction group was the most obviously.Liver function of rats in each group:Compared with the blank group,the ALT and AST levels of the model group increased significantly,and the ALB level decreased significantly(P<0.01).Compared with the model group,the ALT and AST of each treatment group decreased in varying degrees,and the ALB level increased to different degrees(P<0.01).Among them,the BMSCs graft+ Jishengshenqi decoction group was the most obviously.The change of GCSF and EPO in peripheral blood of rats: Compared with the blank group,the GCSF and EPO level of the model group decreased significantly.Compared with the model group,the level of GCSF and EPO in each treatment group increased in varying degrees.Compared with each treatment group,the level of GCSF and EPO in the BMSCs graft+ Jishengshenqi decoction group were significantly higher than those of the BMSCs transplantation group,Jishengshenqi decoction group,and BMSCs graft+ Jishengshenqi decoction+LY294002 inhibitor group.There was no significantly difference among the BMSCs transplantation group,Jishengshenqi decoction group and BMSCs graft+ Jishengshenqi decoction+LY294002 inhibitor group(P>0.05);SDF-1,CXCR4,PI3 K,p-AKT,m TOR protein in liver tissues of rats in each group.After intervention,the expression of SDF-1 and CXCR4 proteins in the liver tissues of each treatment group increased in varying degrees compared with the model group(P<0.01).The protein expression level of PI3 K,p-Akt and m TOR decreased to different degrees.Compared with each treatment group,the expression of SDF-1 and CXCR4 protein in liver tissue of BMSCs graft+Jishengshenqi decoction group were the highest(P<0.01),and the protein expression of PI3 K,p-AKT,and m TOR was the lowest(P<0.01).Conclusions:Entecavir combined with the method of invigorating the kidney and regenerating the marrow can relieve the clinical symptoms of patients with spleen-kidney-yang deficiency syndrome in hepatitis B liver cirrhosis,improve liver function,reduce HBV-DNA load,reduce liver stiffness and shrink the spleen.Animal experiments have shown that Shengshenqi Decoction combined with BMSCs transplantation can promote the migration of BMSCs and effectively improve the liver function and fibrosis of cirrhotic rats.The mechanism of action may be related to the regulation of PI3K/Ak T/m TOR signaling pathway.The expression of genes and proteins exerts therapeutic effects.