PSmad3C^+/- Attenuates The Anti-Hepatic Fibrosis Effect Of Salvianolic Acid B Via Hippo-YAP Signaling Pathway

BackgroundsHepatic fibrosis（HF）is a dynamic parenchymal cell injury or inflammatory scarring response caused by various pathogenic factors,which can progressively develop into cirrhosis and hepatocellular carcinoma,seriously endangering human health and safety.In recent years,traditional Chinese medicine has unique advantages and great potential in anti-liver fibrosis.Salvianolic acid B（Sal B）is a potent active monomer extracted from the traditional Chinese medicine Salvia miltiorrhiza,which is used empirically in liver disease.Smad3 is a key protein in the TGF-β/Smad signaling pathway.Previous experiments have shown that the key cell growth inhibitory signal for liver fibrosis-hepatocellular carcinoma progression is associated with phosphorylation of Smad3 structural domain-specific sites,i.e.,the conversion of Smad3 carboxy-terminal phosphorylation（pSmad3C）to its linkage region phosphorylation（pSmad3L）pro-oncogenic signal.It has also been reported that the Hippo signaling pathway is a relatively conserved serine kinase cascade pathway,and its downstream key effectors Yes-associated protein（YAP）and transcriptional co-activator（TAZ）play key roles in the early regulation of liver fibrosis.In addition,the TGF-β/Smad and Hippo-YAP signaling pathways may interact in the development of liver fibrosis,and our previous studies have shown that Sal B mediates the TGF-β/Smad pathway in mice,delaying the progression of liver fibrosis-hepatocellular carcinoma by promoting the conversion of pSmad3L to pSmad3C.Meanwhile,other members of the group investigated the anti-fibrotic effect of Sal B via Hippo-YAP signaling pathway（not shown in this thesis）.So,what is the effect of the C-terminal phosphorylation site mutation of Smad3 gene(pSmad3C^+/-)on Sal B via Hippo-YAP signaling pathway?It has not been reported.Therefore,in this experiment,pSmad3C^+/-mice were used to investigate the anti-liver fibrosis effect of Sal B via Hippo-YAP signaling pathway in vivo,and to further clarify the effect of pSmad3C^+/-on Sal B-mediated Hippo-YAP signaling pathway.ObjectivesThe pSmad3C^+/-mice that have been established by knock-in ES targeting technology were subjected to expanded breeding,genotype identification,and due to pure lethality,screening of 6 weeks old,♂,HT mice(genotype:pSmad3C^+/-)and their homozygous WT mice(genotype:pSmad3C^+/+),30 mice each in the following experimental groups:WT-Control group,WT-DEN/CCl₄/C₂H₅OH group,WT-DEN/CCl₄/C₂H₅OH+Sal B group,HT-Control group,HT-DEN/CCl₄/C₂H₅OH group,HT-DEN/CCl₄/C₂H₅OH+Sal B group,10 mice/group.Except for Control group,DEN/CCl₄/C₂H₅OH composite modeling method was established for WT/HT-model group and drug administration group mice to induce the formation of spontaneous liver fibrosis model（12 weeks in total）;from the first day of modeling,mice in WT and HT drug delivery groups were given the corresponding dose（15 mg/kg/d）of Sal B solution（by gavage）based on their body weight,and the control mice were given saline solution according to their body weight for simultaneous treatment.At the end of the 12th week of modeling,the mice were anesthetized,and blood was collected from the eyes to determine the serological indexes,and the liver tissues were peeled,fixed with paraformaldehyde,embedded in paraffin,sectioned,stained with HE,MASSON and immunohistochemistry,and observed under a slide scanner to mark the morphological characteristics of the liver tissues and analyze the nature of the lesions.The protein levels ofα-smooth muscle actin（α-SMA）,pSmad3C,p21,pSmad3L,PAI-1 and Hippo-YAP signaling pathway（p MST1,MST1,p YAP,YAP,p TAZ,TAZ,CTGF）related proteins were detected by western blotting to investigate the effect of Sal B on Whether the therapeutic effect on DEN/CCl₄/C₂H₅OH-induced liver fibrosis in mice is mediated through the Hippo-YAP signaling pathway and the effect of pSmad3C^+/-on the Hippo-YAP signaling pathway.Results1.pSmad3C^+/-on the effect of Sal B on the treatment of mouse liver fibrosis.After 12 weeks of modeling,experimental data showed that the liver general,pathology,liver index and serum AST and ALT were normal in WT and HT-Control group mice,and there was no significant difference between the two genotypes of mice.mice in WT and HT-DEN/CCl₄/C₂H₅OH groups showed significantly white granular surfaced on the liver surface and darker liver appearance compared with its Control group.The results of HE and MASSON staining demonstrated that the liver was severely damaged,with more connective tissue and a large amount of inflammatory cell infiltration and collagen fibers;the body weight of the mice was significantly decreased;the liver index was increased;the levels of serum AST,ALT,HA and TGF-β₁were remarkably increased;the results of immunohistochemistry and protein blotting showed that the expression ofα-SMA protein was obviously upregulated.All the above data indicated that the degree of liver pathology in HT mice was much higher than that in WT.After administration of Sal B,the improvement of liver pathology in both WT and HT genotypes was alleviated compared with its model group,and the hepatic fibrosis in WT-Sal B group mice was cured to a greater extent than that in HT.2.Effects of pSmad3C^+/-upon pSmad3C/p21 and pSmad3L/PAI-1 proteins in the anti-liver fibrosis process of Sal B.The expression levels of pSmad3C and p21 proteins in the liver tissues of both WT and HT-DEN/CCl₄/C₂H₅OH mice were elevated compared with their controls;Sal B significantly upregulated the expression levels of pSmad3C and p21 proteins in the liver tissues of WT mice,whereas the extent of upregulation was not significant in HT mice.pSmad3L and PAI-1 protein levels in the liver tissues of both WT and HT-DEN/CCl₄/C₂H₅OH mice were increased,and were more significantly elevated in HT than WT mice;Sal B significantly downregulated the expression levels of pSmad3L and p21 proteins in the liver tissues of both WT and HT mice,but not in HT mice.The levels of pSmad3L and PAI-1 protein in liver tissues of both WT and HT-DEN/CCl₄/C₂H₅OH mice were elevated,and the elevation was more pronounced in HT than WT mice;Sal B significantly downregulated the expression levels of pSmad3L and PAI-1 protein in liver tissues of both WT and HT mice after Sal B administration,and the extent of downregulation was more obvious in WT mice.3.pSmad3C^+/-inhibits the increase in regulation of p MST1,MST1,p YAP,and p TAZ proteins during the hepatic fibrosis phase of Sal B treatment.After modeling by DEN/CCl₄/C₂H₅OH,p MST1 and MST1 protein expression levels were significantly increased and p YAP and p TAZ protein expression levels were significantly decreased in liver tissues of the WT model group,while p MST1,p YAP and p TAZ protein levels were significantly decreased and MST1 protein levels were increased in the HT model group compared with its control group;after Sal B administration,p MST1,MST1,p YAP and p TAZ protein levels were significantly increased in WT mice,p MST1,MST1,p YAP and p TAZ protein levels were significantly increased after Sal B administration,but the MST1 protein expression level was only slightly increased in the HT group,while the changes of p MST1,p YAP and p TAZ protein levels were not significantly different.4.pSmad3C^+/-inhibits the decrease of YAP,TAZ,and CTGF proteins in the hepatic fibrosis treatment phase of Sal B.After DEN/CCl₄/C₂H₅OH modeling,the levels of YAP,TAZ and CTGF proteins were significantly increased in both WT and HT genotypes,and the expression levels of YAP,TAZ and CTGF proteins were significantly higher in the HT model group than in WT mice;after treatment with Sal B administration,the levels of YAP,TAZ and CTGF proteins were significantly downregulated in WT mice,while the levels of TAZ and CTGF proteins in the liver tissues of HT mice were only slightly decreased,and the levels of YAP proteins were not significantly changed compared with their model group.Conclusions1.pSmad3C^+/-exacerbated the extent of pathology in the fibrosis model and weakened the therapeutic effect of Sal B on hepatic fibrosis in mice.2.pSmad3C^+/-inhibited the upregulation of pSmad3C/p21 and the downregulation of pSmad3L/PAI-1 in Sal B-treated mouse hepatic fibrosis.3.pSmad3C^+/-inhibited the upregulation of p MST1,MST1,p YAP,p TAZ proteins and the downregulation of YAP,TAZ,CTGF proteins in hepatic fibrosis in Sal B-treated mice.It was suggested that pSmad3C^+/-attenuated the anti-liver fibrosis effect of Sal B via Hippo-YAP pathway.