| BackgroundHepatocellular carcinoma(HCC)is a primary malignant tumor that is the fifth most common malignant tumor in the world,characterized by high mortality and poor prognosis.The current methods for treating liver cancer have shortcomings.In order to alleviate the pain of patients during treatment,there is still an urgent need to develop new safe treatment strategies to improve treatment outcomes.In recent years,the effectiveness of traditional Chinese medicine in treating and improving HCC has received widespread attention.Salvianolic acid B is a component extracted from Salvia miltiorrhiza.Previous studies have shown that Sal B can effectively delay the progression of mouse liver cancer,inhibit the migration and invasion of liver cancer cells,and induce apoptosis and autophagy.Therefore,further exploration of the molecular mechanism of action of Sal B may provide a theoretical basis for its clinical application.Recent studies have found that some active ingredients of traditional Chinese medicine can induce changes in the TGF-β/Smad and Hippo/YAP signaling pathways.The core of the mammalian Hippo pathway consists of Ste 20 like kinases 3(STK3)and STK4,large tumor suppressor 1/2(LATS1/2),downstream effectors,yes associated proteins(YAP),and transcriptional co activators with PDZ binding motifs(TAZ).It is reported that YAP/TAZ is not only related to liver inflammation and liver fibrosis,but also a signal center in the microenvironment of HCC tumors.Previously,our research team found that Sal B mediates the mouse TGF-β/Smad pathway and delays the progression of liver fibrosis cancer by promoting the transformation of pSmad3L to pSmad3C.However,the impact of Sal B on the Hippo/YAP pathway has not been determined.We suspect that the anti HCC effect of Sal B is related to the regulation of the Hippo/YAP signaling pathway?With these speculations in mind,this study used the HCC model induced by DEN/CC14/C2H5OH(DCC)in C57BL/6J mice and the HepG2 cell model induced by TGF-β1 and XMU-MP-1 to observe the effect of Sal B on the progression of liver cancer from multiple perspectives and investigate its mechanism of action.Objectives1.To observe the effects of Sal B on HCC progression in C57BL/6J mice induced by DCC and explore whether its mechanism of action is related to Hippo/YAP pathway2.To observe the effects of Sal B on HepG2 cell model induced by TGF-β1 and XMU-MP-1 and explore whether the action mechanism is related to Hippo/YAP pathwayMethods1.The effects and mechanisms of Sal B on DCC-induced C57BL/6J mouse liver cancerGroup:control group,model group,Sal B(7.5 mg/kg)group,Sal B(15 mg/kg)group,Sal B(30 mg/kg)group,Colchicine(0.1 mg/kg)group,Except for the control group,the other groups were combined with DEN/CCl4/C2H5OH to induce HCC in mice.The Sal B and Colchicine groups were given the corresponding doses by daily gavage,and the control group and model group were given the corresponding vehicle.Mouse serum and liver tissue samples were collected by eyeball blood collection method to calculate liver index and other physiological indexes.ALT and AST levels were tested at the same time.Histopathological changes were detected by his immune response.The protein levels of pYAP,YAP,pTAZ,TAZ and MST1 were determined by Western blot.The expression and distribution of pYAP,YAP,pTAZ,TAZ and MST1 were determined by cellular immunofluorescence.2.The effects and mechanisms of Sal B on HepG2 cells stimulated by TGF-β1 and XMU-MP-1Group:HepG2 cells were divided into control group,TGF-β1 group,TGF-β1(9pM)group,Sal B(50 μM)+TGF-β1 group,5μM xmu-MP-1(MST1/2 inhibitor)group,Sal B(50μM)+5μM xmu-MP-1 group,10 u M XMU MP-1 group and Sal B group(50μM)10μM XMU MP-1 group were used to detect the proliferation and migration of HepG2 cells by CCK-8 method,EdU method and cell scratch method.The protein levels of pYAP,YAP,pTAZ,TAZ and MST1 were determined by Western blot.The expression and distribution of YAP and TAZ were detected by cell immunofluorescence.Result1.The effects and mechanisms of Sal B on histopathology of DEN/CCl4/C2H5OH-induced HCC in miceThe results of mouse liver weight index showed that after induction by DCC,The mice exhibited symptoms such as weight loss and increased liver index;The test results of serum samples showed that compared with the blank control group,the expression of AST and ALT in the serum samples of the model group increased significantly,reflecting the pathological changes of the mouse liver organogenesis caused by DCC,and the liver function was damaged;Compared with the DCC group,after administration of Sal B,the expression of ALT and AST in mouse serum significantly decreased,indicating that Sal B can slow down the degree of liver damage in mice and restore liver function.2.The effects and mechanisms of Sal B on the levels of YAP,TAZ and their phosphorylated proteins in liver tissue of mice with HCCThe results of Western blot and immunofluorescence analysis were as follows:Compared with the blank control group,the levels of YAP and TAZ proteins in the liver tissue of mice induced by DEN/CCl4/C2H5OH were significantly increased.Compared with DCC group,the expressions of YAP and TAZ protein in liver tissue of mice in all Sal B dose groups were decreased,and the expressions of YAP and TAZ in Sal B(7.5 and 15 mg/kg)groups were significantly decreased.In addition,the protein levels of pYAP and pTAZ increased with the effect of Sal B,and the increase of pYAP and pTAZ protein levels was most obvious in Sal B(15 mg/kg)group.These results suggest that Sal B promotes the expression of pYAP and pTAZ proteins in mouse liver tissue,while inhibits the expression of YAP and TAZ proteins in mouse liver tissue.3.Effects of Sal B on the level of MST1 protein in liver tissue of mice with HCCThe results of Western blot and immunofluorescence experiments showed that the level of MST1 protein in mouse liver tissue was significantly increased under DCC induction compared with blank control group.Compared with mice in DCC group,the expression of MST1 protein in liver tissue of mice in all Sal B dose groups was further enhanced,and the expression of MST1 in Sal B(15 mg/kg)group and Sal B(30 mg/kg)group was significantly enhanced,and the level of MST1 in Sal B(30 mg/kg)group was most significantly increased.4.Effects of Sal B on proliferation and migration in TGF-β1 and XMU-MP-1-induced HepG2 cellsThe results of cell scratch test,CCK-8,and EdU assay showed that compared with the blank control group,the proliferation activity and migration ability of HepG2 cells in the TGF-β1 and XMU-MP-1 groups were significantly increased,indicating that TGF-β1 and XMU-MP-1 promoted the proliferation activity and migration ability of HepG2 cells;As the dosage of XMU-MP-1 increased,the proliferation activity of HepG2 cells significantly increased in a concentration dependent manner.However,the proliferation activity and migration ability of HepG2 cells were significantly reduced after the addition of Sal B.This indicates that the proliferation activity and migration ability of HepG2 cells stimulated by TGF-β1 and XMU-MP-1 can be inhibited by Sal B.5.Effects of Sal B on YAP/TAZ fluorescence intensity in TGF-β1 and XMU-MP-1-induced HepG2 cellsThe effects of Sal B on the expression and distribution of YAP and TAZ proteins in HepG2 cells stimulated by TGF-β1 and XMU-MP-1 was further examined by immunofluorescence assay.Compared with the blank control group,TGF-β1 and XMU-MP-1 stimulation significantly increased the expression of YAP and TAZ proteins in HepG2 cells.Sal B decreased the nuclear expression of YAP and TAZ proteins,and Sal B + XMU-MP-1(5 μM)group and Sal B + XMU-MP-1(10 μM)group showed statistically significant differences(P<0.01).6.Effects of Sal B on the protein expression of YAP/TAZ and its phosphorylated protein in TGF-β1 and XMU-MP-1-induced HepG2 cellsThe results of Western blot showed that:Compared with the blank control group,the protein levels of YAP and TAZ in HepG2 cells of TGF-β1 and XMU-MP-1 groups were significantly increased,and the increase in the 10μM XMU-MP-1 group was more significant than that in the 10μM XMU-MP-1 group(P<0.01).Compared with the model group,the protein expressions of YAP and TAZ in HepG2 cells of Sal B treatment group were decreased,and the expression of YAP and TAZ in Sal B+10μM XMU-MP-1 group was most significantly decreased.7.Effects of Sal B on MST1 protein expression in TGF-β1 and XMU-MP-1-induced HepG2 cellsCompared with the blank control group,the protein level of MST1 in TGF-β1 group and XMU-MP-1 group was significantly decreased.Sal B treatment enhanced the expression of MST1,and Sal B+XMU-MP-1(5 μM)and Sal B+XMU-MP-1(10μM)groups significantly increased the expression of MST1.8.Effects of Sal B on the expression of pSmad3C and pSmad3L proteins in TGF-β1 and XMU-MP-1-induced HepG2 cellsWestern blot assay showed that levels of pSmad3C were decreased in TGF-β1 and XMU-MP-1 groups.Sal B treatment enhanced the expression of pSmad3C,and Sal B+XMU-MP-1(5 μM)and Sal B+XMU-MP-1(10 μM)groups significantly increased the expression of pSmad3C.On the contrary,Sal B treatment decreased pSmad3L expression,and the expression of pSmad3L was significantly reduced in all Sal B administration groups.Conclusion1.Sal B is involved in HCC progression in C57BL/6J mice through Hippo/YAP signaling pathway,Sal B may play an anti-liver cancer role in mice by regulating Hippo/YAP signaling pathway.2.Sal B regulating TGF-β1 and XMU-MP-1-induced YAP and promotes YAP→pYAP transformation to inhibit hepatocellular carcinoma. |
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