Inhibitory Effect And Mechanism Of Ginsenoside Rh4 On Metastasis Of Esophageal Squamous Cell Carcinoma

Esophageal cancer,a highly aggressive malignant tumor with poor prognosis,ranks seventh in incidence and sixth in mortality in the world.Esophageal squamous cell carcinoma(ESCC)accounts for more than 90% of esophageal cancer cases and has a higher mortality rate.Single diagnostic method and limited treatment methods are the main reasons for the high mortality rate.Most patients have already developed lymphatic and other organ metastases at the time of diagnosis.The treatment of esophageal cancer mostly relies on chemotherapy drugs,but its side effects are obvious.Therefore,a safer and more effective drug will provide new ideas for the treatment of esophageal cancer metastasis.Rare ginsenoside Rh4 comes from traditional Chinese medicine ginseng and has high pharmacological activities,including anti-tumor,anti-inflammatory and hypoglycemic.However,the effect of ginsenoside Rh4 on esophageal cancer metastasis has not been reported.Therefore,this study explored the inhibitory effect and effect of ginsenoside Rh4 on ESCC from the aspects of safe concentration,cell migration and invasion,epithelialmesenchymal transition(EMT)and pathway regulation through in vitro and in vivo experiments.mechanism.The main contents are as follows:1.1.The experimental design optimizes the extraction conditions,separates and purifies the triol group ginsenosides by high performance liquid chromatography,and identifies and analyzes the purity of the rare saponins Rh4 monomers obtained by separation.After that,the ginsenoside Rh4 monomer was separated and purified by preparative high performance liquid chromatography,and its purity was 98.75% detected by analytical high performance liquid chromatography.2.MTT assay was used to explore the concentration of Rh4 in inhibiting ESCC metastasis.At this concentration,its efficacy was evaluated by scratch assay,Transwell migration and invasion assay,and Western blotting.The migration and invasion ability of esophageal squamous cell carcinoma cells were significantly inhibited,and ginsenoside Rh4 also inhibited the EMT process of ESCC cells.3.Ginsenoside Rh4 can also effectively inhibit ESCC metastasis in vivo.The KYSE30 mouse footpad lymph node metastasis model was established to explore the inhibitory effect of Rh4 on ESCC metastasis in vivo.The inhibitory effect of Rh4 was evaluated by the size of the lymph node in the popliteal fossa of mice after administration for 28 days.The in vivo toxicity of Rh4 was measured by H&E and serum biochemical indicators.The results showed that ginsenosides could inhibit the distant metastasis of ESCC in vivo without obvious toxicity.4.Ginsenoside Rh4 regulates Wnt/β-catenin signaling pathway.Western blotting and immunohistochemical analysis of Wnt,β-catenin,p-β-catenin protein expression levels showed that ginsenoside Rh4 could significantly reduce the expression of pathway-related proteins.The co-action results of ginsenoside Rh4 and Wnt agonist HLY78 showed that ginsenoside Rh4 could reverse the cell migration ability and protein expression level enhanced by HLY78.This indicated that ginsenoside Rh4 inhibited ESCC metastasis through the Wnt/β-catenin signaling pathway.5.c-Myc is the key factor of ginsenoside Rh4 in regulating ESCC metastasis.Its effect was analyzed by detecting EMT marker protein after c-Myc-siRNA transfection.The results showed that ginsenoside Rh4 mediated the downstream EMT process by regulating the expression of c-Myc.In conclusion,this study proves that ginsenoside Rh4 can regulate the EMT process of ESCC by regulating the expression of c-Myc through the Wnt/β-catenin signaling pathway in vitro and in vivo,thereby inhibiting the metastasis process of ESCC.