Effects Of Invigorating The Spleen And Regulating Stomach On The Growth Of Human Esophageal Carcinoma Cell Line EC9706 Through STAT3 Signaling Pathway

BackgroundEsophageal cancer is one of the most common cancer,studies shown that STAT3signaling pathway was involved in the formation and development of esophageal cancer,affecting its prognosis.Invigorating the spleen and regulating stomach method and the representative fomula Liujunzi decoction could inhibit the proliferation of EC9706 and Eca-109,secretion of IL-6 and VEGF and the expression of STAT3,p-STAT3 in esophageal and endothelial cells.and induce EC9706 cells.can inhibit the formation of blood vessels,proliferation,migration and tubule formation of endothelial cells caused by esophageal cancer cells.Based on the above study,in this study,in order to find the molecular mechanism of invigorating the spleen and stomach in the treatment of esophageal cancer,nude mice transplanted into EC9706 cells and EC9706 cells cultured in vitro were used to investigate the effects on EC9706 cells and transplanted tumor of invigorating the spleen and regulating stomach method and the representative fomula Liujunzi decoction via STAT3 signaling pathway with molecular biology test.In view of the extraction of Liujunzi decoction and the screen of effective part of have been completed by other experimental staff,the part of ethyl acetate with the maximum potency would be uesed in vitro study.ObjectiveTo explore the molecular mechanism of invigorating the spleen and regulating stomach method and the representative fomula Liujunzi decoction in the treatment of esophageal carcinoma,through the observation of the growth of transplanted tumor in nude mice,proliferation of EC9706 cells,and STAT3 pathway related factors and proteins of EC9706 cells and tissues.MethodPart I in vivoThe establishment of animal model of transplanted tumor:inoculate EC9706cells?about 2×10⁶?in logarithmic growth phase in the subcutaneous of nude mice right flank,establish the nude mice model of transplanted tumor.Preparation of experimental drug:transformed into mice dose Liujunzi decoction was concentrated after decocting.Stattic dosage was 25mg/kg,DMSO was diluted after dissolving.Gave drug by groups:nude mice were divided into Control group?C group?,model group?M group?,Liujunzi decoction group?L group?,inhibitor group?S group?,Liujunzi decoction+inhibitor group?L+S group?.C group were gave saline in stomach and injectived saline in enterocoelia.L group were gave Liujunzi decoction and injectived saline.S group were gave saline and injectived Stattic solution.L+S group were gave Six Liujunzi decoction and injectived Stattic solution.Gavage?0.2m L/times?was 1 times a day,intraperitoneal injection?0.2m L/times?was every other day.Observation of indicators:observed the survival state of the nude mice,measured the weight and volume of tumor every other day.Detection of indicators:took blood by enucleating of eyeball blood after 4 weeks,took tumor after death.The level IL-6,VEGF in serum with ELISA,the expression of CD34 and VEGF in tumor with immunohistochemical,expression of JAK2,STAT3,p-STAT3 in tumor with Western blot were detected.Part II The experiment in vitroExperiment 1 The effects on growth of EC9706 cells of ethyl acetate extract of Liujunzi decoction was detected with MTT method.1 The effects on proliferation of EC9706 cells of ethyl acetate extract of Liujunzi decoction,Stattic and IL-6 was deteted with MTT method.The IC₃₅ and IC₅₀ values were used in the following experiments2 The effects on proliferation of EC9706 cells of integrate of ethyl acetate extract and Stattic was detected with MTT method.3 The effects on proliferation of EC9706 cells of removing medium with drug or not,incubating ethyl acetate extract and Stattic for 6h was detected with MTT method.Experiment 1 The effects on expression of STAT3 and p-STAT3 in EC9706 cells of ethyl acetate extract of Liujunzi decoction was detected with Western blot.ResultPart I in vivo1 Compared with model group,the volume of transformed tumor was smaller in Liujunzi decoction group?P<0.05?.The weight of tumor in experiment groups were lighter than that of model group?P<0.05?.2 Compared with model group,The level of IL-6 in serum of experiment group was lower?P<0.05?.The inhibitor group and Liujunzi decoction group were lower than that in model group?P<0.05?.3 Compared with model group,the expression of CD34 in transplanted tumor was lower in experiment groups?P<0.05?.Compared with inhibitor group,the expression of CD34 was slightly higher?P<0.05?.4 The effects on expression of STAT3 and p-STAT3 in transplanted tumor of Liujunzi decoction:compared with model group,the expression of JAK2 reduced in Liujunzi decoction and inhibitor group?P<0.05?.Compared with inhibitor group,the expression of JAK2 reduced in Liujunzi decoction group?P<0.05?,induced in integrate Liujunzi decoction and inhibitor group?P<0.05?.Compared with model group,the expression of STAT3 reduced in Liujunzi decoction and inhibitor group?P<0.05?.Compared with Liujunzi decoction group,the expression of STAT3 induced in integrate Liujunzi decoction and inhibitor group?P<0.05?.Compared with model group,the expression of p-STAT3 reduced in inhibitor group?P<0.05?.Compared with inhibitor group,the expression of p-STAT3 induced in Liujunzi decoction and integrate Liujunzi decoction and inhibitor group?P<0.05?.Part II The experiment in vitro1 Ethyl acetate extract of the Liujunzi decoction could inhibited the proliferation ofEC9706,which showed dose-effect relationship,the inhibition rate increased graduallywiththeincreaseofdose:IC₃₅=34.2?g/m L,IC₅₀=51?g/m L,IC₇₅=71.5?g/m L.2 Stattic could inhibited the proliferation of EC9706 cells,which also showed dose-effect relationship,the inhibition rate increased gradually with the increase of dose:IC₃₅=2.37 M,IC₅₀=2.71 M.3 IL-6 could promoted the proliferation of EC9706 cells,which was not obvious dose-effect relationship.The OD value were higher in concentration of 10⁸0ng/m L IL-6 groups,when compared with control group?P<0.01?.4 When Liujunzi decoction integrated with Stattic,the mean of CDI at the concentration of IC₃₅ and IC₅₀ was 0.423,0.175,less than 0.7.5 Compared with control group,the OD value of experiment groups was higher,when adding IL-6 after removing medium with Liujunzi decoction ethyl acetate extract incubation or Stattic for 6 hours.6 The effects on expression of p-STAT3 of EC9706 cells of Ethyl acetate extract of Liujunzi decoction 35%inhibition rate:compared with control group,the expression of p-STAT3 in Stattic group at 30min and 1h,in Liujunzi decoction at10min⁶h,in L+S group at 10min,30min,6h reduced?P<0.05?.The expression of p-STAT3 in model group at 10min,30min and 6h increased?P<0.05?.Compared with model group,the expression of p-STAT3 in S+IL group in 10min¹h,in L+S and L+S+IL-6 group at 10min,30min and 6h increased?P<0.05?.The effects on expression of STAT3 of EC9706 cells of Ethyl acetate extract of Liujunzi decoction 35%inhibition rate:compared with control group,the expression of STAT3 in S,L,L+S group at 30min,in IL group at 30min,S+IL group at 30min,1h,L+IL group at 1h,L+S+IL group at 1h reduced?P<0.05?.The effects on the ratio of p-STAT3 in STAT3 of EC9706 cells of Ethyl acetate extract of Liujunzi decoction 35%inhibition rate:compared with control group,the ratio of p-STAT3 in S group at 1h,L group at 10min⁶h,L+S at 10min,30min,6h decreased?P<0.05?.Compared with control group,the ratio of p-STAT3 in IL group at10min⁶h increased?P<0.05?.Compared with IL-6 group,the ratio of p-STAT3 in S+IL,at 10min¹h,L+IL,L+S+IL group at 10min,30min and 6h decreased?P<0.05?.Conclusion1 Invigorating the spleen and regulating stomach method and the representative fomula Liujunzi decoction could inhibit the growth of transplanted tumor in nude mice,down regulate the expression of CD34,JAK2 and STAT3 in transplanted tumor.2 Six Gentlemen Decoction of ethyl acetate can inhibit the proliferation of EC9706 cells and down regulate the expression of STAT3 protein in EC9706 cells and its phosphorylation.3 Invigorating the spleen and regulating stomach method and the representative fomula Liujunzi decoction could inhibit the growth of esophageal squamous cell carcinoma cell line EC9706 cells via regulating STAT3 signaling pathway...