Base On Mechanism Of Yiqi Tongluo Jiedu Prescription On Esophageal Cancer Growth Targeting TNF-α/IL-1/NF-κB Inflammatory Signaling Pathway

Background:Esophagus cancer(EC)is one of the most common malignant tumors in the digestive system.Squamous cell carcinoma and adenocarcinoma are the main pathological types.The main pathological type of esophageal cancer in China is esophageal squamous cell carcinoma(ESCC),whose pathological mechanisms are complex and affect each other.Inflammation runs through the entire pathological process of the tumor.Therefore,suppressing the infla mmatory response is of great significance for improving the prevention and treatment level of ESCC.The inflammatory response of ESCC involves different cell types,cytokines,and signaling pathways,forming an ESCC inflammatory molecular network.Studying key cytokines and core signaling pathways in ESCC will better elucidate the mechani sm of action of traditional Chinese medicine in the treatment of ESCC.In this paper,by observing the effect of tumor necrosis factor-α(Tumor Necrosis Factor-α)-induced esophageal squamous carcinoma cell line Eca109 on signaling factor nuclear factor-κB(NF-κB)and secretion of inflammatory factors,the es tablishment of ESCC in vitro inflammation model,to study the regulatory effect of Yiqi Tongluo Jiedu prescriptionon on TNF-α induced Eca109 cell inflammation model in vitro,and to explore the molecular biological mechanism of Chinese medicine in suppressing ESCC inflammation.Objective:Establish TNF-α induced Eca109 cell inflammation model in vitro,observe its effect on NF-κB signaling pathway and secrete inflammatory factors,explore the effect of Yiqi Tongluo Jiedu prescription on NF-κB signaling pathway induced by TNF-α in Eca109 cells The regulatory effect provides an experimental basis for revealing the molecular biological mechanism of Yiqi Tongluo Jiedu prescription in suppressing ESCC inflammation.Methods:1.Establishing TNF-α-induced Eca109 cell inflammation model in vitro1.1 MTT colorimetric method was used to detect the effect of different concentrations of TNF-α induced Eca109 cells for 24 h on cell viability.1.2 Immunofluorescence detection of NF-κBp65 into the nucleus.1.3 ELISA detection of interleukin-1β(IL-1β),interleukin-8(IL-8),matrix metalloproteinase-9(MMP-9)inflammatory factors secretion.2.Observing the effect of Yiqi Tongluo Jiedu prescription on inflammatory microenvironment mediated by NF-κB pathway2.1 MTT colorimetric method to screen the effect of Yiqi Tongluo Jiedu prescription on the growth of Eca109 cells induced by TNF-α,according to the screening results,group intervention.2.2 Immunofluorescence method was used to detect the effect of Yiqi Tongluo Jiedu prescription on TNF-α-induced NF-κBp65.2.3 ELISA to detect the effect of Yiqi Tongluo Jiedu prescription on downstream inflammatory factors.2.4 Western Blot to detect the effect of Yiqi Tongluo Jiedu prescription on the expression of NF-κBp65,p-IκBα,IκBα protein.Results:1.1 The results of MTT showed that TNF-α had different degrees of promotion effect after induction of Eca109 cells for 24 h,and the 20 ng / m L OD value increased most obviously(P <0.05).1.2 Immunofluorescence results showed that the cells in the 20 ng / m L TNF-αgroup showed significant nuclear transfer(P <0.01),NF-κBp65 entered the nucleus,and the NF-κB pathway was activated.1.3 ELISA results showed that the secretion leve ls of IL-8 and MMP-9inflammatory factors increased significantly(P <0.01).2.1 MTT results showed that compared with the TNF-α model group,the Yiqi Tongluo Jiedu prescription group and the NF-κB pathway inhibitor PDTC inhibited the proliferation of Eca109 cells induced by TNF-α(P <0.01).2.2 Immunofluorescence results showed that: compared with the TNF-α model group,the Yiqi Tongluo Jiedu prescription group and the NF-κB pathway inhibitor PDTC group significantly reduced the number of NF-κBp65 cells into the nucleus(P<0.01),inhibit NF-κB pathway activation.2.3 ELISA results showed that compared with the TNF-α model group,the Yiqi Tongluo Jiedu prescription and the NF-κB pathway inhibitor PDTC group both reduced the expression of inflammatory factors IL-8 and MMP-9 secreted by Eca109cells(P <0.01).2.4 Western Blot results showed that compared with the blank control group,the TNF-α group NF-κBp65,p-IκBα,p-IκBα / IκBα protein expression increased,and IκBα protein expression decreased.Compared with the TNF-α model group,the Yiqi Tongluo Jiedu prescription group and the NF-κB pathway inhibitor PDTC group both reduced the expression of NF-κBp65,p-IκBα,IκBα,p-IκBα / IκBα protein.Conclusion:1.TNF-α can promote the proliferation of Eca109 cells,make NF-κBp65 enter the nucleus,activate the NF-κB pathway,can induce cells to secrete IL-8 and MMP-9inflammatory factors,and increase the expression of NF-κBp65 and p-IκBα,degrade IκBα protein,and induce Eca109 cells to establish an in vitro inflammatory model.2.Yiqi Tongluo Jiedu prescription can inhibit the proliferation of Eca109 cells induced by TNF-α,inhibit the activation of NF-κB pathway,down-regulate the levels of IL-8 and MMP-9 inflammatory factors secreted by cells,and reduce the pro tein expression of NF-κBp65,p-IκBα,IκBα expression.Inhibits the cascade activation response of TNF-α to the NF-κB pathway.3.The regulatory effect of Yiqi Tongluo Jiedu prescription on Eca109 cells induced by TNF-α may be related to the inflammatory microenvironment mediated by NF-κB pathway.