幽门螺杆菌介导细胞焦亡重塑胃癌免疫微环境OA

Background and purpose:Helicobacter pylori(H.pylori)chronically colonizes the gastric mucosa and is a strong risk factor for gastric cancer.This study aimed to explore whether H.pylori could induce pyroptosis through the pore-forming protein Gasdermin D(GSDMD),promoting mucosal inflammation and consequently accelerating the progression of gastric cancer.Methods:Pan-cancer transcriptomic profiling of 33 solid tumors from The Cancer Genome Atlas(TCGA)public database was performed to compare GSDMD mRNA expression levels between tumor and normal tissues.The GSDMD protein validation was performed by Western blot and immunohistochemistry in 10 matched gastric cancer tissues and adjacent-normal specimens from Affiliated Changshu Hospital of Nantong University,followed by survival and clinicopathological correlation analyses.This study was approved by the ethics committee of the Changshu Medical Examination Institute(ethical approval number:JYS-LL-2024-011).In vitro,normal gastric epithelial cells(GES-1)and gastric cancer cells(AGS)were co-cultured with H.pylori,and GSDMD mRNA and protein were detected by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot,respectively.Serum interleukin-18(IL-18)and interleukin-1β(IL-1β)were measured by enzyme-linked immunosorbent assay(ELISA)in 16 healthy controls and 16 gastric cancer patients(before and after curative resection)from Affiliated Changshu Hospital of Nantong University,stratified by H.pylori infection status.Flow cytometry,Hoechst 33342 and propidium iodide(PI)double fluorescent staining,lactate dehydrogenase(LDH)release assays,and caspase-1 activity assays were employed to assess pyroptosis of gastric epithelial cells induced by H.pylori infection.The Tumor Immune Estimation Resource(TIMER)database was used to analyze the correlation between GSDMD expression and the infiltration of various immune cells into the tumor microenvironment.Results:In the TCGA database,the mRNA expression of GSDMD in gastric cancer tissues was significantly increased,and it was associated with poor prognosis.The verification results of protein detection in clinical samples showed that,compared with normal tissues,the expression level of GSDMD protein in gastric cancer tissues was significantly increased.In the co-culture model of H.pylori and GES-1 cells,both full-length and cleaved GSDMD(GSDMD-N)mRNA and protein levels were significantly increased compared with those before infection,and it was time-dependent.Similarly,the co-culture model of H.pylori and AGS cells exhibited elevated GSDMD mRNA and protein levels.The level of IL-18 in the serum of clinical gastric cancer patients was increased,particularly in those with positive H.pylori infection.Further analysis revealed that preoperative IL-18 levels were markedly elevated in gastric cancer patients relative to healthy controls,declined postoperatively,but remained above controls;while no significant alteration was observed for IL-1β(P＞0.05).Pyroptosis was markedly elevated in the H.pylori co-culture models with GES-1 and AGS,as evidenced by flow cytometry,LDH release assays,caspase-1 activity and Hoechst 33342/PI double staining,with a progressive increase observed over the course of H.pylori infection.Immune-infiltration profiling further revealed that high GSDMD expression was correlated with increased infiltration of activated dendritic cell(aDC),regulatory T cell(Treg),T helper 2 cell(Th2)and natural killer(NK)cell,accompanied by decreased infiltration of mast cells and central memory T cell(Tcm).This indicated that the expression of GSDMD was related to the regulation of immune cell composition,thus remodeling the tumor immune microenvironment.Conclusion:This study revealed that H.pylori infection could cause local inflammatory responses through GSDMD-mediated pyroptosis and affect the infiltration of immune cells,thereby driving the malignant progression of gastric cancer.This discovery provided an important theoretical basis for formulating new treatment strategies for gastric cancer targeting GSDMD.