幽门螺杆菌感染对胃上皮细胞铁死亡相关脂代谢蛋白表达及脂质过氧化水平的影响

The Effects of Helicobacter pylori infection on the expression of ferroptosis related lipid metabolism proteins and lipid peroxidation in gastric epithelial cells

目的探究幽门螺杆菌(Helicobacter pylori,Hp)感染对胃上皮细胞和C57BL/6小鼠胃组织中铁死亡相关脂代谢蛋白ACSL4、LPCAT3表达及对脂质过氧化物降解产物丙二醛(MDA)和4-羟基壬烯醛(4-HNE)水平的影响。方法用HpGZ7和Hp26695菌株感染正常胃上皮GES-1细胞和胃癌AGS细胞,感染复数为50∶1,6 h、24 h、48 h后分别收集细胞总蛋白和细胞裂解液,Western blot检测ACSL4和LPCAT3的蛋白表达,试剂盒检测细胞裂解液MDA、4-HNE水平。用Hp灌胃感染C57BL/6小鼠1、3、6月,取胃黏膜组织并制备切片,免疫组织化学染色检测ACSL4、LPCAT3蛋白的表达。结果与未感染组比较,Hp感染GES-1(F值分别为18.82、13.07,4.28、13.03)和AGS(F值分别为14.51、25.88,17.90、16.62)细胞ACSL4、LPCAT3蛋白表达水平均增加,并随着感染时间的延长表达呈上升趋势,差异有统计学意义(P<0.05)。与未感染对照组比较,Hp感染C57BL/6小鼠胃组织中ACSL4、LPCAT3的表达水平均显著升高,其中LPCAT3表达水平增加更显著(F值分别为27.11,103.62,P<0.05)。MDA含量均高于未感染组(Hp感染AGS细胞6 h、24 h、48 h后分别为0.27±0.06、1.13±0.14、0.83±0.12,0.56±0.13、0.86±0.11、0.72±0.16。Hp感染GES-1细胞6h、24 h、48 h后分别为0.22±0.08、0.29±0.09、0.80±0.08,0.17±0.06、0.40±0.08、0.63±0.09),差异有统计学意义(F值分别为27.29、10.56,30.85、18.42,P<0.05)。4-HNE含量均高于未感染组(Hp感染AGS细胞6 h、24 h、48 h后分别为0.12±0.03、0.20±0.06、0.25±0.04,0.06±0.02、0.21±0.03、0.30±0.04。Hp感染GES-1细胞6 h、24 h、48 h后分别为0.23±0.06、0.36±0.06、0.63±0.09,0.19±0.04、0.19±0.05、0.41±0.07),差异有统计学意义(F值分别为7.48、32.31,16.83、12.55,P<0.05)。结论Hp感染可引起胃上皮细胞和C57BL/6小鼠胃组织中铁死亡相关脂代谢蛋白ACSL4、LPCAT3表达增加,细胞内脂质过氧化物降解产物MDA与4-HNE积累,提示Hp可能通过影响其感染细胞内的脂代谢水平,诱导胃上皮细胞发生铁死亡。

Objective To investigate the effects of Helicobacter pylori(Hp)infection on the expression of the ferroptosis-related lipid metabolism proteins ACSL4 and LPCAT3 in gastric epithelial cells and gastric tissue from C57BL/6 mice,and on the levels of malondialdehyde(MDA)and 4-hydroxynonenal(4-HNE)in gastric epithelial cells.Methods Normal gastric epithelial GES-1 cells and gastric cancer AGS cells were infected with Hp GZ7 and Hp 26695 strains at a multiplicity of infection of 50:1.Total protein and cell lysates were collected 6,24 and 48 h after infection.Protein expressions of ACSL4 and LPCAT3 were detected by Western blot,and levels of MDA and 4-HNE in cell lysates were detected using kits.C57BL/6 mice were infected by intragastric administration of Hp for 1,3 and 6 months,and gastric mucosal tissues were collected and sectioned.The expression of ACSL4 and LPCAT3 proteins was detected by immunohistochemical staining.Results Compared with the non-infected group,the expression levels of ACSL4 and LPCAT3 proteins in GES-1(F values were 18.82,13.07,4.28,13.03)and AGS(F values were 14.51,25.88,17.90,16.62)cells with Hp infection were significantly increased,and the expression showed an increasing trend with increasing infection time,and the difference was statistically significant(P<o.05).Compared with the uninfected control group,the expression levels of ACSL4 and LPCAT3 were significantly increased in the gastric tissues of C57BL/6 mice with Hp infection,and the expression level of LPCAT3 increased more significantly(F values were 27.11,103.62,P<0.05).The content of MDA in the infected group was higher than that in the uninfected group(AGS cells infected with Hp at 6 h,24 h and 48h were0.27±0.06,1.13±0.14,0.83±0.12,0.56±0.13,0.86±0.11,0.72±0.16.GES-1 cells infected with Hpat 6h,24 h and 48h were 0.22±0.08,0.29±0.09,0.80±0.08,0.17±0.06,0.40±0.08,0.63±0.09).The difference was statistically significant(F values were 27.29,10.56,30.85,18.42,P<0.05).The content of 4-HNE in the infected group was higher than that in the uninfected group(AGS cells infected with Hp at 6 h,24 h and 48 h were 0.12±0.03,0.20±0.06,0.25±0.04,0.06±0.02,0.21±0.03,0.30±0.04.GES-1 cells infected with Hp at 6h,24h and 48h were0.23±0.06,0.36±0.06,0.63±0.09,0.19±0.04,0.19±0.05,0.41±0.07).The difference was statistically significant(F values were 7.48,32.31,16.83,12.55,P<0.05).Conclusion Hp infection can increase the expression of ferroptosis-related lipid metabolism proteins ACSL4 and LPCAT3 in gastric epithelial cells and gastric tissues of C57BL/6 mice,and the accumulation of intracellular lipid peroxide degradation products MDA and 4-HNE.These results suggest that Hp may induce ferroptosis in gastric epithelial cells by affecting lipid metabolism in Hp infected cells.