基于Raf/MEK/ERK通路探讨平胃胶囊含药血清对MNNG诱导的人胃黏膜上皮细胞炎癌转化的影响

Exploring the Effect of Medication-Containing Serum of Pingwei Capsules(平胃胶囊)on MNNG-Induced Inflammatory Cancer Transformation of Human Gastric Mucosal Epithelial Cells Based on Raf/MEK/ERK Pathway

目的从快速加速纤维肉瘤/丝裂原活化的细胞外信号调节激酶/细胞外信号调节激酶(Raf/MEK/ERK)通路探讨平胃胶囊治疗慢性萎缩性胃炎的可能作用机制。方法15只SD大鼠随机分为空白组5只、平胃胶囊组10只,空白组大鼠给予1 ml/100 g的生理盐水灌胃,平胃胶囊组大鼠给予平胃胶囊混悬液0.63 g/(kg·d)灌胃,连续3天后采集血清。采用N-甲基-N-硝基-亚硝基胍(MNNG)诱导人胃黏膜上皮细胞GES-1建立癌前病变细胞模型。将造模成功细胞分为对照组(10%胎牛血清)、空白血清组(10%胎牛血清+10%空白血清)、含药血清组(平胃胶囊含药血清),采用CCK-8法筛选平胃胶囊含药血清干预体积分数及时间。将人胃黏膜上皮细胞GES-1分为正常组、模型组、空白血清组、含药血清组、U0126组、联合组,每组3个复孔。除空白组外其余各组细胞造模成功后,正常组和模型组加入等体积的胎牛血清,空白血清组加入等体积的空白血清,含药血清组加入筛选确认的体积分数的平胃胶囊含药血清,U0126组给予10μmol/L的丝裂原活化的细胞外信号调节激酶1(MEK1)抑制剂U0126,联合组给予等体积的平胃胶囊含药血清+10μmol/L的U0126。培养筛选确认的时间后,ELISA法检测细胞上清中白细胞介素6(IL-6)水平,免疫荧光检测细胞中IL-6、MEK1的表达,RT-qPCR法检测细胞IL-6、Raf、MEK1、ERK mRNA表达,Western Blot法检测细胞IL-6、Raf、MEK1、ERK1/2蛋白表达。结果选取5.35%的体积分数、干预48 h的平胃胶囊含药血清进行后续实验。与正常组比较,模型组、空白血清组细胞上清中IL-6含量,细胞中IL-6、Raf、MEK1、ERK mRNA及IL-6、Raf、MEK1、ERK1/2蛋白表达均升高(P<0.01)。与模型组比较,含药血清组、U0126组、联合组上述各指标均下降(P<0.05或P<0.01)。与含药血清组比较,联合组细胞中IL-6、MEK1表达,IL-6、Raf、MEK1、ERK mRNA表达,IL-6、Raf、MEK1、ERK1/2蛋白表达均降低(P<0.05或P<0.01)。与U0126组比较,联合组细胞中IL-6表达降低,IL-6、MEK1、ERK1/2蛋白表达降低(P<0.05或P<0.01)。结论平胃胶囊含药血清可能通过抑制Raf/MEK/ERK通路改善GES-1细胞的炎癌转化,从而发挥治疗慢性萎缩性胃炎的作用。

Objective To explore the possible mechanism of Pingwei Capsules(平胃胶囊)for chronic atrophic gastritis from rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular-signal-regulated kinase(Raf/MEK/ERK)pathway that influences the activation of fibrosarcoma protein/mitogen.Methods Fifteen SD rats were randomly divided into 5 rats in the blank group and 10 rats in Pingwei Capsules group.The rats in the blank group were given 1 ml/100 g of saline by gavage,and the rats in Pingwei Capsules group were given 0.63 g/(kg·d)of Pingwei Capsule suspension by gavage,and serum was collected for 3 consecutive days.N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)was used to induce human gastric mucosal epithelial cells GES-1 to establish a precancerous lesion cell model.The successful cells were divided into control group(10%fetal bovine serum),blank serum group(10%fetal bovine serum plus 10%blank serum),and medication-containing serum group(serum with medication of Pingwei Capsule),and the volume fraction and time of intervention of Pingwei Capsule-containing serum were screened by CCK-8 assay.Human gastric mucosal epithelial cells GES-1 were divided into normal group,model group,blank serum group,medication-containing serum group,U0126 group,and combined group,with 6 replicate wells in each group.After successful modelling of the cells in all groups except the blank group,an equal volume of fetal bovine serum was added to the normal and model groups,an equal volume of blank serum was added to the blank serum group,a screening volume fraction of Pingwei Capsule-containing serum was added to Pingwei Capsule group,a 10μmol/L mitogenactivated extracellular signal regulated kinase 1(MEK1)inhibitor U0126 was administered in the U0126 group,an equal dose of Pingwei Capsule-containing serum plus 10μmol/L of U0126 was administered to the combined group.After the selected incubation time,the level of interleukin 6(IL-6)was detected in the cells by ELISA,the expression of IL-6 and MEK1 was detected by immunofluorescence,and the expression of IL-6,Raf,MEK1,and ERK mRNA was detected by RT-qPCR,and the expression of IL-6,Raf,MEK1,and ERK mRNA in the cells was detected by Western blot.Results The 5.35%volume fraction,48 h intervention of Pingwei Capsule-containing serum was selected for subsequent experiments.Compared with the normal group,the IL-6 content in cell supernatants and the expression of IL-6,Raf,MEK1,ERK mRNA and ERK1/2 proteins in cells increased in the model group and blank serum group(P<0.01).Compared with the model group,all of the above indexes were improved in medicationcontaining serum group,U0126 group,and combined group(P<0.05 or P<0.01).Compared with medicationcontaining serum group,the expression of IL-6,MEK1 expression,the expression of IL-6,Raf,MEK1 and ERK mRNA,and the expression of IL-6,Raf,MEK1 and ERK1/2 proteins reduced in the cells of combined group(P<0.05 or P<0.01).Compared with the U0126 group,IL-6 expression reduced and IL-6,MEK1 and ERK1/2 protein expression reduced in cells of combined group(P<0.05 or P<0.01).Conclusion The Pingwei Capsule-containing serum may play a role in the treatment of chronic atrophic gastritis by improving the inflammation-cancer transformation of GES-1 cells through inhibiting the Raf/MEK/ERK pathway.