胃复春抑制NF-κB/GSDME介导的细胞焦亡治疗胃癌前病变

Weifuchun Alleviates Gastric Precancerous Lesions by Inhibiting Pyroptosis via NF-κB/GSDME Pathway

摘要:

目的 探讨胃复春（WFC）抑制炎症反应、减轻胃癌前病变（GPL）的作用及分子机制。方法 使用N-甲基-N′-硝基-N-亚硝基胍（MNNG）刺激人胃黏膜上皮细胞GES-1，建立体外GPL细胞模型（MC细胞），使用胱天蛋白酶-3（Caspase-3）抑制剂Z-DEVD-FMK抑制Caspase-3活化。将MC细胞分为空白组（20%空白血清）、WFC治疗（15%、20% WFC含药血清）组、Caspase-3抑制剂组。细胞增殖与活性检测（CCK-8）法检测GES-1或MC细胞活性，Transwell实验检测细胞侵袭，5-乙炔基-2′-脱氧尿苷（EdU）染色检测细胞增殖，流式细胞术检测细胞活性氧（ROS）水平，实时荧光定量聚合酶链式反应（Real-time PCR）检测白细胞介素-1（IL-1）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）水平；基因表达数据库（GEO）公共数据分析细胞焦亡在胃癌进展中作用，蛋白免疫印迹法（Western blot）检测核转录因子-κB（NF-κB）p65、焦孔素E（GSDME）、Caspase-3水平，免疫荧光染色检测NF-κB p65蛋白水平及核转位情况，苏木素-伊红（HE）染色观察临床WFC治疗前后患者胃黏膜病理改变。结果 与正常组比较，MC细胞增殖和侵袭能力显著增强（P<0.01）；与空白血清组比较，WFC含药血清可抑制MC细胞增殖和侵袭（P<0.01），下调IL-1、IL-6、TNF-α mRNA及ROS水平（P<0.05，P<0.01）。胃癌不同时期转录组数据分析显示，细胞焦亡参与胃癌进展，与正常组比较，GSDME在GPL患者中显著升高（P<0.05）。细胞实验证实，与空白血清组比较，WFC含药血清明显抑制NF-κB蛋白水平及NF-κB核转位（P<0.05），WFC含药血清通过抑制Caspase-3对GSDME剪切作用，抑制细胞焦亡；临床患者标本进一步证明，与WFC治疗前比较，WFC治疗后显著下调NF-κB水平及GSDME活化（P<0.01），抑制细胞焦亡，减轻胃黏膜炎症及肠上皮化生。结论 细胞焦亡参与胃癌进展，WFC通过NF-κB/GSDME通路抑制细胞焦亡，减轻胃黏膜炎症，治疗GPL。

Abstract:

Objective To explore the role and molecular mechanism of Weifuchun （WFC） in inhibiting inflammation and alleviating gastric precancerous lesions （GPL）.Method Human gastric mucosal epithelial cells （GES-1） were stimulated with N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） for the modeling of GPL （MC cells）， with Caspase-3 inhibition by Z-DEVD-FMK. MC cells were divided into control （20% blank serum）， WFC （15% and 20% WFC-containing serum）， and caspase-3 inhibitor groups. The cell counting kit-8 （CCK-8） was used to examine the viability of GES-1 cells or MC cells. The Transwell assay and 5-acetylidene-2′-deoxyuridine （EdU） staining were employed to examine cell invasion and proliferation， respectively. Flow cytometry was employed to determine the level of reactive oxygen species. Real-time PCR was conducted to determine the mRNA levels of interleukin （IL）-1， IL-6， and tumor necrosis factor （TNF）-α. Gene Expression Omnibus （GEO） was used to analyze the role of pyroptosis in gastric cancer progression. Western blotting was employed to determine the protein levels of nuclear factor-κB （NF-κB） p65， gasdermin E （GSDME）， and Caspase-3. Immunofluorescence staining was employed to detect the NF-κB p65 protein level and nuclear translocation. Hematoxylin-eosin staining was carried out to observe the pathological changes in the gastric mucosa before and after WFC treatment in the patients.Result Compared with the control group， MC cells presented enhanced proliferation and invasion energy （P<0.01）. Compared with the blank serum group， WFC-containing serum inhibited the proliferation and invasion of MC cells （P<0.01）， down-regulated the mRNA levels of IL-1， IL-6， and TNF-α， and lowered the level of reactive oxygen species （P<0.05， P<0.01）. The transcriptome data at different stages of gastric cancer showed that pyroptosis was involved in gastric cancer progression， and the GSDME level was significantly higher in GPL patients than in the normal group. Compared with the blank serum， WFC-containing serum lowered the level of NF-κB and inhibited the nuclear translocation of NF-κB （P<0.05）， and it inhibited pyroptosis by suppressing the cleavage of Caspase-3 on GSDME （P<0.05， P<0.01）. The analysis of patient specimens further demonstrated that WFC treatment down-regulated the NF-κB level and GSDME cleavage （P<0.01）， inhibited pyroptosis， and alleviated gastric mucosal inflammation and intestinal epithelial metaplasia.Conclusion Pyroptosis is involved in the progression of gastric cancer， and WFC inhibits pyroptosis via the NF-κB/GSDME pathway， thereby alleviating gastric mucosal inflammation in GPL.