基于NOX/ROS信号通路探讨益气化瘀解毒方治疗慢性萎缩性胃炎的机制

Mechanism of Yiqi Huayu Jiedu Decoction on chronic atrophic gastritis based on NOX/ROS signaling pathway

目的:探讨益气化瘀解毒方对慢性萎缩性胃炎(CAG)的作用机制。方法:采用去氧胆酸钠水溶液配合乙醇溶液灌服诱导大鼠CAG模型,造模成功后将大鼠随机分为模型组,益气化瘀解毒方低、中、高剂量组,灌胃给药30d后,测量大鼠胃液分泌量、胃蛋白酶活性及血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)水平;HE染色观察胃组织病理学变化;酶联免疫吸附法检测血清胃蛋白酶原Ⅰ(PGⅠ)、胃蛋白酶原Ⅱ(PGⅡ)及胃泌素(GAS)水平;免疫荧光染色检测胃组织内活性氧(ROS)含量;Western bot和RTPCR检测胃组织NOX/NADPH信号通路的蛋白和基因表达水平。结果:与对照组比较,模型组大鼠胃黏膜层有明显的炎症反应,黏膜腺体萎缩,黏膜下层可见水肿及瘀血,可见肌层增生;胃液分泌量以及胃蛋白酶活性显著降低(P<0.01);血清TNF-α、IL-1β、IL-6含量显著上升(P<0.01),PGⅠ/PGⅡ和GAS含量显著降低(P<0.01);胃组织内ROS含量显著升高(P<0.01),NOX1、NOX2、NOX4的mRNA、蛋白表达显著增加(P<0.01)。与模型组比较,益气化瘀解毒方中、高剂量组大鼠胃黏膜炎症细胞浸润明显减少,胃腺体数量基本恢复正常,无明显肠上皮化生,黏膜下层无明显瘀血水肿;胃液分泌量及胃蛋白酶活性显著升高(P<0.01);血清TNF-α、IL-1β、IL-6含量显著降低(P<0.01),PGⅠ/PGⅡ和GAS含量显著升高(P<0.01);胃组织内ROS含量显著降低(P<0.01),NOX1、NOX2、NOX4的m RNA、蛋白表达显著降低(P<0.01)。结论:益气化瘀解毒方可增加CAG大鼠胃液分泌量和胃蛋白酶活性,抑制炎症因子水平及ROS生成,其可能是通过调控NOX途径来实现的。

Objective:To investigate the mechanism of Yiqi Huayu Jiedu Decoction on chronic atrophic gastritis(CAG).Methods:CAG model was established by deoxycholic acid sodium plus ethanol,rats were randomly divided into the model group,Yiqi Huayu Jiedu Decoction low-dose,medium-dose and high-dose groups after the model was successfully replicated,medication for 30 days,Gastric juice secretion,pepsin activity and serum TNF-α,IL-1β,IL-6 were measured.Gastric histopathological changes were observed by HE staining;the serum PGⅠ,PGⅡand GAS levels were detected by enzyme linked immunosorbent assay;ROS content in gastric tissues was detected by immunofluorescence staining;the protein and mRNA expression levels of NOX1,NOX2,NOX4 in gastric tissues were detected by Western bot and RT-PCR.Results:Compared with the control group,in the model group,the gastric mucosa showed obvious inflammation,mucosal gland atrophy,edema and congestion in the submucosa,and muscular hyperplasia.Gastric juice secretion and pepsin activity were significantly decreased(P<0.01).Serum TNF-α,IL-1β,IL-6 levels were significantly increased(P<0.01),PGⅠ/PGⅡand GAS content were significantly decreased(P<0.01);The ROS content in gastric tissues was significantly increased(P<0.01),and the mRNA and protein expressions of NOX1,NOX2 and NOX4 were significantly increased(P<0.01).Compared with the model group,in the Yiqi Huayu Jiedu Decoction middle and high dose groups,the infiltration of gastric mucositis cells was significantly reduced,the number of gastric glands was basically restored to normal,without obvious intestinal metaplasia,and without obvious blood stasis and edema in the submucosa.Gastric juice secretion and pepsin activity were significantly increased(P<0.01);The serum TNF-α,IL-1β,IL-6 levels were significantly decreased(P<0.01).PGⅠ/PGⅡand GAS content significantly increased(P<0.01).The ROS content in gastric tissue was significantly reduced(P<0.01),and the mRNA and protein expressions of NOX1,NOX2 and NOX4 were significantly decreased(P<0.01).Conclusion:Yiqi Huayu Jiedu Decoction can increase the gastric juice secretion and pepsin activity of CAG rats,inhibit the level of inflammatory factors and the generation of reactive oxygen species,which may be realized by regulating NOX pathway.