基于PI3K/Akt通路探讨芪灵方对慢性萎缩性胃炎伴肠化大鼠的保护作用

Protective Effect of Qiling Prescription on Rats with Chronic Atrophic Gastritis Combined with Gastric Intestinal Metaplasia Via PI3K/Akt Pathway

摘要:

目的 探讨芪灵方干预慢性萎缩性伴肠上皮化生（GIM）的潜在作用机制。方法 将80只SPF级SD大鼠随机分为8组（每组10只）：空白组、空白加芪灵方组、模型组、芪灵方高、中、低剂量组、叶酸组、摩罗丹组，除空白组、空白加芪灵方组外，其余各组大鼠使用0.02 mol·L^-1的N-甲基-N′-硝基-N-亚硝基胍（MNNG）溶液灌胃，并佐以饥饱失常法造模。造模成功后，空白组、模型组予蒸馏水，空白加芪灵方组、芪灵方高、中、低剂量组分别予7.60、15.21、7.60、3.80 g·kg^-1芪灵方水煎剂，叶酸组予0.002 g·kg^-1叶酸悬浊液，摩罗丹组予1.40 g·kg^-1摩罗丹悬浊液灌胃，1次/d，给药8周，期间观察大鼠的一般情况与体质量。麻醉后取大鼠胃组织行苏木素-伊红（HE）染色；免疫组化法（IHC）检测胃组织黏蛋白2（MUC2）、尾部型同源框转录因子2（CDX2）水平；酶联免疫吸附测定法（ELISA）检测血清中白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、胱天蛋白酶-3（Caspase-3）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）水平；蛋白免疫印迹法（Western blot）检测胃组织PI3K、Akt及磷酸化水平。结果 动物实验显示，与空白组比较，参与造模的大鼠体质量自16周起有下降趋势；与模型组比较，芪灵方高、中剂量对大鼠精神状态、体质量均有改善作用。病理结果显示，第24周造模成功，与空白组比较，参与造模的大鼠胃黏膜固有腺体减少，排列相对紊乱；与模型组比较，芪灵方高、中剂量组杯状细胞明显减少或消失，可改善大鼠胃黏膜病理。与空白组比较，模型组胃组织MUC2、CDX2水平升高（P<0.01）；与模型组比较，芪灵方高、中剂量可明显降低胃组织MUC2、CDX2水平（P<0.05，P<0.01）。与空白组比较，模型组大鼠血清中IL-6、IL-1β、TNF-α、Caspase-3、PI3K、Akt水平均提高（P<0.05）；与模型组比较，高剂量芪灵方可显著降低血清中IL-1β、Caspase-3、PI3K、Akt含量（P<0.01），中剂量可显著降低血清中IL-6、IL-1β、TNF-α、Caspase-3、PI3K、Akt含量（P<0.05）。与空白组比较，模型组大鼠胃组织中PI3K、Akt表达显著升高；与模型组比较，高剂量芪灵方显著抑制Akt蛋白表达（P<0.01）。结论 芪灵方可能通过抑制PI3K/Akt通路，抑制促炎因子释放，抑制胃黏膜上皮细胞凋亡，多成分、多靶点、多途径一定程度逆转肠化，延缓炎-癌转化。

Abstract:

Objective To explore the potential mechanism of Qiling prescription in intervening in chronic atrophic gastritis with gastric intestinal metaplasia （GIM）.Method The 80 SPF-grade SD rats were randomly divided into the following eight groups （10 rats per group）： blank group， blank + Qiling prescription group， model group， high-dose Qiling prescription group， medium-dose Qiling prescription group， low-dose Qiling prescription group， folic acid group， and morodan group. Except for the blank and blank + Qiling prescription groups， the other groups underwent modeling by intragastric administration of 0.02 mol·L^-1 N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） solution combined with irregular feeding. After successful modeling， the blank and model groups were given distilled water， the blank + Qiling prescription group， and high， medium， and low-dose Qiling prescription groups were given Qiling prescription water decoction at 7.60， 15.21， 7.60， 3.80 g·kg^-1， respectively， the folic acid group was given folic acid suspension at 0.002 g·kg^-1， the morodan group was given morodan suspension at 1.40 g·kg^-1 by gavage once a day for 8 weeks. The general condition and body weight of the rats were observed during the experiment. Hematoxylin-eosin （HE） staining was performed on gastric tissues. Immunohistochemistry （IHC） was used to detect the levels of mucin 2 （MUC2） and caudal-type homeobox transcription factor 2 （CDX2） in gastric tissues. Enzyme-linked immunosorbent assay （ELISA） was used to measure the serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， cysteine-aspartic protease-3 （Caspase-3）， phosphatidylinositol 3-kinase （PI3K）， and protein kinase B （Akt）. Western blot analysis was performed to detect the expression and phosphorylation levels of PI3K and Akt in gastric tissues.Result Animal experiments showed that compared to the blank group， the rats in the model group had a trend of weight loss starting from week 16. Compared to the model group， high and medium doses of Qiling prescription improved the mental state and body weight of the rats. Pathological results at week 24 showed successful modeling with reduced gastric mucosal glandular cells and disordered arrangement in the model group compared to the blank group. The high and medium-dose Qiling prescription groups showed significantly fewer or absent goblet cells， indicating improved gastric mucosal pathology as compared to model group. Compared to blank group， the model group showed increased levels of MUC2 and CDX2 in gastric tissues （P<0.01）. High and medium doses of Qiling prescription significantly reduced the levels of MUC2 and CDX2 in gastric tissues compared to the model group （P<0.05，P<0.01）. Compared to the blank group， the model group had increased serum levels of IL-6， IL-1β， TNF-α， Caspase-3， PI3K， and Akt （P<0.05）. Compared with the model group， high-dose Qiling prescription significantly reduced the serum levels of IL-1β， Caspase-3， PI3K， and Akt （P<0.01）， medium-dose significantly reduced the levels of IL-6， IL-1β， TNF-α， Caspase-3， PI3K， and Akt （P<0.05，P<0.01）. Compared to the blank group， the model group showed significantly increased expression of PI3K and Akt in gastric tissues. High-dose Qiling prescription significantly inhibited Akt protein expression compared to the model group （P<0.01）.Conclusion Qiling prescription may alleviate GIM and delay inflammation-cancer transformation through multi-component， multi-target， and multi-pathway mechanisms by inhibiting the PI3K/Akt pathway， reducing the release of pro-inflammatory factors， and inhibiting gastric mucosal epithelial cell apoptosis.