基于TGF-β1/PI3K/Akt通路探讨加味沙参麦冬汤对慢性萎缩性胃炎大鼠的影响

Effects of modified Shashen Maidong Decoction in rats with chronic atrophic gastritis based on TGF-β1/PI3K/Akt pathway

目的探讨加味沙参麦冬汤对慢性萎缩性胃炎(CAG)大鼠的保护作用及对转化生长因子-β1(TGF-β1)/人磷脂酰肌醇三羟基激酶(PI3K)/Akt通路的影响。方法将60只大鼠随机分为空白组、模型组、阳性对照组、低剂量组及高剂量组,每组12只。除空白组外,其余各组大鼠采用1-甲基-3-硝基-1-亚硝酸胍(MNNG)构建CAG模型。空白组、模型组大鼠给予等体积生理盐水灌胃,阳性对照组大鼠给予维酶素溶液灌胃,低剂量组及高剂量组分别给予加味沙参麦冬汤5、10 g/kg灌胃,1次/d,共12周。采用苏木精伊红(HE)染色观察胃黏膜组织病理变化;采用酶联免疫吸附测定(ELISA)检测大鼠血清胃动素(MTL)、胃泌素(Gas)、生长抑素(SS)含量及胃组织白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;采用免疫组化检测胃组织增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2(Bcl-2)表达;采用实时荧光定量聚合酶链反应(RT-qPCR)、蛋白质印迹法(Western blot)检测胃组织TGF-β1、PI3K、Akt、p-PI3K、p-Akt mRNA及其蛋白表达水平。结果与空白组比较,模型组大鼠胃组织苍白,褶皱明显减少,胃黏膜变薄、水肿,腺体减少,炎症细胞浸润;血清MTL水平、胃组织IL-6和TNF-α水平、Bcl-2蛋白表达升高,Gas、SS水平和PCNA蛋白表达降低,差异有统计学意义(P<0.01);TGF-β1、PI3K、Akt、p-PI3K、p-Akt mRNA及其蛋白表达水平升高,差异有统计学意义(P<0.01)。与模型组比较,低剂量组及高剂量组随着剂量增加,胃组织颜色变得红润,褶皱增多,胃黏膜各层结构基本恢复,腺体排列较规则,腺体萎缩好转;血清MTL水平、IL-6和TNF-α水平及胃组织Bcl-2蛋白表达降低,Gas、SS水平和PCNA蛋白表达升高,差异有统计学意义(P<0.01);TGF-β1、PI3K、Akt、p-PI3K、p-Akt mRNA及其蛋白表达水平降低,差异有统计学意义(P<0.01)。结论加味沙参麦冬汤可通过抑制TGF-β1/PI3K/Akt通路,上调PCNA基因表达,下调Bcl-2基因表达,抑制炎症因子产生,发挥保护胃组织作用。

Objective To investigate the protective effect of modified Shashen Maidong Decoction in rats with chronic atrophic gastritis(CAG)and its effect on transforming growth factor-β1(TGF-β1)/human phosphatidylinositol trihydroxy kinase(PI3K)/Akt pathway.Methods Sixty rats were randomly divided into blank group,model group,positive control group,low-dose group and high-dose group,with 12 rats in each group.Except for the blank group,the CAG model was constructed using 1-methyl-3-nitro-1-nitrosoguanidine(MNNG)in all groups of rats.The rats in the blank and model groups were given equal volume of saline by gavage,the rats in the positive control group were given vincristine solution by gavage,and the rats in the low-dose group and high-dose group were given 5 g/kg and 10 g/kg modified Shashen Maidong Decoction by gavage,once a day for 12 weeks.The pathological changes of gastric mucosa were observed by hematoxylin eosin(HE)staining;the contents of motilin(MTL),gastrin(Gas)and somatostatin(SS)in serum and the levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in gastric tissue were detected by enzyme-linked immunosorbent assay(ELISA);the expression of proliferating cell nuclear antigen(PCNA)and B-lymphoma-2(Bcl-2)were detected by immunohistochemistry;the real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the mRNA and protein expression levels of TGF-β1,PI3K,Akt,p-PI3K and p-Akt in gastric tissue.Results Compared with the blank group,the gastric tissues of rats in the model group were pale,with reduced folds,thinning and edema of gastric mucosa,reduced glands,infiltration of inflammatory cells;the serum MTL level,gastric tissue IL-6 and TNF-αlevels,Bcl-2 protein expression were significantly increased,while Gas,SS levels and PCNA protein expression were significantly decreased(P<0.01);the mRNA and protein expression levels of TGF-β1,PI3K,Akt,P-PI3K and P-Akt were significantly increased(P<0.01).Compared with the model group,with the increasing dose,the gastric tissue color in the low-dose group and the high-dose group became red and moist,with increased folds,restored structure of each layer of gastric mucosa,the regular glandular arrangement,and the better glandular atrophy;the serum MTL levels,IL-6 and TNF-αlevels and Bcl-2 protein expression in gastric tissue were significantly decreased,while Gas,SS and PCNA protein expression were significantly increased(P<0.01);the TGF-β1,PI3K,Akt,P-PI3K and P-Akt mRNA and theiRprotein expression levels were significantly decreased(P<0.01).Conclusion Modified Shashen Maidong Decoction can protect gastric tissues by inhibiting the TGF-β1/PI3K/Akt pathway,up-regulating the expression of PCNA genes and down-regulating the expression of Bcl-2 genes,and inhibiting the production of inflammatory factors.