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目的:从水通道蛋白4-心房利尿钠钛-非可溶性鸟苷酸环化酶(aquaporin4-atrial natriuretic peptide-particulate guanylate cyclase,AQP4-ANP-p GC)通路探讨附子理中汤温阳健脾祛湿的作用机制。方法:选清洁级Wistar大鼠120只,分为空白组、假手术组、模型组和附子理中汤高、中、低剂量组共6个组,每组20只。空白组常态饲养。假手术组与模型组采用“肩胛骨间棕色脂肪组织(brown adipose tissue,BAT)切除术+高脂饲料喂养+隔日寒冷环境刺激”方法造模(假手术组只找到BAT但不切除)。术后第1天始喂高脂饲料共30 d。在模型组基础上,高、中、低剂量各组分别予附子理中汤40,20,10 g·kg~(-1)·d~(-1)灌胃,其他组给予等体积的生理盐水灌胃。于给药30 d后(其中模型组于造模成功后第1天)取材,苏木素-伊红(HE)染色观察大鼠胃窦与回肠组织病理形态学变化;酶联免疫吸附法(ELISA)检测ANP含量;实时荧光定量PCR(Real-time PCR)法检测AQP4 mRNA表达情况;蛋白免疫印迹法(Western blot)检测AQP4含量。结果:大鼠胃窦与回肠组织HE染色光镜下观察可见,附子理中汤各剂量组大鼠胃肠组织病理学改变均有不同程度的减轻,以高剂量组效果明显。与空白组和假手术组相比,模型组AQP4,ANP含量及AQP4 mRNA相对表达量降低(P<0.05,P<0.01)。与模型组相比,中、高剂量组AQP4,ANP含量及AQP4 mRNA相对表达量升高(P<0.05,P<0.01)。结论:附子理中汤可能通过影响AQP4-ANP-p GC通路中AQP4,ANP含量及AQP4 mRNA表达以参与脾阳虚证大鼠水液代谢的调节。
OBJECTIVE: To investigate the effect of Fuzi Zhongzheng Tang on regulating the spleen and dampness of the spleen and removing dampness from aquaporin 4-atrial natriuretic peptide-particulate guanylate cyclase (AQP4-ANP-p GC) Mechanism. Methods: One hundred and twenty clean Wistar rats were divided into six groups (n = 20 in each group): blank control group, sham operation group, model group and Fuzi Lizhong Tang high, medium and low dose groups. Blank group normal feeding. The sham operation group and model group were modeled by “brown adipose tissue (BAT) resection + high fat diet feeding + cold weather every other day” (sham group only found BAT but not resected). On the first day after operation, high-fat diet was fed for 30 days. On the basis of the model group, the rats in high, middle and low dose groups were treated with 40, 20, 10 g · kg -1 · d -1 of Zili Lizhong Decoction respectively, and the other groups were given the same volume of physiology Saline irrigation. The pathological changes of the gastric antrum and the ileum were observed by hematoxylin and eosin (HE) staining after 30 days of administration, in which the model group was made on the first day after the model was established successfully. Enzyme-linked immunosorbent assay (ELISA) The level of AQP4 mRNA was detected by Real-time PCR. The level of AQP4 was detected by Western blot. Results: Observed by HE staining in gastric antrum and ileum of rats, the pathological changes of gastrointestinal tissues of rats in each dose group of Fuzi Lizhong Decoction were relieved to varying degrees, and the effect was obvious with high dose. Compared with blank group and sham operation group, AQP4, ANP content and relative expression of AQP4 mRNA in model group decreased (P <0.05, P <0.01). Compared with the model group, the contents of AQP4, ANP and the relative expression of AQP4 mRNA in middle and high dose groups increased (P <0.05, P <0.01). Conclusion: Fuzi Lizhong Decoction may participate in the regulation of water-fluid metabolism in rats with spleen-Yang deficiency syndrome by affecting AQP4-ANP content and AQP4 mRNA expression in AQP4-ANP-p GC pathway.