附子理中汤对脾阳虚大鼠AQP4-ANP-pGC轴的影响

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(P0.05,P0.01)。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(P0.05,P0.01)。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。

Effect of Fuzi Lizhongtang on AQP4-ANP-pGC Axis in Rats with Spleen Yang Deficiency Syndrome

Objective: To explore the mechanism of Fuzi Lizhongtang in warming Yang, invigorating the spleen and dispelling dampness through AQP4-ANP-pGC pathway. Method: A total of 120 clean grade Wistar rats were randomly selected and divided into blank control group, sham operation group, model group, and high, middle, low-dose Fuzi Lizhongtang groups, with 20 rats in each group.The blank control group was fed normally.Animal models were established in the sham operation group and the model group through interscapular brown adipose tissue (BAT) resection+high-fat diet+cold environment stimulation at alternate days (the sham operation group only received BAT without resection).The high-fat diet was fed for 30 days since the first day of the operation.In addition to the treatment for the model group, the three Fuzi Lizhongtang groups were respectively treated with high-dose (40 g · kg^-1 · d^-1), middle-dose (20 g · kg^-1 · d^-1) and low-dose (10 g · kg^-1 · d^-1) Fuzi Lizhongtang, while the other groups were given the same amount of normal saline.Materials were collected after 30 days of administration, (the model group was collected on the first day after successful modeling).The morphologic changes in gastric antrum and ileum in rats were observed by HE staining; ANP content was detected by ELISA; the mRNA expression of AQP4 was detected by Real-time PCR.And the AQP4 content was detected by Western blot. Result: HE staining of gastric antrum and ileum in rats was observed under light microscope.Pathological changes in gastrointestinal tissues in rats in each Fuzi Lizhongtang group were alleviated to different degrees, with the best effect in high-dose group.Compared with the control group and the sham operation group, the contents of AQP4, ANP and the relative expression of AQP4 mRNA in the 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 the middle and the high dose groups increased (P<0.05, P<0.01). Conclusion: Fuzi Lizhongtang may regulate body fluid metabolism in rats with spleen Yang deficiency syndrome by changing the contents of AQP4, ANP and the expression of AQP4 mRNA in AQP4-ANP-pGC pathway.