温肾益髓生血方对RA大鼠肾脏PI3K/AKT信号通路影响的研究

Effects of Wenshen Yisui Shengxue Formula on PI3K/AKT Signaling Pathway in Kidney of RA Rats

目的:探讨温肾益髓生血方对RA大鼠贫血的改善作用及其对肾脏PI3K/AKT信号通路的影响。方法:50只雄性SPF级Wister大鼠,按体质量进行随机分层,分为五组:正常组10只,假手术组10只,模型组10只,生血宁组10只,温肾益髓生血组10只。模型组与治疗组每日予180 mg·Kg·d^-1腺嘌呤灌胃及予各治疗组对应的治疗药物,正常组、假手术组与模型组使用等体积去离子水灌胃,每周称取大鼠体质量。饲养12周后,各组大鼠用10%水合氯醛麻醉后取血,测定血清肌酐(Scr)、尿素氮(BUN)、尿酸(UA)等指标;RBC、HGB指标;留取肾组织并用10%福尔马林固定,HE、Mallory染色观察大鼠肾脏病理,免疫组化检测大鼠肾脏EPO、PI3K、AKT、p-AKT、bcl-2蛋白表达水平,原位杂交检测大鼠肾脏EPO、PI3K、AKT、bcl-2mRNA表达水平。结果:与正常组和假手术组比较,模型组体质量明显降低,有极显著性差异(P <00.01),肾脏出现明显病理损害;与模型组比较,温肾益髓生血组大鼠的一般状态有明显改善,各治疗组体质量有所升高,具有极显著性差异(P <0.01),治疗组血清BUN、Scr、UA水平明显降低,RBC、HGB水平升高,具有及显著性差异(P <0.01),肾脏病理损害减少,各治疗组大鼠肾脏EPO、PI3K、AKT、p-AKT、bcl-2蛋白表达水平明显降低,具有极显著性差异(P <0.01),EPO、PI3K、bcl-2mRNA表达水平明显升高,具有极显著性差异(P <0.01),AKTmRNA各组间无明显差异(P>0.05)。结论:温肾益髓生血方能够纠正RA大鼠的贫血状态并可能是通过干预肾脏PI3K/AKT信号通路起到抗肾脏细胞凋亡的作用。

Objective: To explore the effects of Wenshen Yisui Shengxue Formula on improving the anemia of RA rats and its effects on renal PI3K/AKT signaling pathway. Methods: 50 male SPF Wister rats were randomly divided into 5 groups,according to their body weight: normal group, sham operation group, model group, Shengxuening group, and wenshenyisuishengxuefang group, with 10 rats in each group. The model group and treatment group were given 180 mg·Kg·d^-1 adenine, and corresponding therapeutic medicines and adenine for gavage every day. The normal group, the sham operation group and the model group were given equal volume of deionized water for gavage, and the rat body mass was weighed was weekly. After 12 weeks of feeding, rats in each group were anesthetized with 10% chloral hydrate and blood was taken to measure serum creatinine(Scr), urea nitrogen(BUN), uric acid(UA) and other indicators, whole blood red blood cells(RBC), and hemoglobin (HGB). The kidney tissues were preserved and fixed with 10% formalin. And the kidney pathology was observed by HE and Mallory staining. The expression levels of EPO, PI3K, AKT, p-AKT and bcl-2 protein in rat kidney were detected by immunohistochemistry. Results: Compared with the model group and sham operation group, the body mass of the model group was significantly reduced, and there were significant differences(P< 0.01). Significant pathological damage occurred in kidney. Compared with the model group, the condition of Weshen Yisui Shengxue group was significantly improved. The body weight of each group increased, with significant differences(P <0.01). The serum BUN, Scr, UA levels in the treatment group were significantly reduced(P <0.01). And the RBC,HGB levels were elevated and renal pathological damages is also reduced(P <0.01). There were significant differences(P< 0.01). Little pathological damage occurred in kidney. The expression levels of EPO, PI3K, AKT, p-akt and bcl-2 proteins in the kidney of the rats in the treatment group were significantly decreased, with extremely significant differences(P< 0.01). The expression levels of EPO, PI3K and bcl-2 mRNA were significantly increased(P< 0.01).There was no significant difference in AKTmRNA among the groups(P> 0.05). Conclusion: Wenshen Yisui Shengxue Formula can correct anemia in RA rats and may play an anti-apoptosis role by interfering in PI3K/AKT signaling pathway.