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目的探讨哈蟆油(OR)对D-半乳糖所致雌性衰老大鼠肝脏组织p16、p21和细胞周期蛋白D1蛋白表达的影响,进一步探讨哈蟆油延缓雌性大鼠机体衰老机制。方法 SPF级SD雌性青年大鼠40只随机分为D-半乳糖组、维生素E组、哈蟆油高剂量组、中剂量组、低剂量组,每组8只,D-半乳糖颈背部皮下注射42 d,建立亚急性衰老模型。另取雌性青年大鼠8只,同样部位每日注射生理盐水,作为空白对照组。第15天开始灌胃给药,给药时间28 d。给药结束后,免疫组化法检测衰老大鼠肝脏组织p16和p21蛋白的表达情况,蛋白免疫印迹法检测哈蟆油对雌性衰老大鼠肝脏组织细胞周期蛋白D1表达的影响。结果雌性衰老大鼠肝脏组织免疫组化结果表明,p16和p21多为胞浆表达,弥漫性、灶性分布均有。D-半乳糖组p16和p21阳性细胞积分与空白组比较升高,差异有显著性(P<0.01)。与D-半乳糖组相比,哈蟆油中、低剂量组,p16阳性细胞积分降低,差异有显著性(P值均<0.01),哈蟆油各剂量组p21阳性细胞积分降低,差异有显著性(P值均<0.01)。肝脏组织蛋白免疫印迹法结果表明,D-半乳糖组肝脏组织细胞周期蛋白D1蛋白表达与空白组比较降低,差异有显著性(P<0.01)。哈蟆油各剂量组细胞周期蛋白D1蛋白表达与D-半乳糖组比较,表达均升高(P值均<0.01),哈蟆油高剂量组尤为明显。结论哈蟆油可降低肝脏组织p16、p21蛋白的高表达。同时显著提高细胞增殖正性调控因子细胞周期蛋白D1蛋白的表达,促进衰老雌性大鼠肝细胞增殖。哈蟆油延缓雌性衰老作用可能通过调控肝脏p16,p21,细胞周期蛋白D1信号通路来促进有关增殖调控蛋白表达发挥延缓衰老作用。
Objective To investigate the effect of Rana oil (OR) on the expression of p16, p21 and cyclin D1 in the liver tissue of D-galactose-induced female rats, and to further explore the mechanism of Rana oil retarding the aging of female rats. Methods Forty SPF female SD rats were randomly divided into D-galactose group, vitamin E group, high dose of Rana oil, middle dose group and low dose group, with 8 rats in each group. D-galactose subcutaneous After 42 days of injection, a sub-acute aging model was established. Another 8 young female rats, the same site daily injection of saline, as a blank control group. The first 15 days began gavage, dosing time 28 d. Immunohistochemistry was used to detect the expression of p16 and p21 protein in the liver tissue of the aged rats. Western blotting was used to detect the expression of cyclin D1 in the liver tissue of the aging rats. Results The results of immunohistochemistry in the liver tissue of female aging rats showed that p16 and p21 were mostly cytoplasm, diffuse and focal distribution. The positive cells of p16 and p21 in D-galactose group increased compared with the blank group, the difference was significant (P <0.01). Compared with D-galactose group, the positive cells of p16 in Hagia oil were lower, the difference was significant (P <0.01), p21 positive cells in each dose of Hana oil decreased, the difference was Significance (P <0.01). Western blot results showed that the expression of cyclin D1 protein in liver tissue of D-galactose group decreased compared with the blank group, the difference was significant (P <0.01). Compared with D-galactose group, the expression of cyclin D1 protein in each dose of Rana oil increased (P <0.01), especially in the high dose of Rana oil. Conclusion Ovana oil can reduce the high expression of p16, p21 protein in liver tissue. At the same time, it significantly increased the expression of Cyclin D1, a positive regulator of cell proliferation, and promoted the proliferation of hepatocytes in senescent female rats. Oviducts delayed the female senescence effect may be through regulating the liver p16, p21, cyclin D1 signaling pathway to promote proliferation-related protein expression play a role in anti-aging.