归肾丸对卵巢早衰大鼠骨密度及骨微结构的影响及其机制研究

The mechanism and effect of Guishen pill on bone mineral density and microstructure of bone tissue in rats with premature ovarian failure

目的观察归肾丸对卵巢早衰大鼠骨密度及骨微结构的影响,探讨其可能机制,为归肾丸治疗骨质疏松提供实验依据支持。方法将24只Wistar大鼠随机分为正常组、模型组和归肾丸组,每组各8只,其中模型组和归肾丸组通过腹腔注射顺铂的方法(第1、8天腹腔注射顺铂溶液6.0 mg/kg)建立卵巢早衰大鼠模型。造模后正常组、模型组灌蒸馏水,归肾丸组给予归肾丸免煎剂[9.45 g/(kg·d)],第30天处死大鼠,留取动脉血、双侧股骨。Micro CT检查各组大鼠骨密度和骨微结构参数;ELISA检测大鼠血清中RANKL/OPG、IL-10及TGF-β的表达水平;免疫组化检测骨组织RANKL、OPG的表达水平;流式细胞术检测外周血CD4^(+)T细胞中CD25^(+)Foxp3^(+)Treg细胞的比例。结果归肾丸组与模型组比较,Micro CT骨微结构扫描提示BMD、BV/TV、Tb.N、Tb.Th均明显升高(P<0.05);Tb.Sp明显下降(P<0.05);血清中OPG、IL-10的表达较模型组明显升高(P<0.05或P<0.01),RANKL/OPG明显下降(P<0.01),RANKL、TGF-β的表达与模型组比较差异无统计学意义(P>0.05);流式细胞术检测提示归肾丸组外周血CD4^(+)CD25^(+)Foxp3^(+)Treg比例较模型组明显升高(P<0.05);免疫组化检测提示归肾丸组骨组织OPG表达较模型组升高,RANKL表达下降(P<0.05)。直线相关性分析显示外周血Treg细胞的比例与OPG呈显著正相关(r=0.539,P<0.01),与RANKL/OPG比值呈显著负相关(r=-0.578,P<0.01)。结论归肾丸可改善卵巢早衰大鼠骨量减少和保护其骨微结构,其机制可能是通过上调Treg细胞,从而抑制RANKL/OPG途径。

Objective To explore the mechanism and effect of Guishen pill on bone mineral density and microstructure of bone tissues in rats with premature ovarian failure(POF),in order to provide experimental basis for the treatment of osteoporosis.Methods Twenty-four rats were randomly divided into 3 groups:normal group,model group,and the Guishen pill group.Eight rats were included in each group.POF rat model was established by peritoneal injection of cisplatin which was injected at 1st and 8^(th) days with a dose of 6.0 mg/kg.In the normal group and model group,the rats were given distilled water while the rats in Guishen pill group were given Guishen pill granule(9.45 g/kg/day).At 30^(th) day,all the rats were killed.The arterial blood and humerus were collected.Micro CT was applied to detect bone mineral density(BMD)and parameters of microstructure.ELISA was applied to detect the expression of RANKL/OPG,IL-10,and TGF-βin the serum of each group.Immunohistochemical staining was applied to detect the expression of RANKL and OPG in bone tissue.Flow cytometry was used to detect the ratio of T regulatory cells in the peripheral blood.Results The scans in micro CT indicated that BMD,BV/TV,Tb.N,and Tb.Th in the Guishen pill group were higher than those in the POF group(P<0.05).Tb.Sp was lower than that in the POF group(P<0.05).Compared with the POF group,the expressions of serum OPG and IL-10 in the Guishen pill group obviously increased(P<0.05),while the expression of serum RANKL/OPG in the Guishen pill group obviously decreased(P<0.05).Serum RANKL and TGF-βin the Guishen pill group had no significant difference with the model group(P>0.05).The ratio of T regulatory cells in the peripheral blood was significantly higher than that in the model group(P<0.05).The expression of OPG in bone tissue was obviously higher than that of the model group(P<0.05).The expression of RANKL in bone tissue was obviously lower than that of the model group(P<0.05).Conclusion Guishen pill improves BMD and bone microstructure of POF rats.The mechanism may be associated with inhibition of the RANKL/OPG pathway by up-regulating T regulatory cells.