针刺对去卵巢大鼠骨密度及肠黏膜跨膜钙转运相关受体表达的影响

Effect of electroacupuncture on intestinal calcium absorption and expression of transmembrane calcium transport-related receptors in ovariectomized rats

目的:观察针刺对去卵巢大鼠骨密度及钙离子转运相关受体的影响,探讨针灸防治骨质疏松症的机制。方法:3月龄雌性SD大鼠随机分为空白组、假手术组、模型组和电针组,每组8只。模型组和电针组大鼠行双侧卵巢摘除术。电针组交替给予"关元"+"三阴交"(双)和"肾俞"+"后三里"(双)电针干预,每次电针20 min,每日1次,每周连续干预5 d,共12周。12周后用骨密度仪统一测定右侧胫骨、股骨骨密度;采用荧光定量PCR及Western blot法检测肠黏膜内瞬时感受器电位香草酸受体5(TRPV5)、瞬时感受器电位香草酸受体6(TRPV6)、钠钙交换体1(NCX1)和细胞膜钙离子三磷酸腺苷水解酶(PMCA1b)及与钙离子细胞旁转运相关的紧密连接蛋白-1(ZO-1)、封闭蛋白occludin和claudin mRNA和蛋白的表达水平。结果:与空白组、假手术组比较,模型组大鼠骨密度明显降低(P<0.05);与模型组比较,电针组骨密度明显提高(P<0.05)。与空白组、假手术组比较,模型组肠黏膜内TRPV6、NCX1 mRNA表达上调(P<0.01),TRPV5、PMCA1b、ZO-1、occludin和claudin的mRNA表达明显下调(P<0.01);与模型组比较,电针组肠黏膜TRPV6、NCX1 mRNA的表达明显下调(P<0.01,P<0.05),TRPV5、PMCA1b、ZO-1和occludin的mRNA表达水平均上调(P<0.01,P<0.05)。与空白组、假手术组比较,模型组TRPV6、NCX1蛋白表达量升高(P<0.05),TRPV5、PMCA1b、ZO-1、occludin和claudin蛋白表达量显著降低(P<0.05);与模型组比较,电针组TRPV6和NCX1的表达量降低(P<0.05),TRPV5、PMCA1b、ZO-1、occludin和claudin蛋白表达量显著升高(P<0.05)。结论:电针能提高去卵巢大鼠的骨密度,增强参与钙离子吸收跨细胞途径的TRPV5、PMCA1b的mRNA和蛋白表达以及细胞旁途径ZO-1、occludin的mRNA和蛋白表达,这可能是针灸防治骨质疏松症的机制之一。

Objective To observe the effect of electroacupuncture(EA)on bone mineral density(BMD)and expression of calcium transport-related receptors in ovariectomized rats,so as to explore its mechanisms underlying improvement of osteoporosis.Methods Thirty-two three-month female SD rats were randomly divided into control,sham operation,model and EA groups(n=8 in each group).The ovariectomy model was established by resection of bilateral ovaries.EA(2 Hz/15 Hz,2 mA)was applied to"Guanyuan"(CV4)+"Sanyinjiao"(SP6)or"Shenshu"(BL23)+"Housanli"(ST36)alternatively for 20 min,once daily(except the weekends)for 12 weeks.BMD of the tibia and femur were detected by using a bone densitometer.The expression levels of transient receptor potential vanillic receptor 5(TRPV5),transient receptor potential vanillic receptor 6(TRPV6),sodium calcium exchanger 1(NCX1),membrane calcium adenosine triphosphate hydrolase(PMCA1 b),zonula occludens 1(ZO-1),occludin and claudin in intestinal mucosa were detected by quantitative real-time PCR and Western blot,respectively.Results Following modeling,BMD of both tibia and femur,and expression levels of TRPV5,PMCA1 b,ZO-1,occludin and claudin mRNAs were significantly decreased in the model group compared with the control and sham operation groups(P<0.05,P<0.01),while the mRNAs levels of TRPV6 and NCX1 in the model group were increased(P<0.01).After the treatment,BMD of both tibia and femur,and expression levels of TRPV5,PMCA1 b,ZO-1 and occludin mRNAs were significantly increased(P<0.01,P<0.05),and TRPV6 and NCX1 were significantly decreased(P<0.01,P<0.05)in the EA group than those in the model group.Compared with the control and sham operation groups,protein expression levels of TRPV5,PMCA1 b,ZO-1,occludin and claudin in the model group were significantly decreased(P<0.05),while the protein levels of TRPV6 and NCX1 in the model group were increased(P<0.05).After EA intervention,modeling-induced decrease of TRPV5,PMCA1 b,ZO-1,occludin and claudin protein levels and increase of TRPV6 and NCX1 protein levels were all completely reversed in the EA group relevant to the model group(P<0.05).Conclusion EA intervention can improve bone mineral density and up-regulate the mRNA and protein expression of TRPV5,PMCA1 b,ZO-1 and occludin,which may be one of the mechanisms of acupuncture in the treatment of osteoporosis.