金匮肾气丸对自然衰老大鼠逼尿肌细胞M3R、β3—ARmRNA及其蛋白表达的影响(1)
【摘 要】 目的：观察金匮肾气丸对自然衰老大鼠逼尿肌细胞M3R、β3-AR mRNA和蛋白表达的影响。方法：以多尿自然衰老大鼠为“肾虚多尿”模型，给予金匮肾气丸4周，采用酶消化和组织块相结合的方法原代培养逼尿肌细胞，用实时荧光定量PCR法和Westernblotting法观察金匮肾气丸对自然衰老大鼠逼尿肌细胞M3R、β3-AR mRNA及其蛋白表达的影响。结果：与正常对照组比较，自然衰老大鼠逼尿肌细胞M3R mRNA和蛋白的表达明显增加；与模型对照组比较，金匮肾气丸可以抑制自然衰老大鼠逼尿肌细胞M3R mRNA和蛋白的表达；与正常对照组比较，自然衰老大鼠逼尿肌细胞β3-AR mRNA和蛋白表达明显降低；与模型对照组比较，金匮肾气丸可以增加自然衰老大鼠逼尿肌细胞β3-ARmRNA和蛋白的表达。结论：金匮肾气丸可以减少自然衰老大鼠膀胱逼尿肌细胞M3RmRNA及其蛋白的表达和增加自然衰老大鼠膀胱逼尿肌细胞β3-ARmRNA及其蛋白的表达，使M3R和β3-AR之间的平衡状态维持正常的水平。
【关键词】 金匮肾气丸；自然衰老大鼠；M3R；β3-AR
【中图分类号】R285.5 【文献标志码】 A 【文章编号】1007-8517(2016)12-0050-03
Abstract：Objective To observe the impacts of “Jinkui Shenqi pill” on the expression of M3R and β3-AR mRNA and corresponding proteins in the Bladder detrusor cells of Natural aging rat. Methods Natural aging rats were used as “kidney deficiency and urinary” model， then were treated with “Jinkui Shenqi pill”. for 4 weeks and Detrusor cells were cultured by the combination of Enzyme digestion and tissue.The expression of M3R and β3-AR mRNA in the bladder detrusor cells by realtime fluorescence quantitative PCR method was investigated. The expression of M3R and β3-AR proteins in the bladder detrusor cells by Westernblotting was investigated.Results In model rats， the expression of M3R mRNA and corresponding proteins increased and the expression of β3-ARmRNA and corresponding proteins decreased in the Bladder detrusor cells. “Jinkui Shenqi pill” could decreased the expression of M3R mRNA and corresponding proteins，increased the expression of β3-AR mRNA and corresponding proteins in the bladder detrusor cells of Natural aging rat.Conclusion Jinkui Shenqi pill”could make M3R mRNA and corresponding proteins expression of detrusor cells of the natural aging rats decreased，and β3-AR mRNA and corresponding proteins expression of detrusor cells of the natural aging rats increased.
Keywords：Jinkui Shenqi pill； natural aging rat ； M3R；β3-AR
金匮肾气丸由地黄、茯苓、山药、山茱萸、牡丹皮、泽泻、桂枝、牛膝、车前子、附子等药材组成，具有温补肾阳、化气行水的功效，用于肾虚水肿、腰膝酸软、小便不利、畏寒肢冷。膀胱的生理功能是贮存和排尿，尿液在膀胱内贮存到一定限度时，即可及时自主地排出体外[1]。β3受体直接介导逼尿肌松弛，M3受体直接参与了逼尿肌收缩。膀胱逼尿肌的舒张和收缩功能异常可直接导致下尿路功能症状的出现，其发病率随年龄增加而上升[2]。研究在自然衰老大鼠模型上观察M3R、β3-AR mRNA及其蛋白的表达变化及金匮肾气丸对其的影响，以揭示金匮肾气丸影响尿功能的分子机制。
1 仪器与材料
1.1 实验动物 SD自然衰老大鼠(15月龄)，SPF级，雌雄各25只，雌鼠体重320～400g，雄鼠体重440～520g；SD大鼠(3月龄)，SPF级，雌雄各5只，雌鼠体重160～180g，雄鼠体重200～220g，由广州中医药大学实验动物中心提供，许可证号：SCXK(粤)2008-0020，合格证号：0089418，0086829，0086847。
1.2 实验药物 金匮肾气丸(批号：0030377，北京同仁堂科技股份有限公司)，参照Bios氏原法折算大鼠用药剂量，以临床用量折算等效剂量为中剂量，低、中、高剂量分别为0.54、1.08、2.16g/(kg·d)。缩泉胶囊(批号：100302，湖南汉森制药有限公司)，以临床用量折算等效剂量为中剂量，为 0.585 g/(kg·d)。 (鲁湘鄂 吴清和)
【关键词】 金匮肾气丸；自然衰老大鼠；M3R；β3-AR
【中图分类号】R285.5 【文献标志码】 A 【文章编号】1007-8517(2016)12-0050-03
Abstract：Objective To observe the impacts of “Jinkui Shenqi pill” on the expression of M3R and β3-AR mRNA and corresponding proteins in the Bladder detrusor cells of Natural aging rat. Methods Natural aging rats were used as “kidney deficiency and urinary” model， then were treated with “Jinkui Shenqi pill”. for 4 weeks and Detrusor cells were cultured by the combination of Enzyme digestion and tissue.The expression of M3R and β3-AR mRNA in the bladder detrusor cells by realtime fluorescence quantitative PCR method was investigated. The expression of M3R and β3-AR proteins in the bladder detrusor cells by Westernblotting was investigated.Results In model rats， the expression of M3R mRNA and corresponding proteins increased and the expression of β3-ARmRNA and corresponding proteins decreased in the Bladder detrusor cells. “Jinkui Shenqi pill” could decreased the expression of M3R mRNA and corresponding proteins，increased the expression of β3-AR mRNA and corresponding proteins in the bladder detrusor cells of Natural aging rat.Conclusion Jinkui Shenqi pill”could make M3R mRNA and corresponding proteins expression of detrusor cells of the natural aging rats decreased，and β3-AR mRNA and corresponding proteins expression of detrusor cells of the natural aging rats increased.
Keywords：Jinkui Shenqi pill； natural aging rat ； M3R；β3-AR
金匮肾气丸由地黄、茯苓、山药、山茱萸、牡丹皮、泽泻、桂枝、牛膝、车前子、附子等药材组成，具有温补肾阳、化气行水的功效，用于肾虚水肿、腰膝酸软、小便不利、畏寒肢冷。膀胱的生理功能是贮存和排尿，尿液在膀胱内贮存到一定限度时，即可及时自主地排出体外[1]。β3受体直接介导逼尿肌松弛，M3受体直接参与了逼尿肌收缩。膀胱逼尿肌的舒张和收缩功能异常可直接导致下尿路功能症状的出现，其发病率随年龄增加而上升[2]。研究在自然衰老大鼠模型上观察M3R、β3-AR mRNA及其蛋白的表达变化及金匮肾气丸对其的影响，以揭示金匮肾气丸影响尿功能的分子机制。
1 仪器与材料
1.1 实验动物 SD自然衰老大鼠(15月龄)，SPF级，雌雄各25只，雌鼠体重320～400g，雄鼠体重440～520g；SD大鼠(3月龄)，SPF级，雌雄各5只，雌鼠体重160～180g，雄鼠体重200～220g，由广州中医药大学实验动物中心提供，许可证号：SCXK(粤)2008-0020，合格证号：0089418，0086829，0086847。
1.2 实验药物 金匮肾气丸(批号：0030377，北京同仁堂科技股份有限公司)，参照Bios氏原法折算大鼠用药剂量，以临床用量折算等效剂量为中剂量，低、中、高剂量分别为0.54、1.08、2.16g/(kg·d)。缩泉胶囊(批号：100302，湖南汉森制药有限公司)，以临床用量折算等效剂量为中剂量，为 0.585 g/(kg·d)。 (鲁湘鄂 吴清和)