玉米须多糖对D-半乳糖致衰老小鼠肾脏线粒体ATP合成的影响及机制

Study on the effect of polysaccharide from stigma maydison on ATP synthesis in renal mitochondria and its mechanism in D-galactose-induced aging mice

目的探讨玉米须多糖(SMPS)对D-半乳糖(D-gal)致衰老小鼠肾脏线粒体ATP合成的影响,并阐明其可能的作用机制。方法采用颈背部皮下注射D-gal溶液的方法建立衰老小鼠模型。将48只SPF级雄性小鼠随机分为正常对照组(Control组)、D-gal模型组(D-gal组)、玉米须多糖低剂量组(30 mg/kg,SMPS-L组)、玉米须多糖高剂量组(60 mg/kg,SMPS-H组),每组12只。干预42 d后全麻下眼球取血,检测血清中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛;取肾脏组织,荧光素酶法检测各组小鼠肾组织中ATP的含量;实时荧光定量PCR法检测各组小鼠肾组织中线粒体氧化呼吸链中复合体Ⅱ的琥珀酸脱氢酶(SDH)、复合体Ⅲ的细胞色素C还原酶(Cycs)、复合体Ⅳ(COXⅣ)及ATP合酶中的ATP5b mRNA表达水平;Western blot法检测各组小鼠肾组织中线粒体融合蛋白2(MFN2)、动力相关蛋白1(DRP1)和线粒体自噬相关蛋白(P62)的表达水平。结果与Control组比较,D-gal组小鼠肾组织中ATP含量(178±4)×10-4μmol下降(P<0.01),SDH、Cycs、COXⅣmRNA表达水平无明显改变,ATP5b mRNA表达水平(0.67±0.01)下调(P<0.01),MFN2蛋白表达水平(0.29±0.02)降低(P<0.01),DRP1和P62蛋白表达水平(0.31±0.02、0.21±0.01)上升(P<0.01);与D-gal组比较,SMPS干预组小鼠肾组织中ATP含量(193±1)10-4μmol升高(P<0.01),ATP5b mRNA表达和MFN2蛋白表达(0.87±0.05、0.71±0.08)上升(均P<0.01),DRP1和P62蛋白表达(0.20±0.01、0.10±0.01)下调(均P<0.01)。结论SMPS可通过增加抗氧化酶的活性及上调ATP5b mRNA和MFN2蛋白的表达,下调DRP1和P62蛋白表达,改善衰老小鼠肾脏线粒体动态平衡紊乱,促进D-gal致衰老小鼠肾组织线粒体ATP的生成。

Objective To investigate the effect of Stigma Maydis Palysaccharide(SMPS)on ATP synthesis in kidney mitochondria of D-galactose-induced aging mice,and to clarify its possible mechanism.Methods The aging mouse model was established by subcutaneous injection of D-galactose solution in the back of the neck.The 48 SPF male mice were randomly divided into normal control group(control group),D-galactose model group(D-Gal group),SMPS low-dose group and SMPS high-dose group(n=12 for each).The control group was subcutaneously injected with 150 mg/kg normal saline on the back of the neck every day,while the other three groups were subcutaneously injected with 150 mg/kg of D-gal solution on the back of the neck every day.SMPS-L and-H dose groups were given 30 mg/kg and 60 mg/kg of SMPS solution by gavage at the same day of D-Gal injection.The control group and D-GAL group were given the same volume of normal saline daily by gavage for 42 days.Blood samples were collected from the eyeball under general anesthesia after 42 days of intervention for the detection of serum levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and MDA.After harvesting the kidney tissue,various tests were used to detect ATP content,the mRNA expression levels and protein expression levels in kidney.Luciferase assay was used to detect ATP content in renal tissue.Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of succinate dehydrogenase(SDH)of complexⅡ,cytochrome C reductase(Cycs)of complexⅢ,complexⅣ(COXⅣ)and ATP5b in ATP synthase in mitochondrial oxidative respiratory chain.Western blot was used to detect the expression levels of mitochondrial fusion protein 2(MFN2),dynamin-related protein1(DRP1)and mitochondrial autophagy related protein P62 in renal tissues of each group.Results Compared with control group,the activities of serum of SOD(116.53±10.01)U/mg and GSH-Px(127.58±8.74)μmol/L were significantly decreased in D-GAL group(both P<0.01),and serum MDA content(15.42±0.91)μmol/L increased significantly in D-GAL group(P<0.01).Compared with D-GAL group,the activities of SOD(152.80±9.29)U/mg and GSH-Px(274.07±10.73)μmol/L were significantly increased in SMPS intervention group(P<0.01),while the MDA content(8.10±0.66)μmol/L decreased significantly in SMPS intervention group(P<0.01).Compared with control group,the content of ATP(178±4)10-4μmol in D-gal group was significantly decreased(P<0.01),the mRNA expression levels of SDH,Cycs and COXⅣwere not significantly changed in D-gal group,and the mRNA expression level of ATP5b(0.67±0.01)was down-regulated in D-gal group(P<0.01),the expression of MFN2 protein(0.29±0.02)was significantly decreased in D-gal group(P<0.01),and the expression of DRP1 and P62 protein(0.31±0.02 and 0.21±0.01)was significantly increased in D-gal group(both P<0.01).Compared with the D-gal group,the ATP content(193±1)10-4μmol in the kidney tissue of the mice was significantly increased in SMPS intervention group(P<0.01),and the ATP5b mRNA expression and MFN2 protein expression(0.87±0.05 and 0.71±0.08)were significantly increased in SMPS intervention group(both P<0.01),DRP1 and P62 protein expressions(0.20±0.01 and 0.10±0.01)were significantly down-regulated in in SMPS intervention group(both P<0.01).Conclusions SMPS can improve the mitochondrial dynamic homeostasis disorder in aging mice by increasing the activity of antioxidant enzymes,up-regulating the expression of ATP5b mRNA and MFN2 protein,down-regulating the expression of DRP1 and P62 protein,and promoting the generation of mitochondrial ATP in D-gal-induced aging mice kidney tissue.