积雪草酸对糖尿病大鼠肾脏氧化应激及细胞凋亡的影响

Protective effect of asiatic acid on renal oxidative stress and apoptosis in diabetic rats

目的探讨积雪草酸对链脲佐菌素诱导的糖尿病大鼠肾脏氧化应激及细胞凋亡的影响。方法29只清洁级健康雄性sD大鼠（体质量170～210g，2～3月龄）腹腔注射65μg／g链脲佐菌素，建模成功后将存活的24只大鼠按数字表法随机分至糖尿病组（n=6）、10μg·g^-1·d^-1。积雪草酸组（n=6）、20μg·g^-1·d^-1积雪草酸组（n=6）、40μg·g^-1·d^-1积雪草酸组（n=6）。另以6只健康SD大鼠为正常对照组。干预8周后观察各组大鼠尿白蛋白排泄率、血糖、血尿素氮、血肌酐变化；检测。肾皮质中超氧化物歧化酶、丙二醛水平；采用原位末端标记法检测肾脏细胞凋亡情况，免疫组织化学法检测肾脏聚ADP-核糖多聚酶表达水平，Westernblot法检测半胱氨酸蛋白酶3表达水平。应用单因素方差分析和LSD法进行数据统计。结果与正常对照组相比，糖尿病组大鼠尿白蛋白排泄率、血糖、血尿素氮、血肌酐显著升高，肾皮质超氧化物歧化酶活性显著下降，丙二醛含量显著升高，肾脏细胞凋亡率、聚ADP-核糖多聚酶表达和半胱氨酸蛋白酶3蛋白表达水平显著升高（分别为31．0％±5．5％VS5．0％±1．2％、3．9±0．5VS1．7±0．6、1．7±0．4vs0．4±0．3，t值分别为0．84、7．45、6．14，均P〈0．01）。与糖尿病组相比，3个积雪草酸干预组尿白蛋白排泄率、血尿素氮、血肌酐明显降低，超氧化物歧化酶活性明显上升，丙二醛含量明显下降，。肾脏细胞凋亡率明显减少（糖尿病组为31．0％±5．5％，10μg·g^-1·d^-1积雪草酸组为20．6％±4．7％，20μg·g^-1·d^-1积雪草酸组为15．8％±2．6％，40μg·g^-1·d^-1积雪草酸组为10．3％±3．3％），聚ADP-核糖多聚酶表达明显下调（糖尿病组为3．9±0．5，10μg·g^-1·d^-1积雪草酸组为3．0±0．2，20μg·g^-1·d^-1积雪草酸组为2．6±0．4，40μg·g^-1·d^-1积雪草酸组为2．0±0．3），半胱氨酸蛋白酶3蛋白表达下调（糖尿病组为1．7±0．3，10μg·g^-1·d^-1积雪草酸组为1．0±0．2，20μg·g^-1·d^-1积雪草酸组为0．7±0．2，40μg·g^-1·d^-1积雪草酸组为0．5±0．3）。结论积雪草酸对链脲佐菌素诱导的糖尿病大鼠肾脏病变有一定保护作用，其机制可能与氧化应激反应和细胞凋亡被抑制有关。

Objective To evaluate the effect of asiatic acid （AA） on renal oxidative stress and apoptosis in streptozotocin （STZ）-induced diabetic rats. Methods After the diabetic models were successfully established by intraperitoneal injection of 65 μg/g STZ, 24 living SD rats were randomly assigned to 4 groups, including diabetes mellitus group （ DM group） , 10 μg·g^-1·d^-1 AA group, 20 μg·g^-1·d^-1 AA group, and 40 μg·g^-1·d^-1 AA group. Another 6 healthy SD rats were used as normal controls （NC group）. At 8 weeks, urinary albumin excretion rate （UAER）, blood glucose （BG）, blood urea nitrogen （ BUN ） , serum creatinine （ SCr ） , superoxide dismutase （ SOD ） activity, and malondialdehvde （MDA） level were tested. Apoptotic cells in the kidneys were observed by TdT-mediated dUTP nick end labeling （TUNEL）, the expression of poly-ADP-fibose polymerase （PARP） was detected by immunohistochemistry, and the apoptosis-related protein cystein asperate proteinase-3 （Caspase-3） was tested by Western blot. One-way of variance and LSD t test were used for data analysis. Results Compared with the NC group, the levels of UAER, BG, BUN, SCr, and MDA were significantly increased, the activity of SOD was significantly decreased, and the number of apoptotic cells and the expression of PARP anti Caspase-3 were significantly increased in DM group （31.0% ±5.5% vs 5.0% ± 1.2%, 3.9±0. 5 vs 1.7 ± 0. 6, 1.7 ± 0. 4 vs 0.4± 0. 3, respectively; all P 〈 0. 01 ）. Compared with DM group, UAER, BUN and SCr were improved, the activity of SOD was significantly increased, the level of MDA was reduced, and the number of apoptotic cells （DM group： 31.0% ±5.5%; 10 μg·g^-1·d^-1 AA group： 20. 6% ±4. 7%; 20 μg·g^-1·d^-1AA group： 15.8%±2.6%; 40 μg·g^-1·d^-1 AA group： 10.3% ±3.3%） and the expressions ofPARP （DMgroup： 3.9±0.5; 10 μg·g^-1·d^-1 AAgroup： 3.0±0.2; 20 μg·g^-1·d^-1 AA group： 2. 6 ± 0. 4; 40 μg·g^-1·d^-1 AA group： 2. 0 ± 0.3） and apoptosis-related protein Caspase-3 （DMgroup： 1.7 ±0.3; 10 μg·g^-1·d^-1AA group： 1.0±0,2; 20 μg·g^-1·d^-1 AA group： 0.7 ± 0. 2; 40 μg·g^-1·d^-1 AA group： 0. 5 ± 0. 3 ） were reduced in all 3 AA intervention groups. Conclusion AA may play a protective role in STZ-induced diabetic nephropathy, which could be related to the inhibition of oxidative stress and apoptosis.