血糖波动对糖尿病大鼠肾脏氧化应激与凋亡的影响

Effects of oscillating blood glucose level on oxidative stress and apoptosis in the kidney of diabetic rats

目的观察血糖波动对肾小管上皮细胞凋亡的影响及氧化应激的作用。方法体外培养人肾小管上皮细胞（HK-2），给予稳定高糖或波动高糖干预，四甲基偶氮唑盐比色法测定细胞增殖。以SD大鼠建立链脲佐菌素糖尿病模型，并错时腹腔注射胰岛素及葡萄糖，造成血糖波动模型。12周后，测24h尿蛋白、血尿素氮、肌酐、超氧化物岐化酶（SOD）及丙二醛，免疫组化测肾组织NOX-4、Bcl2和Bax蛋白，TUNEL法测。肾小管上皮细胞凋亡，观察超微结构改变。结果与稳定高糖比较，波动高糖更加明显抑制体外肾小管上皮细胞增殖；与稳定高血糖比较，血糖波动大鼠血尿素氮、肌酐、24h尿蛋白、丙二醛升高而SOD下降[（21．50±1．72对12．50±1．85）mmol／L，（97．51±7．84对82．12±11．48）μmol／L，（1．57±0．09对1．04±0．12）mg／24h，（23．50±1．87对14．82±2．96）nmol／ml，（17．22±1．12对21．11±1．80）U／ml，均P〈0．05]，Bcl2蛋白表达减少而Bax、NOX-4增加，凋亡细胞数增加，病理损伤加重。结论与稳定高糖比较，波动高糖明显抑制肾小管上皮细胞增殖，加速。肾小管上皮细胞凋亡，其机制可能与氧化应激水平增高有关。

Objective To explore the effects of oscillating blood glucose on apoptosis in renal tubular epithelial cells of diabetic rats and the role of oxidative stress. Methods Renal tubular epithelial cells （HK-2） were cultured in vitro with stable high glucose or oscillating high glucose, and MTT assay was applied to the measurement of cell proliferation. Streptozotoein induced diabetic model was established with SD rats and the oscillating high blood glucose animal model was induced by intraperitoneal injection of insulin and glucose at different time points every day. 12 weeks later 24 h urine protein （ 24hUP ）, blood urea nitrogen （ BUN ）, serum creatinine （Scr） , superoxide dismutase （SOD）, and malondialdehyde （MDA） were determined. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （ TUNEL）, and immunohisto-ehemistry was used to detect apoptosis associated gene Bax , Bcl-2, and NOX-4 expression in kidney. Changes in uhrastrueture were observed. Results Oscillating high glucose may inhibit renal cells proliferation obviously when comparing with stable high glucose. In the rats with oscillating blood glucose rather than those with stable high blood glucose, there was a significant increase of BUN, Scr, 24hUP, and MDA and a decrease of SOD [ （21.50 ± 1.72 vs 12.50 ± 1, 85 ） mmol/L, （97.51 ±7.84 vs 82.12±11.48 ） μmol/L, （ 1.57 ±0.09 vs 1.04 ± 0.12） mg/24 h, （ 23.50 ± 1.87 vs 14.82± 2.96） nmol/ml, （ 17.22 ± 1.12 vs 21.11 ± 1.80） U/ml, all P〈0.05 ] ; cell apoptosis was intensified with the up-regulation of Bax and NOX-4 protein expression and down-regulation of Bcl-2 in glomerular endothelial cells ; and more severe pathological damages were observed. Conclusion Comparing with stable high blood glucose, oscillating high blood glucose induces more apoptosis and less proliferation of renal tubular epithelial cells and the mechanism may be related to the increased oxidative stress.