维甲酸受体介导全反式维甲酸对糖尿病肾病大鼠肾组织细胞增殖与凋亡的影响

目的 探讨维甲酸受体( RAR-α、RAR-β、RAR-γ)介导的全反式维甲酸(ATRA)对糖尿病肾病大鼠肾组织细胞增殖与凋亡的影响及机制.方法 健康雄性SD大鼠30只,随机选10只为空白对照组；链脲佐菌素(STZ)诱导糖尿病模型,造模成功后随机分为糖尿病模型组(n=10)和ATRA组(n=10).于造模成功第2天起ATRA组大鼠给予ATRA 10 mg·kg-1·d-1灌胃,对照组与糖尿病组均予等体积蒸馏水灌胃.于第8周及第12周观察生化指标及处死各组大鼠,取肾组织行HE染色观察肾脏病理变化；原位末端标记法(TUNEL法)检测各组肾组织细胞凋亡率；间接免疫荧光法检测各组肾组织中维甲酸受体RAR-α、β、γ的表达情况；免疫组化法检测各组肾组织中增殖指标I型胶原(CdI),层粘连蛋白(LN)及凋亡相关因子Smac、caspase-3的蛋白表达.实时定量PCR法检测各组肾组织中Smac、caspase-3 mRNA的表达.结果 与对照组比较,糖尿病组大鼠24h尿蛋白、Scr、BUN、肾质量/体质量比值均显著增加(均P＜0.05),而ATRA组上述指标较糖尿病组减少(均P＜0.05).ATRA组病理改变较糖尿病组减轻.12周时,与对照组比较,糖尿病组细胞凋亡率显著增加(P＜0.01),ATRA组较糖尿病组减少(P＜0.01).间接免疫荧光结果显示,12周时糖尿病组肾组织RAR-α、RAR-β阳性细胞数显著少于对照组(均P＜0.05),而ATRA组较糖尿病组显著增加(均P＜0.01)；各组肾组织中均未见RAR-γ表达变化.免疫组化结果显示,12周时糖尿病组肾组织肾小球系膜区中Col Ⅰ、LN、Smac、caspase-3蛋白的表达显著高于对照组(均P＜0.01),ATRA组较糖尿病组显著减少(均P＜0.01).实时定量PCR结果显示,与对照组比较,糖尿病组Smac、caspase-3 mRNA的表达均显著增多(均P＜0.01),并随时间延长表达增强；ATRA组较糖尿病组显著减少(P＜0.01).结论 全反式维甲酸可能通过其受体介导途径抑制糖尿病肾病肾组织细胞增殖及凋亡,对糖尿病肾病有保护作用.

Influence of retinoic acid receptor-mediated all-trans retinoic acid on renal tissue cell proliferation and apoptosis in rats with diabetic nephropathy

Objective To investigate the influence of retinoic acid receptor (RAR-α, RAR-β and RAR-γ)-mediated all-trans retinoic acid (ATRA) on renal tissue cell proliferation and apoptosis in rats with diabetic nephropathy, and to analysze the possible mechanism. Methods Male SD rats were randomly divided into normal control group (group N, n=10) and diabetic model group (n=20). Diabetes was induced by streptozotocin(STZ) injection. After successful modeling, the model rats were randomly divided into diabetes group (group D, n=10) and ATRA treatment group (group T, n=10). Rats in group T received ATRA 10 mg•kg-1•d-1 by gavage from the 2nd day of successful modeling for 8 or 12 weeks, meanwhile group N and group D received same volume distilled water. In each group, 5 rats were sacrificed respectively at the 8th week or the 12th week, then biochemical markers were measured and kidney pathology was examined. Apoptosis index(AI) of renal tissue cells of each group was tested by TUNEL. The expressions of RAR-α, RAR-β and RAR-γ in renal tissues were tested using indirect immunofluorescence. The expressions of typeⅠ collagen and laminin as proliferation indicators, along with Smac and caspase-3 as the correlated factors of apoptosis in renal tissue of each group were tested by immunohistochemistry staining. The mRNA expressions of Smac and caspase-3 were tested using real-time fluorescence quantitative PCR. Results Compared with group N, 24 h urine protein, serum creatinine, blood urea nitrogen, ratio of kidney weight/body weight increased significantly (P<0.05, respectively) in group D, and further increased with observation time. Compared with the group D, 24 h urine protein and ratio of kidney weight/body weight decreased in group T (P<0.05, respectively). Compared with group D, the group T presented minor pathological changes. TUNEL assay indicated that compared with group N, the group D showed an obvious increase in renal cell apoptosis in time-dependent manner, and the group T showed a decrease compared with the group D (P<0.01, respectively). Compared with group N, the expression of RAR-α and RAR-β positive cells number in group D were decreased (P<0.01, respectively). Compared with group D, the expression of RAR-α and RAR-β positive cells number in group T increased (P<0.01, respectively). Renal tissues of each group did not show expressions of RAR-γ. After 12 weeks, compared with group N, expressions of type-Ⅰ collagen, laminin, Smac and caspase-3 protein in the glomerular mesangial area and basement membrane of renal tissues in group D increased significantly (P<0.01, respectively), and enhanced with time. Compared with the group D, expressions of typeⅠ collagen, laminin, Smac and caspase-3 protein in group T decreased (P<0.01, respectively). Compared with the group N, group D had an obvious increase in the mRNA expressions of Smac and caspase-3, and a significantly decrease in group T (P<0.01, respectively). Conclusions ATRA may prevent the cell proliferation and apoptosis in diabetic renal tissue through its receptor-mediated pathway, and may protect rats against diabetic nephropathy.