糖尿病肾病模型大鼠中存在蛋白降解途径失调

Proteolytic system is dysfunctional in diabetic nephropathy model rats

目的研究蛋白酶体和自噬两条主要蛋白质降解途径在细胞体外及糖尿病肾病（DN）模型大鼠体内中的变化。方法原代培养SD大鼠肾小球系膜细胞，观察该细胞在高糖环境中的增殖情况。分别给予30mmol／L葡萄糖（HG组）、5．4mmol／L葡萄糖（NC组）孵育0h、8h、16h、72h，并用Western印迹法检测蛋白酶体途径蛋白酶体α各亚基（PSMAs）和自噬途径LC3蛋白的表达。以自发性2型DN模型OLETF大鼠和正常对照LETO大鼠作体内实验，每4周动态监测血糖、24h尿蛋白量，于36周龄处死大鼠，取肾组织，观察病理形态学并进行半定量评分；利用Western印迹法分析肾皮质中PSMAs和LC3蛋白的表达。结果与NC组相比，高糖环境下8h蛋白酶体α各亚基、LC3Ⅰ和LC3Ⅱ蛋白表达均降低，差异均有统计学意义（P〈0．05）。其他各时间点两组间差异无统计学意义。与正常对照LETO太鼠相比，OLETF大鼠血糖水平和24h尿蛋白量逐渐升高，差异有统计学意义（P〈0．01）。OLETF大鼠肾小球硬化指数和肾小管间质纤维化指数均显著高于LETO大鼠，差异有统计学意义（P〈0．01）；OLETF大鼠36周龄肾皮质中PSMAs蛋白表达明显减少，与正常对照LETO大鼠差异有统计学意义（P〈0．05），LC3Ⅱ蛋白表达也有降低趋势，但差异无统计学意义。结论蛋白降解途径失调可能在DN的发生、发展中起了一定的作用。

Objective To investigate autophagy and proteasome system alteration in vivo and in vitro of diabetic nephropathy （DN） model rats. Methods Rat glomerular mesangial cells were primaryly cultured, and cell proliferation was tested by MTT assay. The mesangial cells were cultured under different concentrations of glucose （5.4 mmol/L for normal control and 30 mmol/L for high glucose） for 0, 8, 16, 72 hours. The expression of autophagy （LC3） and proteasome （PSMAs） proteins was examined by Western blotting analysis. Spontaneous type 2 diabetes model OLETF and its normal control LETO rats were observed for 36 weeks. The levels of blood glucose and 24 hours urinary protein were evaluated in every 4 weeks. All the rats were sacrificed at the 36th week, and renal pathological changes were semi-quantitively analyzed. The expression of PSMAs and LC3 proteins was also examined in kidney cortex by Western blotting. Results Under high glucose concentrations, the abundance of PSMAs and LC3 proteins significantly reduced in the mesangial cells at 8 hours. There was no significant difference at other time points. The levels of blood glucose and 24 h urinary protein in OLETF rats exhibited progressive increase compared to those in LETO rats （all P〈0.01）. And glomerular sclerosis index and tubulointerstitial injury index were significantly higher than those in LETO rats （all P〈0.01）. The abundance of PSMAs proteins was significantly reduced in renal cortex of OLETF rats compared with LETO rats, while the abundance of LC3 proteins had no significant difference between two groups. Conclusion Proteolytic system dysfunction may play a role in pathogenesis of DN.