益气清热膏通过内质网应激途径保护氨基核苷嘌呤霉素大鼠模型足细胞损伤的机制研究

Yiqiqingregao Protects Podocyte Injury in Puromycin Aminonucleoside Nephrosis Rat Model by Inhibition of Endoplasmic Reticulum Stress

目的:研究益气清热膏对氨基核苷嘌呤霉素大鼠模型(PAN)蛋白尿水平、足细胞损伤的作用,并从调节内质网应激途径的主要分子伴侣:糖调节蛋白78(glucose regulated protein,GRP78)和GRP94的表达探讨益气清热膏的作用机制。方法:24只雄性Wistar大鼠按随机数字表分为假手术组、PAN模型组、益气清热膏组、蒙诺组,每组各6只。除假手术组予等量生理盐水外,其余各组均采用颈静脉插管注射嘌呤霉素氨基核苷(PA)(40 mg/kg)法制备PAN肾病大鼠模型。分别于造模前(0天)、造模后第9天和第14天检测大鼠24 h尿蛋白定量,第15天处死各组大鼠,取肾组织行电镜检查;Western Blot和实时定量逆转录多聚酶链反应(real time-PCR)法检测大鼠肾组织中GRP78和GRP94表达。结果:(1)与假手术组比较,益气清热膏可以降低造模后第9天和第14天大鼠24 h尿蛋白定量(P<0.01)。(2)超微病理:模型组出现广泛足突融合、消失,足突基底部肿胀,足细胞内线粒体肿胀,内皮窗孔凌乱。益气清热膏可以明显改善大鼠足细胞损伤。(3)real-time PCR法和Western blot检测肾皮质GRP78和GRP94的表达。模型组GRP78和GRP94的mRNA水平和蛋白水平显著升高,益气清热膏可以显著降低两者mRNA和蛋白表达水平(P<0.05)。结论:益气清热膏可显著降低PAN模型大鼠尿蛋白水平,修复足细胞损伤,其机制可能与益气清热膏降低肾组织GRP78和GRP94表达,从而抑制内质网应激,修复肾组织蛋白质合成有关,具体机制需要进一步研究证实。

Objective： To investigate the effect of Yiqiqingregao（ YQQRG） on proteinuria and podocyte injury in puromycin aminonucleoside nephrosis（ PAN） rat model,and discuss the mechanism of YQQRG through study of glucose regulated protein 78（ GRP 78） and GRP 94. Methods： 24 male Wistar rats were randomely divided into 4 groups： sham group,PAN model group,Yi Qi Qing Re Gao group（ YQQRG） and fosinopril group,each group with 6 rats. Rats of the experimental groups were performed with injection of puromycin aminonucleoside（ PA） at a dose of 40 mg / kg via the right jugular vein,while rats of the sham group were given an injection of 0. 9% normal saline at the same volume. Twenty- four hour urinary protein levels were measured before injection,9 and 14 days after injection. All rats were sacrificed at day 15. Renal tissue was obtained for electro- microscopic examination. The rest renal cortex tissue was prepared for Western blot and real- time PCR test of glucose regulated protein 78（ GRP78） and GRP94.Results：（ 1） Treatment of YQQRG decreased 24 hour urinary protein remarkably,by Day 9 and 14 after PA injection,compared with PAN model group（ P〈0. 01）.（ 2） Electro- microscopic examination： Diffuse podocyte foot process effacement,swelling of the basal part and mitochondrial of podocyte were observed in model group,and treatment with YQQRG eliminated podocyte damage.（ 3）Molecular biological results： Both mRNA and protein levels of GRP78 and GRP94 were significantly increased in the model group,and YQQRG reduced expression of GRP78 and GRP94 both at mRNA and protein levels. Conclusion： Yiqiqingregao can reduce proteiuria,eliminate podocyte damage in PAN rat model,which may be related to reduction of GRP78 and GRP94 expression and thus inhibition of endoplasmic reticulum（ ER） stress and restoration of normal protein senthesis,further study is needed to explore themechanism of regulatory effect YQQRG on ER stress.