【摘要】 目的:观察大鼠肾缺血再灌注(I/R)后尿中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)浓度、肾组织缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和NGAL的表达以及冬虫夏草(Cordyceps sinensis,C.sinensis)干预后对其的影响,探讨C.sinensis的肾保护作用机制。方法:健康雄性SD大鼠45只随机分为假手术(sham)组、I/R组和C.sinensis组,每组15只。3组组内再按24,48,72 h分成3个亚组,每组5只。用摘除右肾后夹闭左肾蒂1 h恢复再灌注的方法建立肾I/R模型,sham组摘除右肾后未夹闭左肾蒂,术后每日予以生理盐水(2 mL/d)灌胃,I/R组和C.sinensis组分别在术后予以生理盐水(2 mL/d)和人工C.sinensis粉[5.0 g/(kg.d)]灌胃,在相应时间点处死大鼠。检测各组大鼠血尿素氮(BUN)和血肌酐(SCr),比色法检测各组大鼠的尿中N-乙酰-β-D-氨基酸葡萄糖苷酶(NAG)含量,ELISA法检测各组大鼠尿NGAL浓度,HE染色观察大鼠肾小管间质病理变化,并用肾小管间质半定量计分法分析各组大鼠病理损伤情况,RT-PCR检测各组大鼠肾组织中HIF-1αmRNA的表达水平,免疫荧光共聚焦法观察各组大鼠肾组织HIF-1α和NGAL蛋白的表达情况。结果:与sham组相比,I/R组和C.sinensis组大鼠的BUN及SCr水平增高(P<0.05),尿中NAG和NGAL增多(P<0.05)。Sham组大鼠肾小球及小管间质均正常,无明显病理改变;I/R组出现肾小管上皮细胞普遍肿胀,可见小管扩张,管腔管型,刷状缘消失,部分上皮细胞变性坏死等改变,肾小球无明显病理改变。与sham组相比,I/R组和C.sinensis组各时间点大鼠肾组织HIF-1α的mRNA及蛋白、NGAL蛋白的表达均明显升高(P<0.05)。与I/R组相比较,C.sinensis组各时间点的BUN和SCr水平下降(P<0.05),尿中NAG和NGAL浓度降低(P<0.05),肾组织NGAL蛋白的表达下调(P<0.05),HIF-1αmRNA及其蛋白的表达上调(P<0.05)。肾小管病理半定量计分显示C.sinensis组较I/R组小管间质损伤明显改善。结论:I/R大鼠模型病理改变表现为肾小管及肾间质受损,与急性肾损伤病理改变特征相符合。I/R后尿中NGAL水平增高,与尿中NAG及肾小管间质损伤程度成正相关,提示NGAL可能是急性肾损伤时肾小管损伤特异性的生物标志物。C.sinensis可能通过调节肾组织中HIF-1α和NGAL的表达而达到肾保护作用。更多还原

【Abstract】 Objective: To observe the level of urinary neutrophil gelatinase-associated lipocalin(NGAL),the expression of hypoxia inducible factor-1α(HIF-1α) and NGAL in rat kidney after renal ischemia and reperfusion(I/R),before and after the treatment with Cordyceps Sinensis(C.sinensis),and to explore the mechanism of C.sinensis against I/R injury. Methods: A total of 45 healthy male Sprague-Dawley rats were randomly divided into a sham group,a renal I/R model group,and a C.sinensis group(15 in each group).The rats in the sham group and the renal I/R model group were intragastrically administered saline(2 mL/d),and rats in the treatment group were intragastricabby administered of C.sinensis [5.0 g/(kg·d)].The rats were sacrificed at 24,48,and 72 h,respectively after the reperfusion and urinary N-acetyl-β-D-glucosaminidase(NAG) level was measured,renal function in rats was detected,and the pathological changes were observed with HE staining.We determined the urinary NGAL levels in the rats by ELISA,the expression of HIF-1α mRNA by RT-PCR,and the expressions of HIF-1α and NGAL proteins by confocal immunofluorescence. Results: Compared with the sham group,the levels of BUN,SCr,levels of NAG and NGAL in urine were increased in the I/R group and the C.sinensis group,reached a peak at 24 h after the reperfusion and slowly declined at 48 and 72 h.Glomerular and tubulointerstitial areas in the sham group did not show any pathological change.Induced pathological changes included tubular cell necrosis,focal areas of proximal tubular dilation,distal tubular casts,effacement and loss of proximal tubule brush border,etc.Compared with the sham group,the expression of HIF-1α and NGAL in the kidney tissues of the I/R group and the C.sinensis group increased.C.sinensis can lower the level of NAG and NGAL in the urine and the expression of NGAL protein in the kidney tissues.It up-regulated the expression of HIF-1α mRNA and protein in the kidney tissues whilst attenuated the pathological changes. Conclusion: Renal I/R injury in rats can lead to pathological changes in renal tubular epithelial cells and renal interstitial damage,which are consistent with the pathological features of acute kidney injury(AKI).The level of urinary NGAL increases after the I/R,and positively correlates with the level of urinary NAG and pathological changes,suggesting that urinary NGAL may serve as a urinary biomarker for specific detection of tubular injury in AKI.C.sinensis can attenuate the renal I/R-induced AKI.Its mechanism may be associated with up-regulating the expression of HIF-1α and down-regulating the expression of NGAL in the kidney tissues.更多还原