【摘要】 目的 探讨糖蛋白非转移性黑色素瘤B(GPNMB)在糖尿病肾脏疾病(DKD)足细胞衰老和损伤中的作用及机制。方法 将12只6周龄C57BL/6J雄性小鼠，按照随机原则分为对照组和DKD组(每组6只)，通过腹腔注射链脲佐菌素(STZ)，构建DKD模型，采用免疫荧光共染法检测小鼠肾脏组织中GNPMB的表达。体外培养永生化人足细胞，构建高糖刺激诱导的人足细胞损伤模型，将其分为CON组、HG组、HO组、CON+si-VEC组、si-GPNMB组、HG+si-VEC组与HG+si-GPNMB组，采用蛋白质印迹法(Western Blot)检测GPNMB、人足细胞损伤相关蛋白(Desmin、Synaptopodin、ZO-1)、细胞衰老相关蛋白(P53、P21、P16)及衰老相关分泌表型(SASP)炎症因子(IL-1β、IL-6、TNF-α);采用免疫荧光法检测人足细胞线粒体活性氧水平与DNA损伤;采用β-半乳糖苷酶(β-Gal)染色试剂盒检测人足细胞衰老水平。结果 Western Blot结果显示，DKD组小鼠肾小球中GPNMB蛋白表达水平显著高于CON组(P<0.05);免疫荧光共染显示，GPNMB与定位于足突的足细胞标志蛋白Synaptopodin共定位;与CON组比较，DKD组小鼠足细胞内Synaptopodin的荧光信号显著减弱，同一部位GPNMB则明显上调。HG组GPNMB蛋白、mRNA表达水平及蛋白免疫荧光信号均显著高于CON组和HO组; si-GPNMB组GPNMB蛋白及mRNA表达水平均显著低于CON组(P<0.05)。HG组人足细胞损伤相关指标Synaptophysin与ZO-1蛋白表达水平均显著低于CON组和HG+si-GPNMB组，Desmin蛋白表达水平、P53、P21、P16及IL-1β、IL-6、TNF-α蛋白表达水平均显著高于CON组和HG+si-GPNMB组(P<0.05)。免疫荧光染色结果显示，与CON组相比，HG组衰老相关β-Gal(SA-β-Gal)阳性细胞比例升高，线粒体活性氧显著增强，γ-H2AX阳性数量显著增加。结论 GPNMB可能通过影响细胞衰老参与高糖诱导的人足细胞损伤，表明其可作为DKD的潜在治疗靶点。更多还原

【Abstract】 Objective To investigate the role and mechanism of glycoprotein non-metastatic melanoma protein B( GPNMB) in podocyte senescence and injury in diabetic kidney disease( DKD).Methods Twelve 6-week-old male C57BL/6J mice were randomly divided into control group and DKD group,6 mice in each group. The DKD model was established by intraperitoneal injection of streptozotocin( STZ). The expression of GNPMB in the mouse kidney tissue was detected by immunofluorescence costaining. In vitro,an immortalized human podocyte line was subjected to high-glucose-induced injury model.Cells were divided into CON,HG,HO,CON + si-VEC,si-GPNMB,HG + si-VEC and HG + si-GPNMB groups. Protein levels of GPNMB,human podocyte injury-related proteins( Desmin,Synaptopodin,ZO-1),senescence-related proteins( p53,p21,p16) and senescence-associated secretory phenotype( SASP)inflammatory factors( IL-1β,IL-6,TNF-α) were measured by western blotting. Immunofluorescence was used to detect mitochondrial reactive oxygen species levels and DNA damage in human podocytes. β-galactosidase( β-Gal) staining kits were employed to assess senescence levels in human podocytes. Results Western blot analysis revealed significantly higher GPNMB protein expression levels in glomeruli of DKD group mice compared to CON group( P < 0. 05); Immunofluorescence co-staining revealed colocalisation of GPNMB with synaptopodin,a podocyte marker localised to foot processes. Compared with CON group,fluorescence signals for synaptopodin within podocytes were markedly diminished in DKD group,whilst GPNMB expression at the same sites was significantly upregulated. In HG group,GPNMB protein and mRNA expression levels,as well as immunofluorescence signals were significantly higher than those in both CON and HO groups. In si-GPNMB group,GPNMB protein and mRNA expression levels were significantly lower than those in CON group( P < 0. 05). In HG group,the expression levels of the human foot cell damage-related markers Synaptophysin and ZO-1 protein were significantly lower than those in CON group and HG + si-GPNMB group. The expression levels of Desmin protein,P53,P21,P16,IL-1β,IL-6 and TNF-α protein expression levels were significantly higher than those in CON and HG + siGPNMB groups( P < 0. 05). Immunofluorescence staining revealed that,compared with CON group,HG group exhibited an increased proportion of senescence-associated β-Gal( SA-β-Gal) positive cells,significantly enhanced mitochondrial reactive oxygen species and a significantly increased number of γ-H2AX positive cells. Conclusion GPNMB may participate in high-glucose-induced human podocyte injury by mediating cellular senescence,suggesting its potential as a therapeutic target for DKD.更多还原