富亮氨酸α-2糖蛋白1增强间充质干细胞对急性肾损伤的疗效研究

LRG1 enhanced the efficacy of mesenchymal stem cells in the treatment of acute kidney injury

目的探究富亮氨酸α-2糖蛋白1(LRG1)预处理间充质干细胞(MSCs)对急性肾损伤(AKI)疗效的影响。方法8周龄雄性C57BL/6小鼠随机分为4组:假手术组(Sham组)、缺血再灌注损伤组(IRI组)、MSCs组和LRG1-MSCs组。采用左侧肾蒂夹闭30 min,右侧肾切除的方法构建肾单侧IRI模型。Sham组不予治疗,IRI组尾静脉注射等体积磷酸盐缓冲液(PBS),MSCs组尾静脉注射MSCs,LRG1-MSCs组尾静脉注射LRG1预处理的MSCs。术后3 d检测各组小鼠的血清肌酐(Scr)、血尿素氮(BUN)水平。肾脏病理PAS染色后,通过急性肾小管坏死(ATN)评分评估肾损伤情况。Western印迹检测肾组织肾损伤分子-1(KIM-1)蛋白的表达水平。体外实验,将人近端肾小管上皮细胞(HK-2 cells)分为4组:对照组(Control组,正常HK-2细胞)、缺氧/复氧组(H/R组,H/R诱导的HK-2细胞)、MSCs组(H/R诱导的HK-2细胞与MSCs共培养)和LRG1-MSCs组(H/R诱导的HK-2细胞与LRG1-MSCs共培养)。Western印迹检测HK-2细胞凋亡相关蛋白(Bax、Bcl-2)和炎症相关蛋白(TNF-α、IL-6)的表达水平。采用细胞计数试剂盒检测LRG1对MSCs活力影响。细胞划痕实验检测LRG1对MSCs迁移能力的影响。RNA测序探究LRG1预处理MSCs对肾损伤修复影响的作用机制。酶联免疫吸附测定检测LRG1-MSCs培养上清中的前列腺素E2(PGE2)含量。结果IRI组小鼠的Scr、BUN、ATN评分和KIM-1表达水平均明显高于Sham组(P均<0.05);MSCs组和LRG1-MSCs组小鼠的Scr、BUN、ATN评分和KIM-1表达水平均明显低于IRI组(P均<0.05);LRG1-MSCs组小鼠的Scr、BUN、ATN评分和KIM-1表达水平均明显低于MSCs组(P均<0.05)。体外实验结果显示,MSCs组和LRG1-MSCs组HK-2细胞的Bax、IL-6和TNF-α蛋白表达均明显低于H/R组,而Bcl-2蛋白表达高于H/R组(P均<0.05);LRG1-MSCs组HK-2细胞的Bax、IL-6和TNF-α蛋白表达均明显低于MSCs组,而Bcl-2蛋白表达高于MSCs组(P均<0.05)。250 ng/ml LRG1处理24 h能够明显促进MSCs增殖和迁移(P均<0.05)。RNA测序显示,LRG1预处理MSCs后前列腺素内过氧化物合酶2(PTGS2)基因表达上调,LRG1-MSCs培养上清液中PGE2含量明显增高(P均<0.05)。结论LRG1能够促进MSCs分泌PGE2,减轻凋亡和炎症反应,增强了MSCs对AKI的疗效。

Objective To investigate the impact of leucine-richα-2-glycoprotein 1(LRG1)pretreatment of mesenchymal stem cells(MSCs)on the efficacy of MSCs in the treatment of acute kidney injury(AKI).Methods Eight-week-old male C57BL/6 mice were randomly divided into four groups:sham operation group(sham group),ischemia/reperfusion injury(IRI)group,MSCs group,and LRG1-MSCs group.The renal IRI mouse model was established by clamping of the left renal pedicle for 30 min and nephrectomy of the right kidney.The sham group did not receive any treatment,while the IRI group,MSCs group,and LRG1-MSCs group were injected through the tail vein with the same volume of phosphate buffer saline(PBS),MSCs,and LRG1-pretreated MSCs,respectively.These mice were sacrificed at 3 d after surgery,and the serum creatinine(Scr)and blood urea nitrogen(BUN)levels were measured.Renal pathological injury was evaluated by acute tubular necrosis(ATN)score after periodic acid-Schiff(PAS)staining.The protein expression of kidney injury molecule-1(KIM-1)was detected by Western blot in kidney tissues.For the in vitro experiments,HK-2 cells were divided into four groups:control group(normal HK-2 cells),hypoxia/reoxygenation group(HK-2 cells with treatment of hypoxia/reoxygenation),MSCs group(hypoxia/reoxygenation-treated HK-2 cells co-cultured with MSCs),and LRG1-MSCs group(hypoxia/reoxygenation-treated HK-2 cells co-cultured with LRG1-pretreated MSCs).Western blot analysis was used to detect the expression levels of apoptosis-related proteins(Bax and Bcl-2)and inflammation-related proteins(TNF-αand IL-6)in HK-2 cells.Cell proliferation was assessed by the cell counting kit-8(CCK-8)method.The effect of LRG1 on the migration of MSCs was analyzed by the wound healing assay.To explore the mechanism by which LRG1 enhanced the therapeutic effect of MSCs in IRI mice,RNA sequencing was performed.The content of prostaglandin E2(PGE2)in the supernatant of cells was detected by enzyme linked immunosorbent assay(ELISA).Results The levels of Scr,BUN,ATN score,and KIM-1 expression were significantly higher in the IRI group than in the sham group(all P<0.05).In contrast,the Scr,BUN,ATN score,and KIM-1 expression levels in both the MSCs group and LRG1-MSCs group were all significantly lower than those in the IRI group(all P<0.05).Furthermore,the LRG1-MSCs group exhibited significantly lower levels of Scr,BUN,ATN score,and KIM-1 expression than the MSCs group(all P<0.05).In vitro experiments revealed that the protein levels of Bax,IL-6,and TNF-αof both the MSCs group and LRG1-MSCs group were significantly lower than those in the H/R group,while Bcl-2 protein level was higher than that in the H/R group(all P<0.05).Additionally,the expression levels of Bax,TNF-α,and IL-6 proteins in HK-2 cells of the LRG1-MSCs group were lower than those in the MSCs group,while the expression level of Bcl-2 protein was higher than that in the MSCs group.Pretreatment with 250 ng/ml of LRG1 for 24 h significantly promoted the proliferation and migration of MSCs(both P<0.05).RNA sequencing revealed upregulation of the prostaglandin-endoperoxide synthase 2(PTGS2)gene expression in LRG1-pretreated MSCs,and significant increase of PGE2 content in the culture supernatant of LRG1-MSCs group(both P<0.05).Conclusion LRG1 may promote the secretion of PGE2 by MSCs,reduce apoptosis and inflammatory response,and enhance the efficacy of MSCs in the treatment of AKI.