晚期缺血预适应(IPC)联合半乳糖凝集素-9通过上调调节性T细胞减轻小鼠肾脏缺血再灌注损伤

 目的  研究晚期缺血预适应(ischemic preconditioning,IPC)联合半乳糖凝集素-9(galectin-9,Gal-9)对小鼠肾脏缺血再灌注(ischemia reperfusion,IR)损伤的预防作用及可能的机制。方法  75只雄性C57BL/6N小鼠随机分为5组。(1)IR组:d1仅分离两侧肾蒂,不进行预缺血,d5夹闭两侧肾蒂造成缺血35 min。(2)IPC-IR组:d1夹闭两侧肾蒂造成18 min预缺血,d2~d5每天PBS皮下注射,d5给药后立即夹闭两侧肾蒂造成缺血35 min。(3)IPC Gal9 IR组:d1夹闭两侧肾蒂造成18 min预缺血,d2~d5每天给予Gal-9 5 μg皮下注射,并于d5给药后夹闭两侧肾蒂造成缺血35 min。(4)IPC-Gal9-PC61-IR组:d1夹闭两侧肾蒂造成18 min预缺血,d2~d5每天给予Gal-9 5 μg皮下注射,d3和d5分别给予调节性T细胞(regulatory T cells,Treg)特异性拮抗剂PC61腹腔注射200 μg,并在d5给药后夹闭两侧肾蒂造成缺血35 min。(5)sham sham组:不进行预适应,不给药,不给予IR损伤。以上5组于IR或第2次sham术后24 h、72 h和7天取样评价肾功能,并采用流式细胞术比较外周血、脾脏和肾脏中Treg的表达量和肾脏固有免疫系统反应程度及炎性因子的分泌。 结果  缺血再灌注24 h及72 h小鼠血肌酐IPC-IR组显著低于IR组(P<0.01),同时IPC-Gal9-IR组显著低于IPC-IR组(P<0.05),而给予Treg特异性拮抗剂的IPC-Gal9-PC61-IR组与IR组差异无统计学意义(P＞0.05)。缺血再灌注24 h肾小管间质损伤指数IPC-IR组显著低于IR组(P<0.05),同时IPC-Gal9-IR组显著低于IPC-IR组(P<0.05)。IPC-Gal9-IR组肾脏局部中心粒细胞浸润减轻(P＜0.05)且CD4+T细胞分泌的IFN-γ减少(P＜0.05)。IPC-Gal9-IR组外周血、脾脏和肾脏Treg占CD4+T细胞的比例较IR组显著升高(P＜0.05)。结论  晚期IPC联合Gal-9干预对小鼠缺血再灌注肾损伤的保护作用优于单纯缺血预适应,可能与系统上调Treg表达有关。

A combination of delayed ischemic preconditioning (IPC) and galectin-9protects against renal ischemia reperfusion injury in mice througha regulatory T cell dependent mechanism

Objective  To investigate the protective effect and its mechanisms of a combination of delayed ischemic preconditioning (IPC) and galectin-9 (Gal-9) against kidney injury caused by ischemia reperfusion (IR) in mice.Methods   Seventy-five male mice were randomly divided into 5 groups:IR,IPC-IR,IPC-Gal9-IR,IPC-Gal9-PC61-IR and sham-sham groups (n=5).(1) IR group:On day 1,the bilateral renal pedicles were separated without occlusion.Five days after the sham surgeries,mice were subjected to 35 min occlusion of bilateral renal pedicles.(2) IPC-IR group:On day 1,bilateral renal pedicles were clamped for 18 min.A daily injection of 50 μL PBS was subcutaneously administered during day 2 to day 5.Five days after the IPC,mice were subjected to 35 min occlusion of bilateral renal pedicles.(3) IPC-Gal9-IR group:On day 1,bilateral renal pedicles were clamped for 18 min.A daily injection of 5 μg (50 μL) Gal-9 was subcutaneously administered during day 2 to day 5.Five days after the IPC,mice were subjected to 35 min occlusion of bilateral renal pedicles.(4) IPC Gal9-PC61-IR group:On day 1,bilateral renal pedicles were clamped for 18 min.A daily injection of 5 μg (50 μL) Gal-9 was subcutaneously administered during day 2 to day 5.Apart from the IPC,IR procedure and Gal-9 injection,the specific antagonist of regulatory T cells (Treg),200 μg of PC 61,was administered via peritoneal injection on day 3 and day 5 after the IPC.Five days after the IPC,mice were subjected to 35 min occlusion of bilateral renal pedicles.(5) Sham-sham group:On day 1 and day 5,bilateral renal pedicles were separated without occlusion.The mice were sacrificed 24 h,72 h and 7 days after IR.Renal function was assessed based on the serum creatinine (Scr) and renal pathology.The peripheral blood,the spleen and kidney were also collected to evaluate the Treg expression by flow cytometric analysis.Results  The mice Scr 24 h and 72 h after IR in IPC-IR group was significantly lower than that in IR group (P<0.01),the Scr of the IPC-Gal9-IR group was significantly lower than that of the IPC-IR group (P<0.05) as well.While difference was not marked between the IPC-Gal9-PC61-IR group and the IR group (P＞0.05).The tubulointerstitial injury score 24 h after IR in the IPC-IR group was lower than that in the IR group (P<0.05),the IPC-Gal9-IR group was markedly lower than the IPC IR group (P<0.05).The renal neutrophil infiltration and the IFN-γ secreting CD4+T cells in the kidney of the IPC-Gal9 IR group decreased (P＜0.05).Percentage of Treg in the peripheral blood,spleen and kidney of the IPC-Gal9-IR group increased markedly compared with the IR group (P＜0.05).Conclusions   The renal protective effect of delayed IPC in combination with Gal-9 administration was superior to simply IPC in mice,which may related to systematical expansion of Treg.