外源性一氧化碳释放分子3抑制肾脏固有树突状细胞活化的作用机制

Mechanism of exogenous carbon monoxide-releasing molecule 3 in inhibiting activation of renal dendritic cells

目的观察外源性一氧化碳释放分子3(carbon monoxide-releasing molecule 3,CORM-3)对肾固有树突状细胞(renal dendritic cells,rDCs)活化的影响,并探讨其可能的作用机制。方法选取C57BL/6J(H-2kb)小鼠制备肾脏单细胞悬液,采用磁珠分选CD11c+rDCs,并用流式细胞术鉴定rDCs纯度。用CORM-3或无活性的CORM(inactive CO-releasing mol-ecule,iCORM)处理新鲜肾脏分离的rDCs,实时定量RT-PCR检测TLR4基因表达,ELISA法检测rDCs培养液上清中TNF-α蛋白水平。建立TLR4-/-(C3H/HeJ)和TLR4+/+(C3H/HeN)小鼠肾脏热缺血30 min+冷保存24 h模型,在冷保存期间经CORM-3处理之后分选rDCs,抽提RNA,行实时定量RT-PCR检测TNF-α基因表达。结果 CORM-3呈剂量依赖性地抑制小鼠未成熟rDCs的TLR4 mRNA表达(P<0.05)。与iCORM比较,CORM-3能够抑制LPS刺激后的rDCs表达TNF-α(P<0.01)。当TLR4缺陷之后,CORM-3不再抑制rDCs表达TNF-α。结论 CORM-3可显著抑制肾脏未成熟rDCs表达TLR4,也可抑制LPS刺激和缺血损伤时炎症因子的表达,但对于TLR4缺陷小鼠这一抑制作用消失,提示CORM-3对内源性配体介导的rDCs活化过程的抑制作用由TLR4信号通路介导。

Objective To investigate the inhibitory effect of exogenous carbon monoxide-releasing molecule 3（CORM-3） on activation of the renal dendritic cells（rDCs） and the underlining mechanism.Methods Kidneys harvested from C57BL/6J mice were made into single cell suspension.CD11c ＋ rDCs were then sorted by magnetic cell sorting（MACS） and the purity was assessed by flow cytometry.The rDCs were treated by CORM-3 or inactive CO-releasing molecule（iCORM） together with lipopolysaccharides（LPS）. The expression of TLR4 gene was detected by quantitative real-time PCR.TNF-α protein levels in the rDCs culture were determined by ELISA.In addition,TLR4-/-（C3H/HeJ） or TLR4＋/＋（C3H/HeN） mice were subjected to 30 min bilateral kidney ischemia and 24-h cold preservation.The rDCs were then isolated to detect the expression of TNF-α gene in cells by quantitative real-time PCR.Results CORM-3 significantly inhibited the expression of TLR4 mNRA in immature rDCs in a dose-dependent manner（P〈0.05）.Compared with iCORM,CORM-3 significantly suppressed the expression of TNF-α in rDCs after LPS stimulation（P〈0.01）;however,this inhibitory effect of CORM-3 was abrogated in TLR4-/-mice.Conclusion CORM-3 can greatly inhibit TLR4 expression in immature rDCs,and it can also suppress proinflammatory cytokine expression induced by LPS stimulation or ischemia and cold preservation,but not in TLR4 knockout mice,suggesting that CORM-3 suppresses rDCs activation through TLR4 pathway.