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

目的：观察外源性一氧化碳释放分子3（Carbon Monoxide-releasing molecules,CORM-3, CO RM-3）对肾固有树突状细胞(Renal Dendritic Cell，rDC)生长的影响，并探讨其可能的作用机制。方法：采用C57BL/6J，TLR4-/-及TLR4+/+小鼠，制备肾脏单细胞悬液，磁珠分选CD11c + rDC，流式细胞术鉴定rDC纯度。CORM-3处理新鲜分离的rDC，ELISA检测rDC培养液上清中TNF-α蛋白水平；RT-PCR检测TLR4及TNF-α基因表达。另外建立小鼠肾脏热缺血30min + 冷保存24h模型，在冷保存期间经CORM-3处理之后分选rDC，抽提RNA，行RT-PCR检测TNF-α表达。结果：CORM-3明显抑制小鼠未成熟rDC的TLR4表达，下调作用呈剂量相关性。相较于无活性的iCORM (inactive CO-releasing molecules)，CORM-3抑制LPS刺激后的rDC表达TNF-α。当TLR4缺陷之后，CORM-3不再抑制rDC 表达TNF-α。结论：CORM-3可显著抑制肾脏未成熟rDC 表达TLR4，也抑制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rDC活化过程的抑制作用是由TLR4信号通路介导。

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

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.