汉防己甲素通过抑制Mincle/Syk信号介导的巨噬细胞炎性活化减轻小鼠缺血再灌注诱导的急性肾损伤

Tetrandrine attenuates ischemia-reperfusion-induced acute kidney injury in mice through inhibiting inflammatory activation of macrophages mediated by Mincle/Syk signaling pathway

目的:探讨汉防己甲素(Tet)对小鼠缺血再灌注诱导的急性肾损伤(IRI-AKI)的作用及其机制。方法:8周龄雄性C57BL/6小鼠随机分为假手术组、IRI-AKI组、低剂量(20 mg/kg)Tet组、高剂量(40 mg/kg)Tet组和槲皮素(50 mg/kg)阳性对照组,每组6只。采用双侧肾动静脉夹闭45 min后恢复血供的方法建立IRI-AKI模型,Tet和槲皮素组的IRI-AKI小鼠于术前1 h及术后连续3 d给予相应剂量药物腹腔注射,假手术和IRI-AKI组小鼠给予同等体积溶剂注射。实验终点处死动物,收集血清及肾脏组织样本,进行肾功能、病理、mRNA及蛋白等指标检测。在体外,采用脂多糖(LPS;300μg/L)刺激的原代小鼠骨髓来源巨噬细胞(BMDM)进行Tet(1、2和4 mg/L)的抗炎活性研究。结果:(1)与IRI-AKI组相比,低和高剂量Tet干预均能显著降低血清肌酐和血尿素氮水平(P<0.05),并减轻肾小管病理损伤(P<0.05)。(2)Tet干预可以显著抑制IRI-AKI小鼠肾组织中白细胞介素1β(IL-1β)和IL-6的mRNA和蛋白表达及NF-κB信号的活化,减少肾组织巨噬细胞浸润(P<0.05)。(3)在LPS刺激的BMDM中,Tet同样抑制IL-1β和IL-6的mRNA和蛋白表达及NF-κB信号的活化(P<0.05)。(4)进一步实验显示,Tet可以显著降低LPS刺激的BMDM和IRI-AKI小鼠肾组织中Mincle、Syk和p-Syk的表达水平(P<0.05)。结论:Tet可以显著减轻IRI-AKI小鼠肾损伤,减轻肾组织炎症反应,其可能的机制为抑制巨噬细胞Mincle/Syk/NF-κB促炎信号通路。

AIM:To investigate the protective effect of tetrandrine(Tet)on mice with ischemia-reperfusioninduced acute kidney injury(IRI-AKI).METHODS:Eight-week-old male C57BL/6 mice were randomly divided into sham group,IRI-AKI group,low-dose(20 mg/kg)Tet group,high-dose(40 mg/kg)Tet group,and quercetin(Que;50 mg/kg)group,each containing six animals.The IRI-AKI model was induced by occluding bilateral renal blood supply for 45 min followed by reperfusion for 3 d.The mice in Tet and Que groups were intraperitoneally treated with corresponding doses of Tet and Que 1 h before surgery and once daily for 3 consecutive days after surgery.The mice in the sham and IRIAKI groups received an equivalent volume of saline.At the end of the experiment,the animals were sacrificed,and serum and kidney tissues were collected for analysis of renal function,histology,and mRNA and protein expression.In vitro,the anti-inflammatory activity of Tet(1,2 and 4 mg/L)was studied using lipopolysaccharide(LPS;300μg/L)-stimulated primary mouse bone marrow-derived macrophages(BMDM).RESULTS:(1)Both low and high doses of Tet significantly reduced serum creatinine and blood urea nitrogen levels compared with the IRI-AKI model mice,accompanied by attenuated structural injury of the renal tubules(P<0.05).(2)Treatment with Tet significantly reduced the mRNA and protein levels of interleukin-1β(IL-1β)and IL-6,and the activation of NF-κB signaling pathway,with reduced infiltration of macrophages after IRI-AKI(P<0.05).(3)In LPS-stimulated BMDM,Tet also inhibited the mRNA and protein expression of IL-1β and IL-6,and the activation of NF-κB signaling(P<0.05).(4)Further experiments showed that Tet significantly reduced the expression of Mincle,Syk and p-Syk in LPS-stimulated BMDM and IRI-AKI mouse kidney tissues(P<0.05).CONCLUSION:Tetrandrine significantly attenuates renal injury in IRI-AKI mice with reduced renal inflammation.The underlying mechanism might be the inhibition of pro-inflammatory Mincle/Syk/NF-κB signaling pathway in macrophages.