基于煨脓长肉理论探讨紫朱软膏通过NLRP3逆转糖尿病难愈性创面巨噬细胞胞葬逃逸的机制研究

Exploring the Mechanism of Hematite Ointment to Reverse the Macrophage Efferocytosis Escape of Diabetic Refractory Wound through NLRP3 Based on the Theory of Weinong Zhangrou

目的:初步探讨紫朱软膏过NLRP3逆转糖尿病难愈性创面巨噬细胞胞葬逃逸的相关机制。方法:48只C57BL/6小鼠随机分为普通创面模型组、糖尿病创面模型组、紫朱软膏干预组、NLRP3抑制剂组,每组12只。提取创面组织巨噬细胞与凋亡Jurkat细胞共培养,创面拍照并记录创面愈合率,应用RT-qPCR、Western blot、流式细胞仪检测巨噬细胞胞葬能力、NLRP3通路及胞葬相关因子mRNA和蛋白的表达水平。结果:普通创面模型组、紫朱软膏干预组、NLRP3抑制剂组的创面愈合率明显高于糖尿病创面模型组(P<0.01);与普通创面组对比,糖尿病创面模型组巨噬细胞胞葬功能降低明显(P<0.01),NLRP3、Caspase-1 mRNA及蛋白表达水平显著上调(P<0.01),AXL、TYRO3、MFGE8表达水平显著降低(P<0.01);与糖尿病创面模型组相比,紫朱软膏干预组胞葬功能显著提升(P<0.01),NLRP3、Caspase-1 mRNA及蛋白水平显著降低(P<0.01),AXL、TYRO3、MFGE8表达水平显著上调(P<0.05,P<0.01);与糖尿病创面模型组相比,NLRP3抑制剂组巨噬细胞胞葬功能显著提升(P<0.01),NLRP3、Caspase-1 mRNA及蛋白表达水平显著降低(P<0.01),AXL、TYRO3、MFGE8表达水平显著上调(P<0.05,P<0.01)。结论:紫朱软膏可能通过NLRP3通路逆转巨噬细胞胞葬逃逸,从而促进糖尿病创面愈合。

Objective:To investigate the mechanism of hematite ointment reversing macrophage efferocytosis escape in refractory wound of diabetes through NLRP3.Methods:48 C57BL/6 mice were randomly divided into common ulcer model group,diabetes wound model group,hematite ointment intervention group,and NLRP3 inhibitor group.Macrophages were extracted from the wound tissue and co-cultured with apoptotic Jurkat cells.The wound was photographed and the wound healing rate recorded.RT-qPCR,Western blot and flow cytometry were used to detect the efferocytosis capacity of macrophages,NLRP3 pathway and the expression level of mRNA and proteins related to efferocytosis factors.Results:The wound healing rate of the common ulcer model group,hematite ointment intervention group,and NLRP3 inhibitor group was significantly higher than that of the diabetes wound model group(P<0.05).Compared with the common ulcer model group,the efferocytosis function of macrophages in the diabetes wound model group was significantly reduced,the expression levels of NLRP3,Caspase-1 mRNA and protein were significantly upregulated,and the expression levels of AXL,TYRO3,MFGE8 were significantly decreased(P<0.05).Compared with the diabetes wound model group,the hematite ointment intervention group significantly improved the efferocytosis function,significantly reduced the levels of NLRP3,Caspase-1 mRNA and proteins,and significantly upregulated the expression levels of AXL,TYRO3,and MFGE8(P<0.05);Compared with the diabetes wound model group,the efferocytosis function of macrophages in the NLRP3 inhibitor group was significantly increased,the expression levels of NLRP3,Caspase-1 mRNA and proteins were significantly decreased,and the expression levels of AXL,TYRO3,and MFGE8 were significantly upregulated(P<0.05).Conclusion:Hematite ointment,a traditional Chinese medicine for Weinong Zhangrou,may reverse the efferocytosis escape of macrophages through NLRP3 pathway,thus promoting the healing of diabetes wounds.