Galectin-1/RAMP在脓毒血症患者中的表达及对炎性信号通路的调控分子机制研究

Study on the expression of galectin-1/ramp in patients with sepsis and the molecular mechanism regulating inflammatory signaling pathway

目的探究半乳糖凝集素1(galactose lectin 1,Galectin-1)/重组受体活性修饰蛋白(recombinant receptor activity modifying protein,RAMP)在脓毒血症患者中的表达及对炎性信号通路的调控分子机制研究。方法选取河北医科大学第一医院门诊及住院的脓毒血症患者80例,并收集健康志愿者80例作为对照,通过酶联免疫吸附测定法检测2组人群血清抗炎分子Galectin-1和RAMP表达水平。细胞学实验:采用CRISPR-Cas9技术敲除单核细胞Galectin-1基因,将单核细胞TCP-1分为3组,对照组、NC组(转染NC质粒)和试验组(转染CRISPR-Cas9质粒),检测炎症因子白细胞介素6(interleukin 6,IL-6),白细胞介素8(interleukin 8,IL-8)和白细胞介素10(interleukin 10,IL-10)的表达水平,通过qPCR检测炎症通路分子ID3和RAMP mRNA表达水平,Western blot检测ID3和RAMP蛋白表达水平。结果与对照组相比,脓毒血症组患者血清Galectin-1和RAMP表达水平升高(P<0.01);细胞学实验:与对照组相比:敲除Galectin-1组细胞炎症因子分泌增加(P<0.01);qPCR结果表明与对照组相比:敲除Galectin-1组细胞ID3和PAMP mRNA表达水平升高(P<0.01),Western blot实验表明结果表明与对照组相比,敲除Galectin-1组细胞ID3和RAMP蛋白表达水平升高(P<0.01)。结论Galectin-1可能通过抑制炎症信号ID3/RAMP通路影响单核细胞的炎性因子分泌代谢,并参与脓毒血症的发生发展过程。

Objective To investigate the expression of galactose lectin 1(Galectin-1)/recombinant receptor activity modifying protein(RAMP)in patients with sepsis and the molecular mechanism of regulating inflammatory signaling pathway.Methods A total of 80 outpatients and inpatients with sepsis treated in the First Hospital of Hebei Medical University were selected,and 80 healthy volunteers were collected as controls.The expression levels of serum anti-inflammatory molecules Galectin-1 and RAMP in the two groups were detected by ELISA.In terms of cytological experiment,CRISPR-Cas9 technique was used to knock out the galectin-1 gene of monocytes.Monocytes TCP-1 were divided into three groups:control group,NC group(transfected with NC plasmid)and experimental group(transfected with crispr-cas9 plasmid).The expression levels of inflammatory factors interleukin 6(IL-6),interleukin 8(IL-8)and interleukin 10(IL-10)were detected.The expression levels of inflammatory pathway molecules ID3 and RAMP mRNA were detected by qPCR,and the expression levels of ID3 and RAMP proteins were detected by Western blot.Results Compared with the control group,the expression levels of serum Galectin-1 and RAMP in sepsis group were higher(P<0.01).In terms of cytological test,compared with the control group,the secretion of inflammatory factors in the knockout group was increased(P<0.01).QPCR results showed that compared with the control group,the expression levels of ID3 and RAMP mRNA were increased in the knockout group(P<0.01),and Western blot results showed that compared with the control group,the expression levels of ID3 and RAMP protein were increased in the knockout group(P<0.01).Conclusion Galectin-1 may affect the secretion and metabolism of inflammatory factors in monocytes by inhibiting the inflammatory signal ID3/RAMP pathway,and participate in the occurrence and development of sepsis.