高迁移率族蛋白1在子痫前期母胎界面表达及其对巨噬细胞的作用

Expression of high mobility group box 1 at maternal-fetal interface and its effect on macrophages in preeclampsia

目的探究高迁移率族蛋白(HMGB)1在子痫前期(PE)母胎界面的表达及其对巨噬细胞的作用。方法选择2020年6月至2021年12月,在四川大学华西第二医院确诊为PE的24例单胎孕妇为研究对象。按照孕妇PE严重程度,将其分别纳入PE组(n=13)和重度PE(sPE)组(n=11)。按照随机数字表法,选择同期在本院剖宫产术分娩的正常妊娠孕妇纳入对照组(n=13)。采用免疫组织化学(IHC)法、蛋白质免疫印迹法(Western blotting)、实时荧光定量PCR(qPCR)检测3组患者胎盘蜕膜组织HMGB1相对表达水平。采用免疫磁珠富集法分离对照组原代蜕膜巨噬细胞,以不同剂量人重组HMGB1(rHMGB1)对其进行处理48 h,采用流式细胞术检测巨噬细胞表型变化。研究组与对照组PE孕妇年龄、产次等一般临床资料比较,差异均无统计学意义(P>0.05)。本研究遵循的程序符合四川大学华西第二医院伦理委员会规定,通过该伦理委员会审查,并获得批准[审批文号:2021(181)]。所有受试者均签署临床研究知情同意书。结果①HMGB1定位于细胞滋养层细胞、合体滋养层细胞和蜕膜基质细胞的细胞质及细胞核中。②经Western blotting检测sPE组、PE组和对照组HMGB1蛋白相对表达水平分别为3.10(1.50~5.14)、0.63(0.45~2.41)、0.72(0.07~1.49),3组总体比较,差异有统计学意义(χ^(2)=9.222,P=0.010)。③IHC检测结果发现,sPE组、PE组和对照组HMGB1强阳性比例分别为54.6%(6/11)、30.8%(4/13)、0,3组总体比较,差异有统计学意义(χ^(2)=15.024,P=0.001)。④sPE组、PE组和对照组HMGB1 mRNA相对表达水平分别为4.49±1.38、3.01±2.08、1.67±1.48,3组总体比较,差异有统计学意义(F=8.291,P=0.001),其中sPE组相对表达水平分别高于PE组及对照组,并且差异亦有统计学意义(P=0.039、P<0.001)。⑤原代胎盘蜕膜巨噬细胞纯度为97.39%,经rHMGB1处理后,M1型巨噬细胞百分比增加,M1型巨噬细胞标记CD86平均荧光强度(MFI)与rHMGB1含量与呈正相关关系(r=0.808,P<0.001)。结论PE母胎界面HMGB1表达增加,促进蜕膜巨噬细胞M1型极化,可能导致母胎界面炎性微环境。

Objective To investigate the expression of high mobility group box(HMGB)1 at the maternal-fetal interface of pre-eclampsia(PE),and the possible regulatory effect on macrophage polarization.Methods Twenty-four singleton pregnant women admitted to the obstetrics clinic in West China Second University Hospital,Sichuan University from June 2020 to December 2021 were selected as research subjects.They were divided into PE group(n=37)and severe PE(sPE)group(n=11)based on diagnostic criteria for PE.In addition,13 healthy pregnant women were selected as controls by the random number table method.Immunohistochemistry(IHC),Western blotting and quantitative real-time polymerase chain reaction(qPCR)were used to detect placental HMGB1 protein and mRNA levels.Decidual macrophages were isolated using immunomagnetic positive selection.Further polarization characteristics of macrophages were analyzed by flow cytometry(FCM)after being treated with various doses of recombinant human HMGB1(rHMGB1).The procedure followed in this study was in accordance with the regulations of the Ethics Committee of West China Second University Hospital,Sichuan University,which was reviewed and approved[Approval No.2021(181)].Informed consent was obtained from each participant.Results①HMGB1 were observed both in the cytoplasm and nucleus of cytotrophoblast and decidual stromal cells.②Western blotting showed that the relative expression levels of HMGB1 protein in sPE group,PE group and control group were 3.10(1.50-5.14),0.63(0.45-2.41)and 0.72(0.07-1.49),respectively,and the difference was statistically significant(χ^(2)=9.222,P=0.010).③The proportion of sPE,PE and control group strongly positive for HMGB1 was 54.6%(6/11),30.08%(4/13)and 0,respectively,and the difference of HMGB1 was significant(χ^(2)=15.024,P=0.001).④The relative expression levels of HMGB1 mRNA in sPE group,PE group and control group were 4.49±1.38,3.01±2.08 and 1.67±1.48,respectively.Compared with the three groups,the difference was statistically significant(F=8.291,P=0.001).The relative expression level of sPE group was higher than that of PE group and control group respectively,and both the differences were statistically significant(P=0.039,P<0.001).⑤The isolation purity of decidual macrophages was 97.39%.The percentage of M1-related marker(CD86+)increased after rHMGB intervention.The mean fluorescence intensity(MFI)of CD86 was positively correlated with rHMGB dose(r=0.808,P<0.001).Conclusions HMGB1 is significantly up-regulated at the maternal-fetal interface in women with preeclampsia.HMGB1 induces polarization of M1 macrophages and regulates inflammatory cytokines profile at the maternal-fetal interface.