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

doi:10.3877/cma.j.issn.1673-5250.2022.01.004

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.

Results

①HMGB1 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 (χ²=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 (χ²=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). ⑤ The 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.

Key words: High mobility group proteins, Pre-eclampsia, Maternal-fetal interface, Macrophages, Polarization

表1 实时荧光定量PCR引物序列

引物名称	上游引物(5′→3′)	下游引物(3′→5′)	产物长度(bp)
HMGB1	TATGGCAAAAGCGGACAAGG	CTTCGCAACATCACCAATGGA	196
ACTB	GAGAAAATCTGGCACCACACC	GGATAGCACAGCCTGGATAGCAA	177

表1 实时荧光定量PCR引物序列

引物名称	上游引物(5′→3′)	下游引物(3′→5′)	产物长度(bp)
HMGB1	TATGGCAAAAGCGGACAAGG	CTTCGCAACATCACCAATGGA	196
ACTB	GAGAAAATCTGGCACCACACC	GGATAGCACAGCCTGGATAGCAA	177

表2 3组孕妇一般及相关临床资料比较

组别	例数	年龄(岁，±s)	产次[例数(%)]		孕龄(周，±s)	最高收缩压(mmHg，±s)	最高舒张压(mmHg，±s)
组别	例数	年龄(岁，±s)	0次	1次	孕龄(周，±s)	最高收缩压(mmHg，±s)	最高舒张压(mmHg，±s)
sPE组	11	32.3±2.6	9(81.8)	2(18.2)	33.4±2.1	167.4±6.1	106.4±7.2
PE组	13	31.5±2.2	10(76.9)	3(23.1)	37.5±0.8	155.3±3.7	101.7±6.9
对照组	13	30.5±1.7	10(76.9)	3(23.1)	38.4±0.8	129.5±4.4	78.5±5.2
检验值		F＝1.888	χ²＝0.106		χ²＝25.528	F＝200.847	F＝66.498
P值		0.167	0.948		<0.001	<0.001	<0.001
组别	例数	(g/24 h，±s)	最高随机尿蛋白[例数(%)]				新生儿出生体重(g，±s)
组别	例数	(g/24 h，±s)	-	+	++	+++	新生儿出生体重(g，±s)
sPE组	11	4.1±2.5	0(0)	0(0)	7(63.6)	4(36.4)	1 812.6±514.1
PE组	13	1.1±0.6	0(0)	8(61.5)	5(38.5)	0(0)	2 852.3±146.9
对照组	13	0±0	13(100.0)	0(0)	0(0)	0(0)	3 197.7±116.3
检验值		χ²＝31.595	χ²＝31.595				χ²＝30.777
P值		<0.001	<0.001				<0.001

表2 3组孕妇一般及相关临床资料比较

组别	例数	年龄(岁，±s)	产次[例数(%)]		孕龄(周，±s)	最高收缩压(mmHg，±s)	最高舒张压(mmHg，±s)
组别	例数	年龄(岁，±s)	0次	1次	孕龄(周，±s)	最高收缩压(mmHg，±s)	最高舒张压(mmHg，±s)
sPE组	11	32.3±2.6	9(81.8)	2(18.2)	33.4±2.1	167.4±6.1	106.4±7.2
PE组	13	31.5±2.2	10(76.9)	3(23.1)	37.5±0.8	155.3±3.7	101.7±6.9
对照组	13	30.5±1.7	10(76.9)	3(23.1)	38.4±0.8	129.5±4.4	78.5±5.2
检验值		F＝1.888	χ²＝0.106		χ²＝25.528	F＝200.847	F＝66.498
P值		0.167	0.948		<0.001	<0.001	<0.001
组别	例数	(g/24 h，±s)	最高随机尿蛋白[例数(%)]				新生儿出生体重(g，±s)
组别	例数	(g/24 h，±s)	-	+	++	+++	新生儿出生体重(g，±s)
sPE组	11	4.1±2.5	0(0)	0(0)	7(63.6)	4(36.4)	1 812.6±514.1
PE组	13	1.1±0.6	0(0)	8(61.5)	5(38.5)	0(0)	2 852.3±146.9
对照组	13	0±0	13(100.0)	0(0)	0(0)	0(0)	3 197.7±116.3
检验值		χ²＝31.595	χ²＝31.595				χ²＝30.777
P值		<0.001	<0.001				<0.001

表3 3组胎盘蜕膜组织HMGB1蛋白及HMGB1 mRNA相对表达水平比较

组别		例数	Western blotting检测HMGB1结果[M(P₂₅~P₇₅)]	IHC检测HMGB1结果[例数(%)]				HMGB1 mRNA(±s)
组别		例数	Western blotting检测HMGB1结果[M(P₂₅~P₇₅)]	—	+	++	+++	HMGB1 mRNA(±s)
sPE组		11	3.10(1.50~5.14)	0(0)	2(18.2)	3(27.3)	6(54.6)	4.49±1.38
PE组		13	0.63(0.45~2.41)	0(0)	3(23.1)	6(46.2)	4(30.8)	3.01±2.08
对照组		13	0.72(0.07~1.49)	5(38.5)	5(38.5)	3(23.1)	0( 0)	1.67±1.48
总体比较
	检验值		χ²＝9.222	χ²＝15.024				F＝8.291
	P值		0.010	0.001				0.001
两两比较(P值)
	PE组vs对照组		0.140	0.001				0.052
	sPE组vs PE组		0.057	0.353				0.039
	sPE组vs对照组		0.002	<0.001				<0.001

表3 3组胎盘蜕膜组织HMGB1蛋白及HMGB1 mRNA相对表达水平比较

组别		例数	Western blotting检测HMGB1结果[M(P₂₅~P₇₅)]	IHC检测HMGB1结果[例数(%)]				HMGB1 mRNA(±s)
组别		例数	Western blotting检测HMGB1结果[M(P₂₅~P₇₅)]	—	+	++	+++	HMGB1 mRNA(±s)
sPE组		11	3.10(1.50~5.14)	0(0)	2(18.2)	3(27.3)	6(54.6)	4.49±1.38
PE组		13	0.63(0.45~2.41)	0(0)	3(23.1)	6(46.2)	4(30.8)	3.01±2.08
对照组		13	0.72(0.07~1.49)	5(38.5)	5(38.5)	3(23.1)	0( 0)	1.67±1.48
总体比较
	检验值		χ²＝9.222	χ²＝15.024				F＝8.291
	P值		0.010	0.001				0.001
两两比较(P值)
	PE组vs对照组		0.140	0.001				0.052
	sPE组vs PE组		0.057	0.353				0.039
	sPE组vs对照组		0.002	<0.001				<0.001

图1 3组胎盘组织HMGB1蛋白相对表达水平(图1A：HMGB1电泳条带图；图1B：HMGB1相对表达水平柱状图)注：HMGB1为高迁移率族蛋白1，sPE为重度子痫前期，PE为子痫前期

图2 3组胎盘组织HMGB1 mRNA相对表达水平柱状图注：HMGB1为高迁移率族蛋白1，sPE为重度子痫前期，PE为子痫前期

图3 HMGB1表达及细胞定位(IHC)[图3A~3D: sPE组、PE组、对照组、阴性对照组胎盘蜕膜组织HMGB1的表达(×100)；图3E：连续组织切片(蜕膜基质细胞标记波形蛋白表达定位，×200)；图3F：连续组织切片(滋养层细胞标记角蛋白7表达定位，×200)；图3G：连续组织切片(HMGB1表达定位，×200)]注：HMGB1为高迁移率族蛋白1，IHC为免疫组织化学染色

图4 原代胎盘蜕膜巨噬细胞鉴定流式细胞术散点图(图4A：根据FSC和SSC，筛选了表达CD14的均质细胞群；图4B：巨噬细胞同时表达CD68及CD14占97.39%；图4C：阴性对照，CD14单染、CD68单染对照及CD14、CD68双染对照)注： FSC表示正向散射，SSC表示侧向散射

图5 rHMGB1对蜕膜巨噬细胞M1型标记(CD86)诱导作用的流式细胞术示意图(图5A：空白处理；图5B：25 ng/mL rHMGB1处理；图5C：50 ng/mL rHMGB1处理；图5D：100 ng/mL rHMGB1处理；图5E：200 ng/mL rHMGB1处理；图5F：rHMGB1处理前)(红色矩形区域所示为M1型巨噬细胞阳性细胞比例)注： rHMGB1为重组高迁移率族蛋白1，FSC表示正向散射，SSC表示侧向散射

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