基于卡介苗感染过程中MAPK-Mcl-1信号通路和巨噬细胞极化调控机制的生物信息学分析及其实验验证

doi:10.13481/j.1671-587X.20260216

Abstract

Abstract:

Objective To discuss the role of mitogen-activated protein kinase （MAPK）-myeloid cell leukemia sequence 1 （Mcl-1） signaling axis in the polarization process of macrophages infected with Bacillus Calmette-Guérin （BCG）， and to clarify its possible molecular mechanism. Methods The transcriptome sequencing data of GSE89391 and GSE51029 were downloaded from Gene Expression Omnibus （GEO） database， and the MAPK signaling pathway-related gene sets were downloaded from Molecular Signatures Database （MSigDB）. The gene set variation analysis （GSVA） package was used to perform single-sample gene set enrichment analysis （ssGSEA） on the macrophage data in GEO database， and the activity scores of MAPK signaling pathway were calculated. The infected samples were divided into Mcl-1 high expression group and Mcl-1 low expression group according to the median of Mcl-1 expression level， and gene set enrichment analysis （GSEA） was performed. Spearman correlation analysis was used to assess the correlation between Mcl-1 expression level and MAPK pathway activity in macrophages in GEO database. The extracellular signal-regulated kinase （ERK） pathway blocker PD98059 （50 μmol·L?1）， c-Jun N-terminal kinase （JNK） pathway blocker SP600125 （30 μmol·L?1）， and p38 pathway blocker SB203580 （60 μmol·L?1） were prepared.The Raw264.7 cells were divided into control group， various blocker-treated groups， BCG group， and various blocker-treated groups after BCG infection. BCG bacterial solution was prepared and used to infect the cells in corresponding groups， and the corresponding blockers were added to the cells in various groups. The cell supernatants and cell samples in various groups were collected at 0， 12， and 24 h after treatment. Enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of Mcl-1， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， interleukin-10 （IL-10）， and transforming growth factor-β （TGF-β） in supernatant of the macrophages in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of M1 marker inducible nitric oxide synthase （iNOS） mRNA and M2 marker found in inflammatory zone 1 （Fizz1） mRNA in the macrophages in various groups. Results Based on GEO databases， compared with uninfected control macrophages， the level of Mcl-1 gene in the macrophages infected with Mycobacterium tuberculosis（MTB） was significantly increased （P<0.05）. The ssGSEA analysis results showed that compared with uninfected control macrophages， the MAPK signaling pathway activity score in MTB-infected macrophages was significantly increased （P<0.05）. The GSEA analysis results revealed that differentially expressed genes in Mcl-1 high expression groups were significantly enriched in the MAPK pathway （P<0.05）. The Spearman correlation analysis results indicated there was a positive correlation between the expression level of Mcl-1 gene and MAPK signaling pathway activity score （P<0.05）. At 12 and 24 h after treatment， compared with BCG group， the levels of Mcl-1 in supernatant of the macrophages in various blocker-treated groups were significantly decreased （P<0.05）. At 12 h after treatment， compared with control group， the levels of IL-6 and TNF-α in supernatant of the macrophages in BCG group were significantly increased （P<0.05）. Compared with BCG group， the levels of IL-6 and TNF-α in supernatants of the macrophages in BCG+SP and BCG+PD+SP groups were significantly decreased （P<0.05）. At 24 h after treatment， compared with control group， the level of TNF-α in supernatant of the macrophages in BCG group was significantly increased （P<0.05）. Compared with BCG group， the levels of IL-6 in supernatants of the macrophages in BCG+PD， BCG+SP， BCG+SB， and BCG+PD+SP groups were significantly decreased （P<0.05）， and the levels of TNF-α in supernatants of the macrophages in all blocker-treated groups were significantly decreased （P<0.05）. At 12 h after treatment， compared with control group， the level of TGF-β in supernatant of the macrophages in BCG group was significantly increased （P<0.05）. Compared with BCG group， the levels of TGF-β in supernatants of the macrophages in BCG+SP， BCG+PD+SP， BCG+PD+SB， BCG+SP+SB， and BCG+PD+SP+SB groups were significantly increased （P<0.05）. At 24 h after treatment， compared with control group， the level of IL-10 in supernatant of the macrophages in BCG group was significantly increased （P<0.05）. Compared with BCG group， the levels of IL-10 and TGF-β in supernatants of the macrophages in BCG+PD， BCG+SP， BCG+PD+SP， BCG+PD+SB， and BCG+SP+SB groups were significantly decreased （P<0.05）. At 0 h after infection， compared with control group， the expression levels of iNOS and Fizz1 mRNA in the macrophages in BCG group were significantly increased （P<0.05）. At 12 h after treatment， compared with control group， the expression level of iNOS mRNA in the macrophages in BCG group was significantly decreased （P<0.01）， while the expression level of Fizz1 mRNA was significantly increased （P<0.01）. Compared with BCG group， the expression levels of iNOS mRNA in the macrophages in all blocker-treated groups were significantly increased （P<0.01）， and the expression levels of Fizz1 mRNA in the macrophages in BCG+PD， BCG+PD+SP， BCG+PD+SB， BCG+SP+SB， and BCG+PD+SP+SB groups were significantly increased （P<0.01）. At 24 h after treatment， compared with control group， the expression levels of iNOS and Fizz1 mRNA in the macrophages in BCG group were significantly increased （P<0.05）. The expression levels of iNOS mRNA in the macrophages between BCG group and all blocker-treated groups showed no statistically significant difference （P>0.05）. Compared with BCG group， the expression level of Fizz1 mRNA in the macrophages in BCG+PD+SP group was significantly decreased （P<0.01）. Conclusion MAPK signaling pathway may mediate the polarization process of macrophages infected with BCG by regulating Mcl-1 activity， in which the JNK pathway plays a core regulatory role， and the p38 and ERK pathways are synergistically involved in the regulation.

Key words: Bioinformatics, Bacillus Calmette-Guérin, Macrophage polarization, Myeloid cell leukemia sequence 1, Mitogen-activated protein kinase signaling pathway, c-Jun N-terminal kinase

CLC Number:

R363.2

Ruihan GE,Chen LI,Shengpeng WANG,Yang LU,Caixia TAN,Haotian CUI,Xinmin WANG,Le ZHANG. Bioinformatic analysis on regulatory mechanism of MAPK-Mcl-1 signaling pathway and macrophage polarization during Bacillus Calmette-Guérin infection and its experimental validation[J].Journal of Jilin University(Medicine Edition), 2026, 52(2): 440-450.

Figures/Tables 9

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References 23

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Gene	Sequence (5'-3')	Length (bp)
iNOS	F:TGATGTGCTGCCTCTGGTCT	20
	R:GAGCTCCTGGAACCACTCGT	20
Fizzl	F:GAGATCCAGAGTGGAGATACTTGC	24
	R:TCTTAGGACAGTTGGCAGCAG	21
β-actin	F:GTGACGTTGACATCCGTAAAGA	22
	R:GCCGGACTCATCGTACTCC	19

Group	Mcl-1 level
Group	(t/h) 0	12	24
Control	0.48±0.02	1.04±0.03	1.51±0.14
BCG	0.46±0.06	0.76±0.01^*	1.06±0.04^*
BCG+PD	-	0.57±0.04^△	0.57±0.02^△
BCG+SP	-	0.42±0.05^△	0.47±0.06^△
BCG+SB	-	0.36±0.12^△	0.52±0.08^△
BCG+PD+SP	-	0.41±0.06^△	0.57±0.02^△
BCG+PD+SB	-	0.39±0.03^△	0.47±0.01^△
BCG+SP+SB	-	0.34±0.07^△	0.43±0.02^△
BCG+PD+SP+SB	-	0.39±0.08^△	0.45±0.02^△

Group	IL-6			TNF-α
Group	(t/h) 0	12	24	0	12	24
Control	84.14±20.02	166.27±7.46	51.77±1.22	68.08±2.52	161.55±13.85	158.81±4.73
BCG	81.64±7.21	290.38±9.21^*	59.89±3.17	74.82±1.88	194.20±5.66^*	177.32±2.87^*
BCG+PD	-	190.40±15.20	44.75±2.94^△	-	160.45±41.96^△	97.91±1.93^△
BCG+SP	-	145.61±13.49^△	42.28±2.45^△	-	161.56±20.18^△	81.86±2.72^△
BCG+SB	-	205.32±10.09	47.47±2.44^△	-	165.15±5.54^△	86.03±2.07^△
BCG+PD+SP	-	183.54±16.04^△	43.15±1.22^△	-	165.91±19.06^△	64.18±3.37^△
BCG+PD+SB	-	170.24±18.08	56.92±1.21	-	163.68±18.87^△	50.81±2.95^△
BCG+SP+SB	-	254.15±16.04	53.49±3.64	-	195.58±12.93	58.43±7.20^△
BCG+PD+SP+SB	-	224.85±17.00	82.50±1.20^△	-	200.75±7.20	69.23±2.32^△

Group	IL-10			TGF-β
Group	（t/h） 0	12	24	0	12	24
Control	34.69±11.47	27.89±3.42	53.58±5.07	72.74±4.73	314.72±9.84	424.23±7.36
BCG	43.57±10.66	31.47±5.47	71.77±2.68^*	79.52±5.69	379.25±17.08^*	464.49±3.65
BCG+PD	-	38.65±1.85	52.26±5.95^△	-	396.45±18.38	289.72±7.69^△
BCG+SP	-	32.45±5.77	56.22±2.03^△	-	424.21±16.88^△	368.48±9.90^△
BCG+SB	-	35.38±2.26	66.16±7.03	-	394.31±9.83	477.11±18.24
BCG+PD+SP	-	33.42±1.07	63.54±4.16^△	-	411.40±23.13^△	320.59±11.41^△
BCG+PD+SB	-	38.66±6.24	65.09±2.80^△	-	426.28±31.43^△	348.98±7.53^△
BCG+SP+SB	-	37.01±3.60	64.42±3.24^△	-	436.96±16.86^△	348.98±3.76^△
BCG+PD+SP+SB	-	37.34±1.07	56.35±1.65^△	-	493.91±16.65^△	469.92±14.51

Group	iNOS			Fizz1
Group	（t/h） 0	12	24	0	12	24
Control	1.00±0.10	1.00±0.10	1.00±0.10	1.00±0.10	1.00±0.10	1.00±0.10
PD	-	1.33±0.13	0.54±0.05^*	-	1.24±0.66	0.31±0.04^*
SP	-	1.60±0.13	0.84±0.11^*	-	1.49±0.64	0.26±0.10^*
SB	-	2.31±0.17^**	0.48±0.08^*	-	4.43±2.13^*	0.41±0.11^*
PD+SP	-	2.02±0.88^**	1.00±0.14	-	8.27±0.64^*	0.77±0.12^*
PD+SB	-	2.15±0.04^**	0.74±0.09^*	-	2.69±0.89^*	0.22±0.01^*
SP+SB	-	1.35±0.09	0.61±0.04^*	-	6.59±0.27^*	0.18±0.08^*
PD+SP+SB	-	0.76±0.23	0.61±0.06	-	2.44±0.90^*	0.83±0.20^*
BCG	1.34±0.07^*	0.50±0.03^**	1.86±0.10^*	1.43±0.21^*	2.42±0.48^**	32.32±1.12^*
BCG+PD	-	1.57±0.14^△△	0.64±0.09	-	3.48±0.36^△	24.24±3.66
BCG+SP	-	1.59±0.15^△△	0.87±0.03	-	1.45±0.63	29.51±0.41
BCG+SB	-	1.94±0.13^△△	0.52±0.06	-	1.49±0.40	24.65±2.17
BCG+PD+SP	-	2.19±0.06^△△	1.19±0.18	-	15.98±0.91^△△	2.87±0.32^△△
BCG+PD+SB	-	2.56±0.12^△△	0.91±0.02	-	8.16±0.76^△△	32.88±1.76
BCG+SP+SB	-	1.35±0.22^△△	0.37±0.03	-	3.52±0.32^△	14.74±2.40
BCG+PD+SP+SB	-	1.17±0.15^△△	0.94±0.08	-	7.60±0.79^△△	22.78±1.76

Bioinformatic analysis on regulatory mechanism of MAPK-Mcl-1 signaling pathway and macrophage polarization during Bacillus Calmette-Guérin infection and its experimental validation

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