ALKBH5通过TRAF1/NF⁃κB通路减轻脓毒症心肌损伤的机制

doi:10.3969/j.issn.1006-5725.2024.17.005

摘要/Abstract

摘要：

目的探讨ALKBH5减轻脓毒症心肌损伤（SIMD）的分子机制。方法采用逆转录荧光定量聚合酶链反应（RT-qPCR）的方法检测50例SIMD患者及50例健康人血液中ALKBH5和TRAF1表达水平，并通过Pearson分析两者表达水平的相关性；细胞体外实验中，根据不同的处理方式（过表达TARF1和干扰ALKBH5表达）将心肌细胞H9C2分为7组，通过CCK8、免疫吸附实验（ELISA）、Western blot等实验方法研究ALKBH5靶向TRAF1调控脂多糖（LPS）诱导的心肌细胞损伤的分子机制；大鼠体内实验中，根据处理方式不同（过表达TARF1和干扰ALKBH5），将LPS诱导的大鼠分为6组，通过比色法、ELISA、Western blot、HE染色、免疫组化等实验方法进一步研究ALKBH5靶向TRAF1通过NF-κB通路减轻心肌细胞损伤的作用机制。结果 ALKBH5和TRAF1在SIMD患者中表达水平下调，且Pearson分析显示两者呈正相关（P < 0.001）；细胞体外实验表明，过表达TRAF1促进细胞的增殖，抑制炎症因子和NF-κB通路相关蛋白的表达，而敲低ALKBH5得到了相反的结果的；大鼠体内实验结果显示，敲低ALKBH5促进心肌细胞的损伤、炎症因子和NF-κB相关通路蛋白表达以及NF-κB p65蛋白的核易位，而过表达TRAF1得到了相反的结果。结论 ALKBH5通过减少TRAF1的甲基化增加TRAF1的稳定性，从而抑制NF-κB通路，进而减轻SIMD。

关键词: 脓毒症心肌损伤, ALKBH5, TRAF1, NF-κB

Abstract:

Objective To investigate the molecular mechanism of ALKBH5 reducing sepsis-induced myocardial dysfunction （SIMD）. Methods The expression levels of ALKBH5 and TRAF1 in the blood of 50 SIMD patients and 50 healthy individuals were detected using reverse transcription fluorescence quantitative polymerase chain reaction （RT-qPCR）， and the correlation between their expression levels was analyzed by person analysis； In vitro experiments， H9C2 myocardial cells were divided into 7 groups according to over expression of TARF1 and knockdown ALKBH5. The molecular mechanism of ALKBH5 targeting TRAF1 to regulate lipopolysaccharide （LPS）induced myocardial cell damage was studied through experiments such as CCK8， ELISA， and Western blot； In the in vivo experiment of rats， LPS induced rats were divided into 6 groups according to over expression of TARF1 and knockdown ALKBH5. Experimental methods such as colorimetry， ELISA， Western blot， HE staining， and immunohistochemistry were used to study the mechanism of ALKBH5 targeting TRAF1 through NF- κB pathway in reducing myocardial cell damage. Results The expression levels of ALKBH5 and TRAF 1 were downregulated in SIMD， and the Pearson analysis showed a positive correlation between them（P < 0.001）； In vitro experiments showed that overexpression of TRAF1 promotes cell proliferation， inhibits the expression of inflammatory factors and proteins involved in the NF- κB pathway， and knockdown ALKBH5 obtain the opposite resulst； In vivo experiments in rats showed that knockdown ALKBH5 promotes injury in cardiomyocytes， expression of inflammatory factors and NF- κB-related pathway proteins， and nuclear translocation of NF-κB p65 protein， but the overexpression of TRAF 1 yielded the opposite results. Conclusion ALKBH5 increases the stability of TRAF1 by reducing its methylation， thereby inhibiting NF- κB pathway， thereby reducing SIMD.

Key words: sepsis -induced myocardial dysfunction, ALKBH5, TRAF1, NF-κB

中图分类号:

R364.5

刘敏,陈喜云,吕建磊,冯洁. ALKBH5通过TRAF1/NF⁃κB通路减轻脓毒症心肌损伤的机制[J]. 实用医学杂志, 2024, 40(17): 2381-2389.

Min LIU,Xiyun CHEN,Jianlei LÜ,Jie. FENG. ALKBH5 reduce septic-induced myocardial dysfunction by regulating the TRAF1/NF-κB pathway[J]. The Journal of Practical Medicine, 2024, 40(17): 2381-2389.

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时间	Control	LPS	LPS+sh-NC	LPS+sh-ALKBH5
0 h	1.01 ± 0.03	1.03 ± 0.14	0.98 ± 0.15	0.98 ± 0.18
24 h	2.03 ± 0.32	1.42 ± 0.28	1.35 ± 0.35	1.12 ± 0.29
48 h	3.01 ± 0.24	2.15 ± 0.25	2.23 ± 0.27	1.58 ± 0.32
72 h	4.12 ± 0.42	3.19 ± 0.31	3.35 ± 0.26	2.24 ± 0.35
t值	3.086^a		4.410^b
P值	0.037^a		0.012^b

组别	TNF-α	IL-6	IL-8
Control	5.67 ± 1.05	2.56 ± 1.28	2.88 ± 1.35
LPS	20.03 ± 2.09	15.95 ± 1.32	16.05 ± 2.32
t值	10.634	12.613	8.500
P值	< 0.001	< 0.001	0.001
LPS+sh-NC	19.83 ± 2.85	15.23 ± 1.45	16.55 ± 2.23
LPS+sh-ALKBH5	28.45 ± 2.07	25.98 ± 3.56	26.92 ± 3.34
t值	4.239	4.844	4.472
P值	0.013	0.008	0.011

组别	Control	LPS	LPS + pcDNA	LPS + pcDNA-TRAF1	LPS + pcDNA-TRAF1 + sh-NC	LPS + pcDNA-TRAF1 + sh-ALKBH5	LPS + pcDNA-TRAF1 + NF-κΒ activator 1
0 h	1.01 ± 0.02	1.03 ± 0.05	0.99 ± 0.11	0.98 ± 0.12	1.03 ± 0.05	0.95 ± 0.15	1.03 ± 0.05
24 h	2.05 ± 0.28	1.45 ± 0.16	1.38 ± 0.27	1.83 ± 0.34	1.85 ± 0.28	1.39 ± 0.32	1.42 ± 0.24
48 h	3.04 ± 0.22	2.14 ± 0.28	2.25 ± 0.24	2.96 ± 0.23	2.84 ± 0.16	2.22 ± 0.24	2.24 ± 0.21
72 h	4.15 ± 0.32	3.15 ± 0.21	3.46 ± 0.27	4.86 ± 0.25	4.89 ± 0.34	3.23 ± 0.23	3.35 ± 0.14
t值	4.525^a		6.600^b		8.311^e		0.772^d
P值	0.011^a		0.003^b		0.001^c		> 0.05^d

分组	TNF-α	IL-6	IL-8
Control	5.58 ± 1.08	2.66 ± 0.84	3.45 ± 1.32
LPS	20.15 ± 2.59	16.35 ± 2.28	16.25 ± 2.22
t值	8.993	9.759	6.305
P值	< 0.001	< 0.001	0.003
LPS + pcDNA	20.83 ± 1.67	16.23 ± 1.98	16.45 ± 1.23
LPS + pcDNA-TRAF1	15.46 ± 2.57	11.98 ± 1.56	11.86 ± 1.52
t值	3.035	2.920	4.066
P值	0.038	0.043	0.015
LPS + pcDNA-TRAF1 + sh-NC	15.35 ± 1.35	11.34 ± 1.54	11.45 ± 2.24
LPS + pcDNA-TRAF1 + sh-ALKBH5	20.42 ± 1.43	16.43 ± 1.34	16.82 ± 1.83
t值	2.962	3.748	3.611
P值	0.041	0.020	0.023
LPS + pcDNA-TRAF1 + NF-κΒ activator 1	21.45 ± 2.67	16.34 ± 2.21	17.02 ± 2.56
t值	0.589	0.048	0.110
P值^a	> 0.05	> 0.05	> 0.05

组别	TNF-α	IL-6	IL-8	cTnI
Control	5.58 ± 1.08	2.66 ± 0.84	3.45 ± 1.32	20.54 ± 3.45
LPS	20.15 ± 2.59	16.35 ± 2.28	16.25 ± 2.25	65.43 ± 6.65
t值	8.993	9.766	8.499	18.877
P值	0.001	0.001	0.001	< 0.001
LPS+pcDNA	20.83 ± 2.67	16.23 ± 2.98	16.45 ± 3.23	64.53 ± 5.65
LPS+pcDNA-TRAF1	10.46 ± 1.57	8.98 ± 1.47	8.92 ± 1.52	22.56 ± 3.21
t值	5.799	3.779	3.653	11.187
P值	0.004	0.020	0.022	< 0.001
LPS+pcDNA-TRAF1+sh-NC	9.35 ± 1.35	8.34 ± 1.54	8.45 ± 1.24	23.24 ± 2.84
LPS+pcDNA-TRAF1+sh-ALKBH5	20.42 ± 2.43	16.23 ± 2.34	16.82 ± 2.45	62.45 ± 7.23
t值	6.898	4.878	5.280	8.743
P值	0.002	0.008	0.006	0.001