H2S 通过抑制海马 ROCK2 在 Thr436 和 Ser575 位点的表达和激活，保护大鼠脑缺血再灌注损伤。

IF 4.2 3区医学 Q1 PHARMACOLOGY & PHARMACY

European journal of pharmacology Pub Date : 2024-10-30 DOI:10.1016/j.ejphar.2024.177079

Fang Fang , Yi-Ning Guan , Mei-Jing Zhong , Ji-Yue Wen , Zhi-Wu Chen

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引用次数: 0

摘要

背景：H2S是一种内源性气体信号分子，它通过在Tyr722处磷酸化rho相关的含线圈蛋白激酶2（ROCK2），抑制ROCK2蛋白的表达和活性，从而保护脑缺血/再灌注（I/R）损伤。我们曾报道 H2S 通过抑制 ROCK2 在 Thr436 和 Ser575 处的磷酸化保护大鼠神经元免受体外缺氧/再缺氧损伤，但目前尚不清楚这两个位点是否参与了 H2S 对脑 I/R 损伤的保护作用：方法：在大鼠海马中转染野生型和突变型 ROCK2 真核质粒，通过结扎双侧颈总动脉建立 I/R 模型。通过水迷宫实验检测大鼠的行为缺陷，并用酶联免疫吸附法测定ROCK2、乳酸脱氢酶（LDH）、神经特异性烯醇化酶（NSE）和活性氧（ROS）。采用 Western-blot 分析法检测 ROCK2 的表达，采用 RT-qPCR 检测 bcl-2 和 Bax mRNA：结果：NaHS（4.8mg/kg）能明显抑制ROCK2wild-pEGFP-N1转染大鼠I/R后血清LDH、NSE和ROS的升高，但对ROCK2T436A-pEGFP-N1-和ROCK2S575F-pEGFP-N1-转染大鼠无明显影响；与 ROCK2wild-pEGFP-N1 转染大鼠相比，NaHS 对 I/R 增加的逃逸潜伏期和 I/R 减少的目标象限距离占总距离的百分比的抑制作用在 ROCK2T436A-pEGFP-N1- 或 ROCK2S575F-pEGFP-N1 转染大鼠中明显减弱或消失；NaHS明显抑制了ROCK2wild-pEGFP-N1-转染大鼠海马ROCK2和GFP-ROCK2蛋白、Bax mRNA和ROCK2活性的升高以及海马bcl-2 mRNA的降低，但对ROCK2T436A-pEGFP-N1-或ROCK2S575F-pEGFP-N1-转染大鼠无明显影响。结论H2S 通过 Thr436 和 Ser575 位点抑制海马 ROCK2 的表达和激活，从而保护大鼠的脑 I/R 损伤。

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H2S protects rat cerebral ischemia-reperfusion injury by inhibiting expression and activation of hippocampal ROCK2 at the Thr436 and Ser575 sites

Background

H₂S is an endogenous gas signal molecule, which protects cerebral ischemia/reperfusion (I/R) injury by phosphorylating rho-associated coiled coil-containing protein kinase 2 (ROCK₂) at Tyr722, and inhibiting ROCK₂ protein expression and activities. We previously reported that H₂S protected rat neurons from hypoxia/reoxygenation injury in vitro through inhibiting phosphorylation of ROCK₂ at Thr436 and Ser575, but it is unclear whether these two sites are involved in protection of H₂S against cerebral I/R injury.

Method

Rats transfected with wild-type and mutant eukaryotic plasmids of ROCK₂ in hippocampus were used to establish I/R model by ligating bilateral common carotid artery. Rat behavioral deficit was detected by water maze assay, and ROCK₂, lactate dehydrogenase (LDH), nerve-specific enolase (NSE) and reactive oxygen species (ROS) were determined by ELISA. ROCK₂ expressions was examined by western-blot assay, and bcl-2 and Bax mRNAs were examined by RT-qPCR.

Results

NaHS (4.8 mg/kg) significantly inhibited the I/R-increased serum LDH, NSE and ROS in the ROCK₂^wild-pEGFP-N1-transfected rats, but had no obvious effect in the ROCK₂^T436A-pEGFP-N1- or the ROCK₂^S575F-pEGFP-N1-transfected rats; inhibitions of NaHS on the I/R-increased escape latency and the I/R-decreased percentage of target quadrant distance to total distance were markedly attenuated or abolished in the ROCK₂^T436A-pEGFP-N1- or the ROCK₂^S575F-pEGFP-N1-transfected rats compared with those in the ROCK₂^wild-pEGFP-N1-transfected rats; NaHS obviously inhibited the I/R-increased hippocampal ROCK₂ and GFP-ROCK₂ proteins, Bax mRNA, and ROCK₂ activity, as well as the I/R-decreased hippocampal bcl-2 mRNA in the hippocampus of the ROCK₂^wild-pEGFP-N1-transfected rats, but had no significant effect in the ROCK₂^T436A-pEGFP-N1- or the ROCK₂^S575F-pEGFP-N1-transfected rats.

Conclusion

H₂S protects cerebral I/R injury in rats by inhibiting expression and activation of hippocampal ROCK₂ via the Thr436 and Ser575 sites.

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来源期刊

European journal of pharmacology 医学-药学

CiteScore

9.00

自引率

0.00%

发文量

572

审稿时长

34 days

期刊介绍： The European Journal of Pharmacology publishes research papers covering all aspects of experimental pharmacology with focus on the mechanism of action of structurally identified compounds affecting biological systems. The scope includes: Behavioural pharmacology Neuropharmacology and analgesia Cardiovascular pharmacology Pulmonary, gastrointestinal and urogenital pharmacology Endocrine pharmacology Immunopharmacology and inflammation Molecular and cellular pharmacology Regenerative pharmacology Biologicals and biotherapeutics Translational pharmacology Nutriceutical pharmacology.