Fang Fang, Guan Yi-Ning, Zhong Mei-Jing, Wen Ji-Yue, Chen Zhi-Wu
Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, 230032, PR China; Department of Pharmacy, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, PR China.
Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, 230032, PR China.
Eur J Pharmacol. 2024 Dec 15;985:177079. doi: 10.1016/j.ejphar.2024.177079. Epub 2024 Oct 30.
HS is an endogenous gas signal molecule, which protects cerebral ischemia/reperfusion (I/R) injury by phosphorylating rho-associated coiled coil-containing protein kinase 2 (ROCK) at Tyr722, and inhibiting ROCK protein expression and activities. We previously reported that HS protected rat neurons from hypoxia/reoxygenation injury in vitro through inhibiting phosphorylation of ROCK at Thr436 and Ser575, but it is unclear whether these two sites are involved in protection of HS against cerebral I/R injury.
Rats transfected with wild-type and mutant eukaryotic plasmids of ROCK 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 ROCK, lactate dehydrogenase (LDH), nerve-specific enolase (NSE) and reactive oxygen species (ROS) were determined by ELISA. ROCK expressions was examined by western-blot assay, and bcl-2 and Bax mRNAs were examined by RT-qPCR.
NaHS (4.8 mg/kg) significantly inhibited the I/R-increased serum LDH, NSE and ROS in the ROCK-pEGFP-N1-transfected rats, but had no obvious effect in the ROCK-pEGFP-N1- or the ROCK-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 ROCK-pEGFP-N1- or the ROCK-pEGFP-N1-transfected rats compared with those in the ROCK-pEGFP-N1-transfected rats; NaHS obviously inhibited the I/R-increased hippocampal ROCK and GFP-ROCK proteins, Bax mRNA, and ROCK activity, as well as the I/R-decreased hippocampal bcl-2 mRNA in the hippocampus of the ROCK-pEGFP-N1-transfected rats, but had no significant effect in the ROCK-pEGFP-N1- or the ROCK-pEGFP-N1-transfected rats.
HS protects cerebral I/R injury in rats by inhibiting expression and activation of hippocampal ROCK via the Thr436 and Ser575 sites.
硫化氢（HS）是一种内源性气体信号分子，它通过在Tyr722位点磷酸化含rho相关卷曲螺旋的蛋白激酶2（ROCK），并抑制ROCK蛋白表达及活性，从而保护脑缺血/再灌注（I/R）损伤。我们之前报道过，HS在体外通过抑制ROCK在Thr436和Ser575位点的磷酸化来保护大鼠神经元免受缺氧/复氧损伤，但尚不清楚这两个位点是否参与HS对脑I/R损伤的保护作用。
采用海马区转染ROCK野生型和突变型真核质粒的大鼠，通过结扎双侧颈总动脉建立I/R模型。采用水迷宫试验检测大鼠行为缺陷，采用酶联免疫吸附测定法（ELISA）检测ROCK、乳酸脱氢酶（LDH）、神经特异性烯醇化酶（NSE）和活性氧（ROS）。采用蛋白质免疫印迹法检测ROCK表达，采用逆转录定量聚合酶链反应（RT-qPCR）检测bcl-2和Bax mRNA。
硫氢化钠（NaHS，4.8mg/kg）显著抑制ROCK-pEGFP-N1转染大鼠I/R诱导升高的血清LDH、NSE和ROS水平，但对ROCK-pEGFP-N1-或ROCK-pEGFP-N1转染大鼠无明显影响；与ROCK-pEGFP-N1转染大鼠相比，NaHS对ROCK-pEGFP-N1-或ROCK-pEGFP-N1转染大鼠I/R诱导延长的逃避潜伏期及I/R诱导降低的目标象限距离占总距离百分比的抑制作用明显减弱或消除；NaHS明显抑制ROCK-pEGFP-N1转染大鼠海马区I/R诱导升高的ROCK和绿色荧光蛋白（GFP）-ROCK蛋白、Bax mRNA及ROCK活性，以及I/R诱导降低的海马区bcl-2 mRNA，但对ROCK-pEGFP-N1-或ROCK-pEGFP-N1转染大鼠无显著影响。
HS通过Thr436和Ser575位点抑制海马区ROCK的表达与激活，从而保护大鼠脑I/R损伤。
