Role of CSE-Produced H2S on Cerebrovascular Relaxation via RhoA

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Role of CSE-produced H2S on cerebrovascular relaxation via RhoAROCK inhibition and cerebral ischemia-reperfusion injury in mice Jiyue Wen, Shanshan Gao, Fanglin Chen, Shuo Chen, Mei Wang, and Zhiwu Chen ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00533 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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ACS Chemical Neuroscience

Role of CSE-produced H2S on cerebrovascular relaxation via RhoA-ROCK inhibition and cerebral ischemia-reperfusion injury in mice

Ji-Yue Wen1*, Shan-Shan Gao1*, Fang-Lin Chen1*, Shuo Chen2, Mei Wang3#, Zhi-Wu Chen1# 1Department

of Pharmacology, Anhui Medical University, Hefei, Anhui 230032, China

2Department

of Physiology, Anhui Medical University, Hefei, Anhui 230032, China

3Department

of pharmacy, Children’s Hospital of Soochow University, Suzhou, Jiangsu 215025, China

Running title: CSE-produced H2S induced cerebrovascular relaxation and cerebral protection

*: contributed equally to this work.

To whom correspondence should be addressed Zhi-Wu Chen, E-mail: [email protected], Phone: 86-0551-6516-1133 Mei Wang,

E-mail: [email protected]

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Abstract Role of CSE-produced H2S on cerebrovascular relaxation and cerebral ischemia-reperfusion (I/R) injury were investigated using CSE knockout (CSE-/-) and wild-type (CSE+/+) mice. The relaxation of cerebral basilar artery (BA) to CSE-produced H2S and its mechanism were detected. The results revealed that both NaHS, donor of exogenous H2S and ROCK inhibitor Y27632 could induce significant relaxation of BA, but the relaxation of BA to NaHS was significantly attenuated by Y27632. In addition, removal of endothelium could reduce the relaxation of BA to Y27632; CSE knockout also significantly attenuated Y27632-induced BA relaxation with endothelium rather than without endothelium. By contrast, the contraction of the BA from CSE-/- mice to RhoA agonist LPA or U46619 was stronger than that from CSE+/+ mice. Furthermore, RhoA activity and ROCK protein expression remarkably increased in the BA VSMCs from CSE-/- mouse, which were inhibited by NaHS pretreatment. These findings revealed that the CSE-produced H2S induced cerebrovascular relaxation is generated from endothelial cells and the mechanism of vascular relaxation may relate to inhibition of RhoA-ROCK pathway. We next sought to confirm the protective effect of CSE-produced H2S on cerebral I/R injury produced by middle cerebral artery occlusion and bilateral common carotid artery occlusion in mice. We investigated the changes of neurological deficit, cerebral infarct, brain water content, LDH decrease, MDA increase as well as impairment of learning and memory function. The results showed that the cerebral injury became more grievous in CSE-/- mice than that in CSE+/+ mice, which could be remarkably alleviated by NaHS pretreatment. Keywords: Hydrogen sulfide; RhoA-ROCK pathway; ischemia/reperfusion; cerebral basilar artery; learning and memory function INTRODUCTION Cerebral ischemic stroke is one of main causes responsible for long-term disability and death over the world. Cerebral ischemia-reperfusion (I/R) occurs when blood supply returns following a period of ischemia. Inflammation and lipid peroxidation have been reported to be involved in cerebral I/R injury, which can induce various degrees of nerve injury, and affect brain function, especially in learning and memory function1. Autoregulation of cerebral blood vessels after I/R is of great importance to protect nerve cell against ischemia injury2, 3. Hydrogen sulfide, a chemical compound with the formula H2S, is regarded as a pollutant and hazardous toxic gas for a long time. Currently, endogenous H2S has been found to be involved in various 1


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physiological and pathological processes such as the regulation of vascular tone and blood pressure, and has been considered as the third gaseous signal molecule besides nitric oxide (NO) and carbon monoxide 4, 5.

In mammals, endogensous H2S is generated from L-cysteine by cystathionine β-synthas (CBS) and

cystathionine-γ-lyase (CSE), and from β-mercaptopyruvic acid in mitochondria by 3-mercaptopyruvate sulfurtransferase (3-MST)5. In vasculature, the endogenous H2S is primarily produced by CSE in endothelium from L-cysteine 6. Our previous studies have demonstrated that vascular H2S mediated the hyperpolarization and dilation of rat cerebral arteries including the basilar artery (BA) and the middle cerebral artery (MCA)7, 8. But the role and mechanism of CSE-produced H2S on cerebrovascular function and the potential protective role of CSE-produced H2S against ischemia injury are still unclear. The small G protein RhoA and its downstream effectors of Rho-associated coiled coil-forming kinase (ROCK) have important roles in many cardiovascular pathogenesises. ROCK is ubiquitously expressed, including in VSMCs. RhoA-ROCK signaling pathway is involved in regulation of vascular tension, and participates in vascular smooth muscle cells contraction in many vascular beds 9, 10. RhoA-ROCK signaling pathway is activated when cerebral I/R injury occurs. Inhibition of ROCK could increase cerebral blood flow after MCA occlusion in mice and exert the protective effect on neuron against ischemia injury11, 12. These findings indicated that blocking RhoA-ROCK pathway could produce cerebrovascular dilation. Moreover, ROCK inhibitor Y-27632 could enhance recovery of rabbit from ischemia by preventing arteriolar vasoconstriction after cerebral I/R injury13. Furthermore, it has been pointed out that vasodilation to endothelial NO is partly produced by inhibiting the RhoA-ROCK signaling pathway14, 15. All of obove findings confirmed that inhibition of the RhoA-ROCK signaling pathway could produce the cerebrovascular relaxation, which is very benefial to exert cerebral protection against ischemia injury. Like endothelial NO, the endothelial CSE-produced H2S is already an important vascular relaxing factor, whether RhoA-ROCK pathway also participates in its cerebral vasodilatation and subsequent cerebroprotection? Hence, in the present study, the role and mechanism of CSE-produced H2S in cerebrovascular function were investigated and the cerebral protection of CSE-produced H2S against I/R injury was also further confirmed. RESULTS AND DISCUSSION The neuroprotective therapy of ischemic stroke depends on a functional vasculature. Therefore, vascular protection is regarded as a therapeutic approach to limiting stroke-induced damage16, 17. 2



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Previous studies have demonstrated that the H2S possess the therapeutically beneficial role in the treatment of moderate cerebral I/R injury18. However, the effect of endogenous H2S in cerebral I/R injury is unclear, and the potentially protective mechanism of endogenous H2S from vasculature to neuron is especially needed to explore. In view of the fact that the endogenous H2S in vasculature is primarily produced by CSE6. In addition, accumulating evidences have suggested that RhoA-ROCK pathway participates in the contraction of smooth muscle cells in various vascular beds19-21. Hence, the present study firstly focused on cerebrovascular artery to explore the role of CSE-produced H2S on vascular relaxation and determine whether RhoA-ROCK pathway is associated with the changes of cerebrovascular function induced by CSE-produced H2S. Exogenous H2S induced relaxation of BA First of all, we investigated the role of NaHS, exogenous H2S donor on the precontracted BA. As shown in Fig. 1, 1×10-6 ~ 1×10-3 mol/L NaHS, exogenous H2S donor induced a concentration-dependent relaxation in the BA from CSE+/+ mice, this relaxation was partially but markedly attenuated by pretreatment with 3.0 μmol/L Y27632, ROCK inhibitor22 prior to adding vasoconstrictor, the Emax being reduced from 92.4 ± 1.9% to 82.7 ± 3.0. The result suggested that H2S-induced relaxation of mice BA was partially involved in inhibition of the RhoA-ROCK pathway. Role of RhoA-ROCK pathway in H2S-induced relaxation of BAs We next explored the role of CSE-produced H2S on vascular relaxation and determined whether RhoA-ROCK pathway is associated with the changes of cerebrovascular function induced by CSE-produced H2S. Role of CSE-produced H2S in LPA- and U46619-induced vasoconstriction As shown as in Fig. 2A, compared with the vehicle group, RhoA agonist LPA (10 ~ 160 μmol/L) or U46619 (1×10-9 ~ 1×10-6 mol/L) caused significant and concentration-dependent constriction with Emax of 0.30 ± 0.01 mN (LPA) and 1.41 ± 0.10 mN (U46619) in the BAs from CSE+/+ mice, respectively. Fig. 2B showed that the contractions of BAs to LPA or U46619 from CSE-/- mice were profoundly increased than those from CSE+/+ mice, the Emax being increased to 0.44 ± 0.05 mN (LPA) and 2.13 ± 0.17 mN (U46619), respectively. These results indicated that activation of RhoA could produce constriction of mouse BA, and the BA constriction to RhoA agonist LPA or U46619 was more significant from CSE knockout mouse than that from wild-type mouse, suggesting that RhoA-ROCK pathway may involve in CSE-produced H2S-induced relaxation of mouse cerebral artery. 3


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Role of CSE-produced H2S in Y27632-induced vasodilation And then, we investigated the role of CSE-produced H2S in Y27632-induced vasodilation to confirm the relationship between the RhoA-ROCK pathway and CSE-produced H2S-induced relaxation of mouse cerebral artery. The result is shown in Fig 3A, at the ranges of 1×10-7 ~ 1×10-5 mol/L, Y27632, ROCK inhibitor induced a significant and concentration-dependent relaxation in the BA from CSE+/+ mice with Emax of 103.9 ± 16.8%, and the relaxation to Y27632 at the concentration of 1×10-6 and 1×10-5.5 mol/L was significantly decreased in the BA from CSE-/- mice, but the maximal relaxation (Emax: 95.2 ± 18.0%) was not significantly reduced compared to that in the BA from CSE+/+ mice. The results revealed that CSE knockout could decrease Y27632-induced relaxation of mouse BA. These findings are consistent with prior work showing that CSE-produced H2S participate in ROCK inhibition mechanism of vasorelaxation. Role of vascular endothelium in Y27632-induced relaxation It was well known that endothelium releases H2S and other relaxing factors such as NO and PGI2 to relax VSMC23.Therefore, we next sought to demonstrate the further effect of vascular endothelium in Y27632-induced relaxation by endothelial removal. As shown in Fig. 3B, Y27632-induced relaxation of the BA from CSE+/+ mice or CSE-/- mice was significantly weakened by removing vascular endothelium, but the relaxation in endothelium-denuded BA is still significant compared with that in the vehicle group, suggesting non-endothelial mechanism partially involved in Y27632-induced vasodilatation. Unlike the relaxation of the BA with endothelium, there is no significant difference between the relaxation of endothelium-denuded BAs from CSE+/+ mice and CSE-/- mice. These results suggested that CSE-produced H2S induced cerebrovascular relaxation is generated from endothelial cells, and CSE in VSMC did not mediate Y27632-induced relaxation. That is to say, endothelial CSE-produced H2S or/and other endothelial relaxing factors participated in inhibition of the RhoA-ROCK pathway and subsequent relaxation in VSMC.This is agreement with the previous study which revealed that CSE protein predominantly localized in endothelium, with faint expression in VSMC6. Hence, VSMC might not have enough CSE-produced H2S to mediate the Y27632-induced relaxation. CSE in endothelial cell, but not in VSMC, is associated with the RhoA-ROCK pathway inhibition-induced vasodilatation of the BA. Combing with fact that pretreatment of Y27632 partially but markedly attenuated relaxation of NaHS (Fig.1), these findings revealed that the dilation of the BA induced by endothelial CSE-produced H2S was at least partially involved to RhoA-ROCK pathway. 4



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Effect of CES-produced H2S on RhoA activity and ROCK proteins expression Due to that RhoA-ROCK pathway inhibition and subsequent relaxation occur in the VSMC24, primary cultured VSMC of rat BA was used in the present study. As shown in the Fig. 4A, RhoA activity was significantly increased by the application of LPA (10 μmol/L) in the BA VSMCs from CSE+/+ mice. Compared with the vehicle group of CSE+/+ mice, the RhoA activity also profoundly increased in the vehicle group of CSE-/- mice, but incubation with 100 or 200 μmol/L NaHS for 24 h could remarkably inhibit the increase of RhoA activity. This result suggested that endothelial CSE-produced H2S could inhibit activation of RhoA in mouse BA VSMCs. Furhermore, exposure to 10 μmol/L LPA significantly increased expressions of both ROCK1 and ROCK2 proteins in the BA VSMCs from CSE+/+ mice (Fig. 4B). Similarly to RhoA activity, ROCK1 and ROCK2 proteins markedly increased in the BA VSMCs from CSE-/- mice (p < 0.05), but the increases were remarkably inhibited by 100 µmol/L or 200 µmol/L NaHS treatment, and Y27632 had similarly inhibitory effect. The results showed that absence of CSE could increase expression of ROCKs, whereas supplement of H2S could inhibit the expressions. It was observed that knocking out CSE caused significant increase of RhoA activity as well as elevations of ROCK1 and ROCK2 protein expression in the BA VSMCs. Nevertheless, NaHS could remarkably inhibit the increase of RhoA activity and the elevations of ROCK1 and ROCK2 protein expression. This indicates that CSE-produced H2S can inhibit RhoA-ROCK pathway in mouse cerebral artery VSMCs, which not only accord with the aforementioned partial involvement of RhoA-ROCK pathway in CSE-produced H2S-induced dilation of mouse BA, but also explain the decrease of dilation of the BA to Y27632 in CSE knockout mice because the increased RhoA-ROCK pathway could lead to weaken inhibitory effect of Y27632. Effect of CSE knockout on MCAO-induced cerebral I/R injury in mice The vasorelaxation in mouse cerebral artery is very beneficial to protect cerebral I/R injury in mice17. Hence, we next sought to confirm the neuroprotective effect of CSE-produced H2S on cerebral I/R injury using mouse MCAO model, which is commonly used to assess the focal cerebral injury. Cerebral infarct is always used as a direct indicator of stroke outcome. In addition, the neurological deficits, vasogenic edemia are also relatively reliable indicators of cerebral injury25, 26. In present study, obvious aggravation of CSE knockout on cerebral I/R injury was indicated by that MCAO/reperfusion-induced neurological deficit, cerebral infarct and brain water content were significantly increased in CSE-/- mice than those in 5


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CSE+/+mice (Fig. 5). These findings suggested that CSE is involved in mouse cerebral I/R injury. Effect of CSE-produed H2S on Bilateral CCAs occlusion and reperfusion-induced injury in mice Furthermore, the bilateral CCA occlusion (2-VO) was used as a model of global cerebral ischemia, which was widely used in the previous studies27, 28and associated with a disruption of learning and memory function18. Cerebral I/R injury could produce large amounts of oxygen-free radicals in neurocytes and subsequently cause lipid peroxidation and cerebral damage. Hence, like LDH leakage, MDA, a product of lipid peroxidation, have also been applied to assess cerebral I/R injury29. As shown in Fig. 6A, compared with the sham group, there are significant decrease of LDH activity and increase of MDA content in CSE+/+ mouse or CSE-/- mouse cerebral tissues of the model groups. Interestingly, the LDH activity decrease and the MDA content increase were more significant in the model group of CSE-/- mice than those in the model group of CSE+/+ mice. The results indicated that cerebral I/R could cause significant cerebral injury, and the injury was more evident in CSE-/- mice than that in CSE+/+ mice. Whereas, H2S donor NaHS 2.4 and 4.8 mg/kg pretreatment obviously inhibited cerebral I/R-induced decrease of LDH activity and increase of MDA content in cerebral tissues of CSE+/+ mice and CSE-/- mice (Fig. 6B and C). The observations revealed that the 2-VO followed by reperfusion led to a significant injury in cerebrum of CSE+/+ mice and CSE-/- mice. This injury occurred more grievous in CSE-/- mice than that in CSE+/+ mice. Together with treatment effect of H2S donor NaHS on the injury suggested that CSE-produced H2S could inhibit cerebral I/R-induced LDH leakage and lipid peroxidation. This supports the aforementioned result that CSE-produced H2S could inhibit cerebral I/R injury. Effect of CSE-produed H2S on cerebral I/R-caused mouse learning and memory deficits Step down test in mice is a classical method to study learning and memory function30. As shown in Table 1, the examination of step down test showed that cerebral I/R significantly increased learning latency and mistake numbers of learning and memory, and decreased memory latency in both CSE+/+ mice and CSE-/- mice. Table 1 also showed that CSE deletion (CSE-/-) could markedly aggravate cerebral I/R-caused learning and memory deficits by comparison of the model group of CSE-/- mice and the model group of CSE+/+ mice. However, 2.4 and 4.8 mg/kg NaHS significantly alleviated the cerebral I/R-caused learning and memory deficits in CSE+/+ mice, and 4.8 mg/kg NaHS also reduced the deficits in CSE-/- mice. The resulted suggested that CSE-produced H2S may be related to alleviation of cerebral I/R-caused learning and memory deficits. 6



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Effects of CSE-produed H2S on impairment of mouse place navigation and spatial probe ability Morris water maze (MWM) test is a well-validated method for evaluating spatial learning memory ability in rodents31.As shown as in Fig. 7A, mice in each group almost exhibited the same situation in locating the platform on day 1 trainning, there was no significant difference among the average escape latencies of all groups. The performance of all mice got improvement with training, the average escape latencies on successive days decreased from day 2 to day 4. Regardless of CSE+/+ mice or CSE-/- mice, the latency of the model group was significantly longer than that of the sham group on day 3 and day 4, suggesting a cerebral I/R-caused impairment of place navigation ability. Fig. 7A also showed that the latency was significantly longer in the model group of CSE-/- mice compared with that in the model group of CSE+/+ mice, suggesting that CSE knockout could deteriorate cerebral I/R-caused impairment of place navigation ability. But 2.4 and 4.8 mg/kg NaHS pretreatment markedly inhibited the prolongations of latency in CSE+/+ mice and CSE-/- mice (Fig. 7B and C). Cerebral I/R-caused impairment of spatial probe ability was indicated by decreases in number of entry, time and distance crossing platform location compared with those of the sham group of CSE+/+ mice or CSE-/- mice (Table 2). The effect of CSE deletion on mice spatial probe ability was similar to that on the place navigation ability, number of entry and both proportions of time and distance in the model group of CSE-/- mice decreased significantly compared to those in the model group of CSE+/+ mice. However, 2.4 and 4.8 mg/kg NaHS pretreatment had significant treatment in CSE+/+ mice and CSE-/- mice. Our results showed that cerebral I/R significantly reduced learning memory function in aforementioned mouse step down test and spatial learning memory in MWM test. CSE knockout markedly aggravated the reductions of learning memory function and spatial learning memory ability in mice. Combining with the result that treating effect of NaHS on the impairments of learning memory function and spatial learning memory ability in mice subjected to cerebral I/R injury, it can be concluded that CSE-produced H2S could protect cerebral I/R-induced impairment of learning and memory functions in mice. Here, we have for the first time demonstrated that (1) CSE-produced H2S could induce relaxation of cerebral blood vessels; (2) RhoA-ROCK pathway inhibition may be at least partially involved to the H2S-induced cerebral vasodilatation; (3) CSE-produced H2S has a protective effect on mouse cerebral I/R injury, especially at the impairment of learning and memory function; (4) The protective effect may 7


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be related to CSE-produced H2S-induced cerebral vasodilatation in mice. Further studies are of crucial importance to elucidate the direct relationship between the cerebrovascular relaxation induced by CSE-produced H2S and ischemia protection. Although accumulated studies demonstrated that exogenous H2S has protective effect on ischemic stroke. But until now, exogenous H2S donor NaHS is not used in clinical medicine. It may related to the following reasons: increasing reports revealed that the protection of exogenous H2S in ischemia stroke depends on its concentration, for H2S was considered to be toxic at higher concentration and beneficial at low concentration32, 33, hence, the effective dose of NaHS is limited to narrow range; In addition, the speed of H2S generation is another concern, the commonly used H2S donors cause a large and rapid release of H2S. Therefore, further study is of pressing importance to demonstrate how to reach a defined and sustainable concentration range of H2S donors and assess those potential clinical applications. METHODS Reagents Lysophosphatidic acid (LPA), 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F2α (U46619), NaSH, acetylcholine (ACh), Y27632 and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma Chemical (St. Louis, USA); Malondialdehyde (MDA) and lactate dehydrogenase (LDH) assay kits were purchased from Nanjing Jiancheng Biological Co (Nanjing, China); G-LISA RhoA activation assay biochemistry kit was purchased from Cytoskeleton (Denver, USA); Mouse monoclonal antibodies against ROCK1 and ROCK2 were purchased from Nanjing Enogene Biological Co (Nanjing, China); Physiological salt solution (PSS) comprised the following (mM): NaCl 118, KCl 4.7, CaCl2 1.6, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 5.5, EDTA 0.026, adjusted pH to 7.4 with NaOH, and the solution was bubbled with 95% O2 and 5% CO2. NaHS was dissolved in distilled water just before the experiment and kept in the dark. Animals Adult wild type (CSE+/+) and CSE knockout (CSE-/-) C57BL/6J mice (with 20~24g body weight and aged 12~15 weeks, female to male=1:1) were provided by Shanghai biomodel organism science & technology development Co., Ltd. The animals were housed in the Animal Center of Anhui Medical University, with a controlled temperature 22 ± 2℃ and relative humidity 54±2%, with free to food and water. All experimental procedures were approved by the Ethics Review Committee of Anhui Medical University, which comply with the Guide for the Care and Use of laboratory Animals published by the 8



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US National Institutes of Health (NIH Publication no. 85-23, revised 2011). And every effort was taken to minimize pain or discomfort of mice. Experimental protocol In brain, CSE-produced H2S is generated in cerebrovascular tissue. Therefore, isolated cerebral artery was used to investigate CSE-produced H2S-induced cerebral vasodilatation and its relation to RhoA-ROCK pathway. Finally, together with application of NaHS, primary vascular smooth muscle cells (VSMCs) of cerebral artery from both wild type mice and CSE knockout mice were utilized to examine activity of the RhoA-ROCK pathway, illustrating inhibition of CSE-produced H2S on RhoA-ROCK pathway in the VSMCs. In animal model experiments, MCA occlusion in mice was applied to evaluate the role of CSE in cerebral I/R injury. The role of CSE was further to be confirmed in bilateral common carotid arteries (CCA) occlusion-induced cerebral I/R in mice, and exogenous H2S donor NaHS was given in CSE knockout mice to supplement deficiency of endogenous H2S so as to demonstrate role of CSE-produced H2S in cerebral I/R injury. Blood vessels experiment The blood vessels experiment was performed as previously described23, 34. Briefly, mice were anesthetized with 3.5% chloral hydrate (10 ml/kg) by intraperitoneal injection and killed by decapitation. The brain was rapidly harvested and put in ice-cold Krebs solution. Then, BA was carefully isolated from the brain and immersed in ice-cold PPS immediately. The artery was cut into segments of 3 mm in length, and the segment was inserted into two tungsten wires and mounted on a wire myograph in 5-ml chambers (DMT, Aarhus, Denmark) containing PSS at 37°C and continuously aerated with a mixture of 95% O2 and 5% CO2. Changes in isometric tension were measured by using the software. After equilibration under 0 tension resting state for 30 min, the segment was stretched progressively to determine an optimal resting tension (1~3 mN, according to different vessel diameter), which was automatically calculated by PowerLab Chart software. Then, after equilibration under the optimal resting tension, artery segment was precontracted with 60 mM KCl to check reproducible contractility of the individual BA segment. The artery was precontracted by adding 1×10 –7 mol/L U46619 until a stable contraction was obtained, followed by relaxation with NaHS or other relaxant agents. In order to examine endothelium dependency, the luminal surface of some artery segment was rubbed by a human hair to remove the endothelium. The functional denudation of the endothelium was confirmed by dilation to ACh < 30% at the beginning of each experiment. 9


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Primary cell culture Primary VSMCs were isolated from the BA utilizing tissue explantation and trypsin enzymatic digestion as described previously35. The VSMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum at 37˚C in a 5% CO2 incubator. When the cultures reached 80~90% confluency, Serum free medium containing LPA, Y27632, NaHS and vehicle was, respectively, added into the culture medium. After 24 h, the cultured cells were collected to extract proteins by using the method previously described36, 37 for measurements of RhoA activity and expressions of ROCK1 and ROCK2 proteins. Measurement of RhoA activity RhoA activity of primary VSMCs was measured by using luminescence-based G-LISA activation assay Biochem kitTM and detected at 490 nm using a microplate spectrophotometer38. Absorbance values were normalized by protein content using a colorimetric assay. Western blotting The total proteins extracted from primary VSMCs were performed as described previously36, 37. The total protein (30 μg) was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membranes. Membranes were cutted into three sections and blocked by Tris-buffer saline containing 5% skim milk and 0.05% Tween 20 at room temperature for 2 h, and then followed by incubation overnight at 4°C with the same buffer with the primary antibodies of ROCK1 or ROCK2 (Enogene, China) or monoclonal antibody against β-actin (Bioworld). After incubated with the appropriate secondary antibody for 1 h at 37°C, the bands were visualized using an enhanced chemiluminescence kit (Thermo, USA). β-actin in the same protein extracts were used as internal control, relative intensity of the band was determined by densitometry. Model of MCA occlusion in mice MCA occlusion (MCAO) of CSE+/+ and CSE-/- mice was performed as previously described 23, 34, with eight animals in each group . Mice were anesthetized with 3.5% chloral hydrate (10 ml/kg) by intraperitoneal injection and and fixed on a heated operation table to maintain body temperature at around 37.5 °C. The right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were isolated through a midline neck incision, respectively. After the ECA being ligated, a fish thread with a diameter of about 0.185 mm was gently inserted from the CCA to the ICA until it passed the MCA origin (approximately 15 mm) so as to occlude the MCA. After 2 h of occlusion, 10



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the thread was withdrawn and the MCA was reperfused for 16 h. After the reperfusion, abnormal neurological symptom in each mouse was examined and scored to evaluate the neurological function. Mouse was then sacrificed under anesthesia by an overdose of chloral hydrate. Brain was quickly harvested and sliced into 2 mm thick coronal sections. After being weighed to measure wet weight of brain. The slices from one brain were incubated in 2% TTC solution at 37°C for 30 min and subsequently fixed with 4% poly formaldehyde. Normal brain tissue was stained orange red, and infarct tissue was stained white. The slices were weighed, and the white infarction tissues were carefully separated and weighed. After that the total brain tissues including the normal and the infarct parts were dried at 105 °C for 48 h to measure dry weight of brain. Percentage of cerebral infarct and brain water content were calculated as follows, respectively: Percentage of cerebral infarct = weight of infract tissues / weight of all slices× 100%

(1)

Brain water content = (wet weight - dry weight) / wet weight × 100%.

(2)

Model of Bilateral CCAs occlusion in mice Mice were subjected to 20 min of bilateral CCAs occlusion by fastening as described previously 18.

Briefly, mice were anesthetized with 3.5% chloral hydrate by intraperitoneal injection (10 ml/kg).

Bilateral CCAs were exposed by a midline incision in the neck, and loose threads were placed around the vessels. Then, the vessels were fully ligated for occlusion. After 20 min, the ligation was loosened to restore the blood flow and the skin was sutured. Mice were randomly assigned to 7 groups: the sham group of CSE+/+ mice, the sham group of CSE mice-/-, the model group of CSE+/+ mice, the model of CSE-/- mice, the 1.2, 2.4 and 4.8 mg/kg NaHS-treated CSE-/- mice groups,with eight animals in each group. The mice in the sham group or the model group were daily administered 0.9% saline solution via intraperitioneal injection for 3 days, the NaHS-treated CSE-/- mice were intraperitioneally injected daily for 3 days at a dose of 1.2, 2.4 and 4.8 mg/kg, respectively. 1 h after the third administration, a cerebral I/R injury was established by occlusion of bilateral CCAs in mice in the model group and the NaHS treatment groups. Mice in the sham group underwent exposure of bilateral CCAs but not ligation. The mice were respectively given each dose of NaHS or 0.9% saline solution at 10 h after bilateral CCAs occlusion. Step down test At 2 h after the last administration of NaHS or saline solution, mouse was placed on the floor of which was made of parallel stainless steel bars spaced 0.4 cm apart in a jumping apparatus 39, 40. After 11


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adapting 5 min, mouse received a mild electric foot shock (36 V) from the floor. The latency of mouse stepping onto the elevated rubber platform and the frequency of it stepping down the platform to expose electric shock within 5 min were respectively recorded for assessing as learning latency and number of learning errors. 24 h later, mice were placed onto the platform, and the latency and frequency of it stepping down the platform to expose electric shock within 5 min were assessed as the memory latency and the number of memory errors, respectively. Morris water maze test MWM consists of a circular pool (diameter 150 cm and height 60 cm) and a platform. The pool was divided into four equal quadrants (NE, SE, SW and NW) and filled with water to a depth of 21 cm. The platform was located in the center of the SW quadrant and submerged 1 cm below the water surface. The water was maintained at 22 ± 1℃ and made into milk-white by adding nontoxic titanium dioxide powder. Movement of mice in the maze was monitored by a computerized video tracking system connected to a computer39, 41, 42. The task consisted of two phases: place navigation and spatial probe. In place navigation phase, mice were subjected to four trials per day for four consecutive days. The mice were released into the water respectively from one of four starting points at the midpoint of the four quadrants perimeter. The mouse was allowed to swim freely until it reached and stayed on the platform. The time required to reach the platform was recorded as escape latency. If a mouse failed to locate the platform within 60 sec, it was gently guided to the platform and allowed to remain there for 15 sec before the start of next trial, and its escape latency was recorded as 60 sec. Subsequent starting points carried out in a clockwise direction for the next trials. The interval between trials given each day was approximately 30 secs. The result was expressed as average escape latency by calculating from four trials. For the second phase, the platform was removed from the pool. The mice were respectively released into water from the starting point of the NE quadrant and allowed to swim for 60 secs. Number of entry, time and distance of it crossing the SW quadrant (platform location) were measured. Biochemical measurements After the experiment, mice were sacrificed and the brains were harvested. The hemispheres were washed twice with cold saline solution, and then homogenized. After centrifuged, supernatants of the homogenization were collected. MDA content and lactate LDH activity were respectively detected at 532 nm and 450 nm by spectrophotometry according to the procedures provided by the assay kit. 12



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Statistical Analysis Blood vessel data is presented as mean ± SEM. The other data are expressed as mean ± SD. Statistical analyses were performed by one-way ANOVA followed by the Duncan test to determine the difference between groups. A value of p < 0.05 was to be considered statistically significant.

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Figure legends: Figure 1. NaHS-induced relaxation of the BAs from CSE+/+ mice and effect of Y27632 (3.0 µmol/L) on the relaxation (mean ± SEM, n = 8). **P

Role of CSE-Produced H2S on Cerebrovascular Relaxation via RhoA

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