Effects of fasudil hydrochloride on ROCK2 protein and ferroptosis in hippocampus during early brain injury in rats with subarachnoid hemorrhage / 中华行为医学与脑科学杂志

Objective:To investigate the effects of fasudil hydrochloride(FH) on Rho-associated kinase 2(ROCK2) protein and ferroptosis in hippocampal area during early brain injury in rats with subarachnoid hemorrhage(SAH).Methods:Total 36 SPF grade Sprague-Dawley rats were divided into 3 groups by random number table method: Sham group, SAH group and SAH+ FH (a ROCK2 protein inhibitor) group (FH goup) with 12 rats in each group.SAH animal model was established by internal carotid artery perforation.The rats in FH group were injected intraperitoneally with FH(15 mg/kg) 30 minutes after successful modeling, and rats in Sham group and SAH group were injected intraperitoneally with the same volume of 0.9% sodium chloride solution.Twenty-four hours after the intervention, shuttle box test was used to observe the learning and memory ability of rats.The Fe 2+ content in rat hippocampus tissue was detected by colorimetry, and the protein levels of ROCK2 and ferroptosis-related long-chain acyl-CoA synthetase 4(ACSL4) and glutathione peroxidase 4(GPX4) in hippocampus were detected by immunohistochemistry and Western blot.Statistical analysis was performed by SPSS 20.0 software.One-way ANOVA was used for multigroup comparison, and LSD test was used for further pairwise comparison. Results:(1)In the shuttle box test, there were statistically significant differences in the number of avoidance reactions and avoidance reaction time of rats among the three groups( F=20.348, 22.316, both P<0.05). The number of avoidance reaction in SAH group was less than that in Sham group ((17.92±2.94) times, (27.13±3.48) times, P<0.05), the time of avoidance reaction in SAH group was longer than that in Sham group ((9.15±2.87) s, (3.68±1.09) s, P<0.05), while the number of avoidance reaction in FH group ((21.63±4.11) times) was more than that in SAH group, and the time of avoidance reaction ((6.08±1.76) s) was shorter than that in SAH group (both P<0.05). (2) The colorimetry results showed that there was a statistically significant difference in the content of Fe 2+ in hippocampus of rats among the three groups( F=7.965, P<0.05). The Fe 2+ content in SAH group was significantly higher than that of Sham group((0.091±0.032) nmol/mg, (0.038±0.024) nmol/mg, P<0.05), and the Fe 2+ content in the FH group ((0.065±0.021) nmol/mg) was lower than that of SAH group ( P<0.05). (3) There were significant differences in the number of ROCK2, ACSL4 and GPX4 positive cells in hippocampus of rats among the three groups in immunohistochemistry ( F=7.602, 14.171, 36.077, all P<0.05). The positive cells of ROCK2 and ACSL4 in SAH group ((21.63±4.72), (55.13±19.41)) were significantly higher than those of Sham group ((11.63±3.62), (23.38±3.74)) (both P<0.05), and the positive cells of ROCK2 and ACSL4 in FH group ((15.88±6.64), (44.75±8.29)) were both lower than those of SAH group(both P<0.05), while the number of GPX4 positive cells in SAH group (25.38±6.30) was significantly lower than that of Sham group (60.25±10.36) ( P<0.05), and the number of GPX4 positive cells in FH group (45.13±7.51) was higher than that of SAH group( P<0.05). (4)The results of Western blot showed that there were significant differences in the expression levels of ROCK2, ACSL4 and GPX4 proteins in the hippocampus of rats among the three groups( F=4.812, 12.573, 10.849, all P<0.05). The protein expression levels of ROCK2 and ACSL4 in SAH group were significantly higher than those in Sham group(both P<0.05), and the protein expression levels of ROCK2 and ACSL4 in FH group were lower than those in SAH group (both P<0.05), while the expression level of GPX4 protein in SAH group (0.27±0.09) was significantly lower than that in Sham group( P<0.05), and the expression level of GPX4 protein in FH group was higher than that of SAH group ( P<0.05). Conclusion:FH can inhibit ferroptosis in the hippocampus and improve the learning and memory ability of rats, and the mechanism may be related with down-regulation of ROCK2 protein.

Rho-Associated Kinase 2 Polymorphism in Patients With Vasospastic Angina

BACKGROUND AND OBJECTIVES: Recent studies indicate that in response to vasoconstrictor stimuli, the small GTPase RhoA and its down-stream effector, Rho-associated kinase 2 (ROCK)/Rho-kinase, are associated with hypercontraction of the vascular smooth muscle of coronary arteries through augmentation of myosin light chain phosphorylation and Ca2+ sensitization. Expression of ROCK/Rho-kinase mRNA was significantly increased and up-regulated in the spastic coronary artery in a porcine model, and a specific inhibitor of ROCK/Rho-kinase inhibited coronary artery spasm in humans. We therefore explored the role of ROCK2 polymorphisms in the pathogenesis of vasospastic angina (VA). SUBJECTS AND METHODS: We studied 106 patients with VA who exhibited spontaneous or provoked coronary spasm during coronary angiography and compared the prevalence of ROCK2 polymorphisms between this group of patients with VA and controls whose angiograms were normal, and in whom the ergonovine test did not cause spasm (n=107). Five single nucleotide polymorphisms (SNPs) of the ROCK2 gene were selected. SNPs were genotyped by high-resolution melting. Linkage disequilibrium and haplotype analyses were performed using the SHEsis program. RESULTS: The prevalence of genotypes of the 5 interesting SNPs in patients with VA was not different from that in the control group. In haplotype analysis, the haplotype G-T-C-T-G (in order of rs978906, rs2271621, rs2230774, rs1515210, and rs3771106) was significantly associated with a decreased risk of VA (p=0.007). CONCLUSION: The haplotype G-T-C-T-G in the ROCK2 gene had a protective effect against VA, suggesting the involvement of ROCK2 in VA pathogenesis.

The relationship of serum RhoA/ROCK-2 signal transduction pathway in patients with OSAHS and pulmonary hypertension / 中国综合临床

Objective To investigate the role of RhoA/Rho associated kinase-2 (RhoA/ROCK-2) in the development of pulmonary hypertension (PH) in patients with obstructive sleep apnea-hypopnea syndrome (OS-AHS). Methods Thirty patients diagnosed as OSAHS by polysomnograshy(PSG) test in our sleep laberatoty were recruited as the observation group, and fifteen healthy subjects matched in gender, age and body mass index (BMI) were recruited as the controls. Pulmonary arterial pressure was measured by echocardiography. Serum RhoA/ROCK-2 levels were measured. Results The level of PAP was (47.30±12.85)mm Hg in OSAHA patients complicated with PH, (22.31±3.07)mm Hg in OSAHA patients without PH, which were significantly higher than that in the controls (19.47±1.92) mm Hg (W=175.50, P < 0.05). The serum RhoA and ROCK-2 in OSAHA patients with-out PH (10.43±3.10 and 22.31±16.10 μ/L, respectively) were significantly higher those in the controls (2.94±1.20)μg/L and (6.04±0.28)μg/L, respectively) (W=120.00, W= 121.00, respectively, P<0.05), whereas significantly lower than that in OSAHA patients complicated with PH(14.85±8.49)μg/L, (36.81±12.69) μg/L, respectively) (H =29.172, H =30.242, respectively, P <0.05). There was a positive correlation between PAP and AHI in patients with OSAHS, whether complicated with PH or not(r_s=0.793, r_s=0.887,P <0.05), and there was a negative correlation between PAP and LSaO2 in patients with OSAHS (r_s=-0. 562,r_s = -0.751, P <0.05). There were positive correlations between the level of RboA/ROCK-2 and PAP in patients with OSAHS(r_s = 0.793,r_s = 0.887,P < 0.05). Finally, there was a positive correlation between the level of RhoA and ROCK-2 in patients with OSAHS (r_s = 1.000,r_s = 1.000,P < 0.05). Conclusions OSAHS is an inde-pendent risk factor for pulmonary hypertension. The levels of serum RhoA/ROCK-2 in OSAHS patients with PH were increased in the development of the disease. It may play an important role in the process of pulmonary hyper-tension in patients with OSAHS.