简介：摘要目的探讨双氢睾酮（DHT）对人外周血早期内皮祖细胞（PB-EPCs）增殖、迁移功能的影响及RhoA/ROCK信号通路在其中的作用。方法取健康成人外周血分离、培养出早期EPCs并鉴定。分别以不同浓度DHT(1、10、100 nmol/L)干预，得出最佳浓度和最佳作用时间后用于后续干预实验。分组：对照组、DHT、RhoA抑制剂C3 exoenzyme+DHT组、ROCK抑制剂Y-27632+DHT组。检测各组EPCs的增殖、迁移能力。ELISA法检测各组细胞上清液液中VEGF蛋白含量。结果DHT在一定范围内呈浓度-时间依赖性促进EPCs增值、迁移功能，分别在浓度为10 nmol/L和24 h达到最大作用效果。与单纯10 nmol/L的DHT刺激组相比，C3 exoenzyme[(0.22±0.02) vs (0.26±0.05),P>0.05]和Y-27632[(0.21±0.04) vs (0.26±0.05), P>0.05]能够减弱DHT诱导的EPCs增殖，但差异无统计学意义。DHT对EPCs迁移能力的促进作用可被C3 exoenzyme[(35.26±4.27) vs (46.92±5.46), P<0.05]和Y-27632[(33.61±5.33) vs (46.92±5.46), P<0.01]抑制。DHT对EPCs分泌VEGF能力的促进作用可被C3 exoenzyme[(116.75±7.42) vs (156.80±21.74), P<0.05]和Y-27632[(121.73±5.33) vs (156.80 ±21.74), P<0.01]抑制。结论DHT呈一定的浓度-时间依赖性促进EPCs的增殖、迁移以及VEGF的表达，其中RhoA/ROCK信号通路参与调控了此过程。

简介：摘要目的探讨RhoA/Rho激酶1(ROCK1)下调抑制肌球蛋白轻链(MLC)磷酸化在主动脉夹层(AD)形成过程中的作用。方法检测正常(NA)和AD主动脉原代平滑肌细胞(SMC)中RhoA/ROCK1的表达与MLC的磷酸化程度。免疫荧光观察SMC中MLC磷酸化程度和结构。β-氨基丙腈(BAPN)联合应用ROCK1抑制剂Fasudil，验证RhoA/ROCK1下调在AD形成中的作用。组间比较采用t检验或χ2检验，Kaplan-Meier生存曲线采用非参数检验。结果NA组和AD组年龄(t＝－1.439，P>0.05)、性别(χ2＝0.900，P>0.05)差异无统计学意义，AD组高血压患者多于正常组(χ2＝5.562，P<0.05)。AD主动脉SMC中RhoA(NA：42 782±31 339，AD：6 975±3 130，t＝2.585，P<0.05)和ROCK1表达下调(NA：64 626±38 822，AD：13 851±961，t＝2.977，P<0.05)，MLC磷酸化减少(NA：230 193±27 749，AD：51 142±48 151，t＝6.561，P<0.01)。免疫荧光显示AD组SMC和Fasudil处理的SMC中MLC磷酸化降低，结构受损。Fasudil联合BAPN的夹层形成率(100%)高于BAPN的夹层形成率(50%，χ2＝22.780，P<0.01)。结论RhoA/ROCK1下调抑制MLC磷酸化促进了AD的发生。

简介：AbstractBackground:Dysuria is one of the main symptoms of genitourinary syndrome of menopause, which causes serious disruption to the normal life of peri-menopausal women. Studies have shown that it is related to decrease of detrusor contractile function, but the exact mechanism is still poorly understood. Previous results have suggested that the sphingosine-1-phosphate (S1P) pathway can regulate detrusor contraction, and this pathway is affected by estrogen in various tissues. However, how estrogen affects this pathway in the detrusor has not been investigated. In this study, we detected changes of the S1P/RhoA/Rho associated kinases (ROCK)/myosin light chain (MLC) pathway in the detrusor of ovariectomized rats in order to explore the underlying mechanism of dysuria during peri-menopause.Methods:Thirty-six female Sprague-Dawley rats were randomly divided into SHAM (sham operation), OVX (ovariectomy), and E groups (ovariectomy + estrogen), with 12 rats in each group. We obtained bladder detrusor tissues from each group and examined the mRNA and protein levels of the major components of the S1P/RhoA/ROCK/MLC pathway using quantitative real-time polymerase chain reaction and Western blotting, respectively. We also quantified the content of S1P in the detrusor using an enzyme linked immunosorbent assay. Finally, we compared results between the groups with one-way analysis of variance.Results:The components of the S1P pathway and the RhoA/ROCK/MLC pathway of the OVX group were significantly decreased, as compared with SHAM group. The percent decreases of the components in the S1P pathway were as follows: sphingosine kinase 1 (mRNA: 39%, protein: 45%) (both P < 0.05), S1P (21.73 ± 1.09 nmol/g vs. 18.86 ± 0.69 nmol/g) (P < 0.05), and S1P receptor 2/3 (S1PR2/3) (mRNA: 25%, 27%, respectively) (P < 0.05). However, the protein expression levels of S1PR2/3 and the protein and mRNA levels of SphK2 and S1PR1 did not show significant differences between groups (P > 0.05). The percent decreases of the components in the RhoA/ROCK/MLC pathway were as follows: ROCK2 (protein: 41%, mRNA: 36%) (both P < 0.05), p-MYPT1 (protein: 54%) (P < 0.05), and p-MLC20 (protein: 47%) (P < 0.05), but there were no significant differences in the mRNA and protein levels of RhoA, ROCK1, MYPT1, and MLC20 (all P > 0.05). In addition, all of the above-mentioned decreases could be reversed after estrogen supplementation (E group vs. SHAM group) (all P > 0.05).Conclusion:In this study, we confirmed that ovariectomy is closely associated with the down-regulation of the S1P/RhoA/ROCK/MLC pathway in the rat detrusor, which may be one mechanism of dysuria caused by decreased contractile function of the female detrusor during peri-menopause.