Effects of hydrogen on expression of ROCK1 and mDia1 in intestinal tissues of septic mice / 中华麻醉学杂志

Objective To evaluate the effects of hydrogen on the expression of Rho-associated protein kinase 1(ROCK1)and mammalian diaphanous-related formin 1(mDia1)in intestinal tissues of septic mice.Methods Ninety male C57BL/6 mice,weighing 20-25 g,aged 6 weeks,were divided into 3 groups(n=30 each)using a random number table:control group(group C),sepsis group(group S)and sepsis plus hydrogen group(group SH).Sepsis was produced by cecal ligation and puncture(CLP).Group SH inhaled 2% hydrogen for 1 h starting from 1 and 6 h after CLP.Twenty mice in each group were selected and observed for 7-day survival rate.Ten mice in each group were sacrificed at 24 h after CLP,and blood samples were obtained from hearts to measure the activity of serum diamine oxidase(DAO)and to count the colony-forming units after bacterial culture.The small intestinal samples were obtained for microscopic examination of pathological changes and for determination of ROCK1 and mDia1 positive cell rates(using immunohistochemical staining)and expression of intestinal epithelial junctional protein E-cadherin(by immunofluorescent staining).Intestinal damage was assessed and scored.The ratio of ROCK1 to mDia1 positive cell rates(ROCK1/mDia1 ratio)was calculated.Results Compared with group C,the survival rate was significantly decreased,the serum DAO activity,colony counts and intestinal damage scores were increased,ROCK1 and mDia1 positive cell rates were increased,and the expression of E-cadherin was down-regulated in S and SH groups,ROCK1/mDia1 ratio was increased in group S(P0.05).Compared with group S,the survival rate was significantly increased,the serum DAO activity,colony counts and intestinal damage scores were decreased,the ROCK1 positive cell rate was decreased,the mDia1 positive cell rate was increased,ROCK1/mDia1 ratio was decreased,and the expression of E-cadherin was up-regulated in group SH(P<0.05).Conclusion The mechanism by which hydrogen improves intestinal barrier function may be related to down-regulation of ROCK1 expression,up-regulation of mDia1 expression and correction of the imbalance in ROCK1/mDia1 ratio in intestinal tissues of septic mice.

Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivo / 中华妇产科杂志

Objective To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA(shRNA) on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism.Methods Human ovarian cancer cell line HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer.Tumor-bearing nude mice were assigned randomizely to three groups:LentiRhoA-sh group,Lenti-negative control (NC) group and phosphate buffered saline (PBS) group.lentivirus mediated RhoA shRNA,negative control lentivirus and PBS were respectively injected in the three groups.Effects of treatment were observed by tumor growth curve,tumor volume,tumor weight,and tumor inhibition rate.Xenograft tissues and liver,spleen,lung,and renal tissues were examined by hematoxylin and eosin (HE) staining or were detected by streptavidin-perosidase (SP)immunochemical method.The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR,immunochemistry and Western blot assay.Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) method and apoptotic index (AI) were counted.Results Compared with Lenti-NC group and PBS group,the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days(P ＜ 0.01).Tumor volume (338 ± 114) mm3 decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group(1190 ± 332)mm3 and PBS group (1101 ± 396) mm3 (P ＜ 0.01).Tumor weight (0.23±0.11)g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group (0.79 ± 0.19)g and PBS group (0.74 ± 0.17)g (P ＜ 0.01).Real-time qPCR result shown that the expression of RhoA mRNA (0.30 ± 0.05) decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group (0.95 ±0.06) and PBS group(1.00 ±0.11 ; P ＜0.01).Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P ＜ 0.01).TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) ％ compared with those in Lenti-NC group(5.2 ±±2.0)％ and PBS group(6.0 ±2.1)％ (P ＜0.01).Conclusion Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA,inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.

Expression of RhoA and Rho kinase in junctional zone of human adenomyosis and its relationship with dysmenorrheal / 中华妇产科杂志

Objective To investigate the expression of RhoA and Rho kinase in junctional zone (JZ) of adenomyosis and normal myometrium and explore its relationship with severity of dysmenorrheal.Methods From Mar.to Dec.2012,32 cases with adenomyosis undergoing hysterectomy were enrolled as adenomyosis group including 18 cases with proliferative endometrium and 14 cases with secretory endometrium matched with 29 cases with hysterectomy due to cervical disease and ovarian tumor as control group including 12 cases with proliferative endometrium and 17 cases with secretory endometrium in Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University.JZ smooth muscle cells were isolated and cultured immediately after the operation.The expression of mRNA and protein of RhoA and ROCK Ⅰ in JZ in two groups were measured by real-time fluorescence quantitative RT-PCR and western blot.Results (1) The mRNA expression of RhoA and ROCK Ⅰ in JZ of adenomyosis group did not show cyclic change.In proliferative phase,the expression of RhoA and ROCK Ⅰ (1.41 ±0.16,1.05 ±0.15) was not significantly higher than that in secretory phase (1.17 ± 0.25,0.98 ± 0.10) (P ＞ 0.05).While JZ in control group,it showed obviously cyclic change.The expression level of them in proliferative phase(0.93 ±0.10,1.00 ± 0.18) was significantly higher than that in secretory phase (0.48 ± 0.03,0.55 ± 0.05) (P ＜0.05) ; It also showed that expressions of RhoA and ROCK Ⅰ in adenomysis group were significant higher than those in the control (P ＜ 0.05).(2) The mRNA and protein expression of RhoA and ROCK Ⅰ was positively correlated in each of two groups (r =0.48,P ＜ 0.01 ; r =0.67,P ＜ 0.01).(3) The expression of RhoA and ROCK Ⅰ were 1.66 ±0.19,1.32 ±0.11 in severe dysmenorrheal,1.28 ±0.12,1.09 ±0.08 in moderate dysmenorrheal and 0.93 ± 0.09,0.81 ± 0.06 in mild dysmenorrheal,it all reached statistical difference when compared the other group.(All P ＜ 0.05).Conclusions The expressions of RhoA and ROCK Ⅰ in JZ in adenomyosis group were higher than those in control group,and positively correlated with the severity of dysmenorrheal in adenomysis group,but it does not change with the menstrual cycle.High expression of RhoA and ROCK Ⅰ might be involved in abnormal contraction of uterine myometrium and correlated with the dysmenorrheal in adenomysis.

Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

Previously, we demonstrated that the p190 Rho guanine nucleotide exchange factor (p190RhoGEF) was induced following CD40 stimulation of B cells. In this study, we examined whether p190RhoGEF and a downstream effector molecule RhoA are required for B cell differentiation. Expression of p190RhoGEF positively correlated with the expression of surface markers and transcriptional regulators that are characteristic of mature B cells with plasma cell (PC) phenotypes. Moreover, either the overexpression of p190RhoGEF or the expression of a constitutively active RhoA drove cellular differentiation toward PC phenotypes. B cell maturation was abrogated in cells that overexpressed p190RhoGEF and a dominant-negative form of RhoA simultaneously. CD40-mediated maturation events were also abrogated in cells that overexpressed either dominant-negative p190RhoGEF or RhoA. Together, these data provide evidence that p190RhoGEF signaling through RhoA in CD40-activated B cells drives the induction of the PC differentiation.

Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing alpha-smooth muscle actin (alpha-SMA) via transforming growth factor-beta1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced alpha-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced alpha-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced alpha-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-beta1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-beta1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-beta1/Smad2 signaling pathway.

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47 PHOX. Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47 PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Changes in Myogenic Tone in Spontaneously Hypertensive Rat: Role of RhoA and Protein Kinase C

BACKGROUND AND OBJECTIVES: The myogenic response was originally described as a contraction of a blood vessel that occurred following an increase in intravascular distending pressure. Conversely, a reduction in intravascular pressure produces myogenic vascular relaxation. Recent attention has focused on the potential role of this myogenic mechanism in the control of tone in the resistance vasculature, and in particular on how this mechanism may contribute to the increased vascular resistance seen in hypertension. Therefore, in the present study, we investigated the role of myogenic tone in the generation and/or maintenance of hypertension. MATERICAL AND METHODS: Myogenic tone was developed by stretching of the basilar arteries of WKY (istar Kyoto rat) and SHR (spontaneously hypertensive rats). Contractile responses, PKC (protein kinase C) immunoblots and translocation of PKC and RhoA were measured. In the presence of extracellular Ca2+ the stretching of the resting vessel evoked a myogenic contraction in the basilar arteries of SHR and WKY. Myogenic tone was significantly greater in SHR than in WKY. However, in the absence of extracellular Ca2+, stretching evoked a myogenic contraction in SHR, but not in WKY. The stretch-induced myogenic tone was inhibited by nifedipine. The effect of nifedipine was similar in both SHR and WKY rats. H-7, calphostin C and Y-27632, also inhibited stretch-induced myogenic tone in both SHR and WKY. The inhibitory effects of these drugs were greater in SHR than in WKY. Immunoblotting showed rho A and PKC alpha were translocated from the cytosol to the cell membrane with stretching in both SHR and WKY. PKC beta, however, was translocated to the cell membrane with stretching in SHR, but not in WKY. CONCLUSION: These results suggest that stretch-induced myogenic tone is significantly greater in SHR than in WKY. Furthermore, the increase in amount and/or activity of PKC beta and ROK (rhoA-associated kinase) may be a key mechanism accounting for the enhanced myogenic tone in SHR.