Role of RhoA/ROCK2 signaling pathway in multiple exposures to sevoflurane-induced long-term cognitive impairment in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the role of RhoA/ROCK2 signaling pathway in multiple exposures to sevoflurane-induced long-term cognitive impairment in neonatal rats.Methods:Sixty SPF healthy neonatal Sprague-Dawley rats of either sex, aged 6 days, weighing 12-20 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), multiple exposures to sevoflurane group (group S) and RhoA/ROCK2 signaling pathway inhibitor Y-27632 group (group Y). Group S and group Y inhaled 3% sevoflurane for 2 h at days 6, 7 and 8 after birth.In group Y, Y-27632 5 mg/kg was intraperitoneally injected before sevoflurane anesthesia.The spontaneous activity was evaluated by open field test on day 35 after birth.The cognitive function was detected by Morris water maze test at day 36 after birth.The rats were sacrificed after Morris water maze test, and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons and cytoplasmic calcium concentration ([Ca 2+ ] i) (by flow cytometry) and expression of phosphorylated RhoA (p-RhoA), ROCK2 and cleaved-caspase-3 (by Western blot) and for microscopic examination of the ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:There was no significant difference in movement speed, distance and time of stay in the open field center in the open field test among the three groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal neurons and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological injury to hippocampal neurons was found in group S. Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, the apoptosis rate of hippocampal neurons and [Ca 2+ ] i were decreased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was down-regulated ( P<0.05), and the pathological injury to hippocampal neurons was attenuated in group Y. Conclusions:The mechanism by which multiple exposures to sevoflurane induces long-term cognitive impairment is related to activation of RhoA/Rock2 signaling pathway and induction of apoptosis rate of hippocampal neurons in neonatal rats.

Role of RhoA/ROCK2 signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective:To evaluate role of Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil protein kinase 2 (ROCK2) signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion (I/R) injury in rats.Methods:Eighty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 230-280 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group S), cerebral I/R group (group IR), cerebral I/R plus trilobatin group (group T) and cerebral I/R plus trilobatin plus RhoA/ROCK2 signaling pathway agonist AA group (group A). The model of focal cerebral I/R injury was developed by middle cerebral artery occlusion in anesthetized animals.Trilobatin 15 mg/kg was given by gavage twice a day for 3 consecutive days in T and A groups.RhoA/ROCK2 signaling pathway agonist AA 10 mg/kg was intraperitoneally injected before each administration by gavage in group A. Neurobehavioral score was assessed at 24 h of reperfusion, then the rats were sacrificed, and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons (by flow cytometry), cerebral infarction volume (by TTC staining), and expression of phosphorylated RhoA (p-RhoA), ROCK2 and cleaved caspase-3 (by Western blot) and for microscopic examination of ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:Compared with group S, the neurobehavioral score, apoptosis rate of hippocampal neurons, and cerebral infarction volume were significantly increased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in group IR.Compared with group IR, the neurobehavioral score, apoptosis rate of hippocampal neurons and cerebral infarction volume were significantly decreased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was down-regulated ( P<0.05), and the pathological damage to hippocampal neurons was alleviated in group T. Compared with group T, the neurobehavioral score, apoptosis rate of hippocampal neurons, and cerebral infarction volume were significantly increased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in group A. Conclusions:RhoA/ROCK2 signaling pathway is involved in trilobatin-induced reduction of cerebral I/R injury, which may be related to inhibition of apoptosis in hippocampal neurons of rats.

Effect of lidocaine on Rho/ROCK signaling pathway during endotoxin-induced acute lung injury in rats / 中华麻醉学杂志

Objective To evaluate the effect of lidocaine on Ras homologue (Rho)/Rho kinase (ROCK) signaling pathway during endotoxin-induced lung injury (LI) in rats.Methods Forty SPF male Wistar rats,aged 5-8 weeks,weighing 200-250 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),lipopolysaccharide (LPS) group (group LPS)and lidocaine at 3 different doses groups (L1-3 groups).LI was induced by intraperitoneal LPS 5 mg/kg (0.1 ml).The equal volume of normal saline was given intraperitoneally in group C.Lidocaine 2,4 and 8 mg/kg was intraperitoneally injected at 1 h before LPS administration in L1-3 groups,respectively.The animals were sacrificed at 6 h after LPS or normal saline administration.Broncho-alveolar lavage fluid (BALF) was collected for determination of concentrations of interleukin-1 beta (IL-1β),IL-6 and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay).The lung tissues were obtained for examination of the pathological changes which were scored and for measurement of the wet/dry weight ratio (W/D ratio) and activities of myeloperoxidase (MPO) and for determination of the expression of Rho,ROCK1,ROCK2,myosin phosphatase target protein 1 (MYPT1),phosphorylated MYPT1 (p-MYPT1)and ZO-1 (by Western blot).The phosphorylation of MYPT1 was calculated.Results Compared with group C,the activity of MPO,lung injury score,W/D ratio and concentrations of IL-1β,IL-6 and TNF-αin BALF were significantly increased,the expression of Rho,ROCK1 and ROCK2 was up-regulated,the phosphorylation of MYPT-1 was increased,and the expression of ZO-1 was down-regulated in the other four groups (P＜0.05).Compared with group LPS,the activity of MPO,lung injury score,W/D ratio and concentrations of IL-1β,IL-6 and TNF-α in BALF were significantly decreased,the expression of Rho,ROCK1 and ROCK2 was down-regulated,the phosphorylation of MYPT-1 was decreased,and the expression of ZO-1 was up-regulated in L1-L3 groups (P＜0.05).Conclusion Lidocaine can inhibit activation of Rho/ROCK signaling pathway during endotoxin-induced LI in rats,and the effect may be related to the antiinflammatory mechanism of lidocaine.

Role of RhoA/ROCK2 signaling pathway in propofol-induced neuroapoptosis in hippocampus of newborn rats / 中华麻醉学杂志

Objective To evaluate the role of Ras homolog family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling pathway in propofol-induced neuroapoptosis in the hippocampus of newborn rats.Methods Experiment Ⅰ Primarily cultured hippocampal neurons were seeded in 6-well culture plates at a density of 1×106 cells/ml and divided into 2 groups (n=6 each) using a random number table:solvent control group (C group) and propofol group (P group).Propofol was added with the final concentration of 60 μg/ml in group P.Dimethyl sulfoxide was added with the final concentration of 0.04％ in group C.The expression of RhoA and ROCK2 in hippocampal neurons was measured by Western blot at 24 h of incubation.Experiment Ⅱ Primarily cultured hippocampal neurons were seeded in 6-well culture plates at a density of 1 × 106 cells/ml and divided into 3 groups (n =6 each) using a random number table:solvent control group (C group),propofol group (P group) and propofol plus specific RhoA/ROCK2 signaling pathway blocker Y27632 group (P+Y group).Propofol was added with the final concentration of 60 μg/ml in group P.Propofol at the final concentration of 60 μg/ml and Y27632 at the final concentration of 10 μmol/L were added in group P+Y.Dimethyl sulfoxide was added with the final concentrauon of 0.04％ in group C.At 24 h of incubation,the neuroapoptosis in hippocampi was detected by flow cytometry,and the expression of activated caspase-3 in hippocampal neurons was measured by Western blot.The apoptotic rate was calculated.Results Experiment Ⅰ Compared with group C,the expression of RhoA and ROCK2 in hippocampal neurons was significantly up-regulated in group P (P＜0.05).Experiment Ⅱ Compared with group C,the apoptotic rate of hippocampal neurons was significantly increased,and the expression of activated caspase-3 was up-regulated in P and P+Y groups (P＜0.05 or 0.01).Compared with group P,the apoptotic rate of hippocampal neurons was significantly decreased,and the expression of activated caspase-3 was down-rcgulatcd in group P + Y (P＜ 0.05).Conclusion Activation of RhoA/ROCK2 signaling pathway is involved in propofol-induced neuroapoptosis in hippocampi of newborn rats.

Effect of inhalation of hydrogen gas on Rho/ROCK pathway in lung tissues of septic mice with acute lung injury / 中华麻醉学杂志

Objective To evaluate the effect of inhalation of hydrogen gas (H2) on Rho/Rho kinase (ROCK) pathway in lung tissues of septic mice with acute lung injury.Methods Forty male C57BL/6 mice,weighing 20-25 g,aged 6 weeks,were randomly divided into 4 groups (n=10 each) using a random number table:sham operation group (group Sh),sham operation + inhalation of H2 group (group H2),sepsis group (group S),and sepsis+ inhalation of H2 group (group S+H2).Sepsis was produced by cecal ligation and puncture (CLP).Both H2 and S+H2 groups inhaled 2％ H2 for 1 h starting from 1 and 6 h after CLP.The mice in each group were sacrificed at 24 h after CLP.The bronchoalveolar lavage fluid (BALF) was collected for determination of protein concentrations,polymorphonuclear neutrophil (PMN) count,and concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay).The lung tissues were obtained for examination of the pathological changes which were scored and for determination of the wet/dry weight ratio (W/D ratio),activities of myeloperoxidase (MPO) and superoxide dismutase (SOD),malonaldehyde (MDA) level,the expression of Rho,ROCK1,ROCK2 and activated caspase-3,and phosphorylation of myosin phosphatase target protein 1 (MYPT-1) (by Western blot).Results Compared with group Sh,the concentrations of protein,PMN count,and concentrations of TNF-α and IL-1β in BALF,lung injury score,W/D ratio,and levels of MPO and MDA in lung tissues were significantly increased,the activity of SOD in lung tissues was significantly decreased,the expression of Rho,ROCK1,ROCK2 and activated caspase-3 was significantly upregulated,and the phosphorylation of MYPT-1 in lung tissues was significantly increased in S and S+H2 groups (P＜0.05),and no significant change was found in the parameters mentioned above in group H2 (P＞0.05).Compared with group S,the concentrations of protein,PMN count,and concentrations of TNF-α and IL-1β in BALF,lung injury score,W/D ratio,and levels of MPO and MDA in lung tissues were significantly decreased,the activity of SOD in lung tissues was significantly increased,the expression of Rho,ROCK1,ROCK2 and activated caspase-3 in lung tissues was significantly down-regulated,and the phosphorylation of MYPT-1 in lung tissues was significantly decreased in group S+H2 (P＜0.05).Conclusion The mechanism by which inhalation of H2 attenuates acute lung injury is related to inhibition of Rho/ROCK pathway activation in lung tissues of septic mice.

Effect of propofol on RhoA/ROCK1 signaling pathway in human gastric cancer cells / 中华麻醉学杂志

Objective To preliminarily evaluate the role of Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) signaling pathway in propofol-induced inhibition of metastasis of human gastric cancer cells.Methods Human gastric cancer MKN-45 cells cultured in vitro,with the concentration of 1.0× 106 cells/ml,were seeded in culture plates,and incubated for 24 h.The plates were then randomly divided into 4 groups using a random number table:control group (group C),propofol group (group P),lysophosphatidic acid group (group L) and propofol + lysophosphatidic acid group (group PL).Group C received no administration.In group P,propofol at the final concentration of 16 μg/ml was given.In group L,lysophosphatidic acid at the final concentration of 1 μmol/L was administered.In group PL,propofol and lysophosphatidic acid were given with the final concentration of 16 μg/ml and 1 μmol/L,respectively.All the cells were then incubated for another 24 h.The migration of cells was determined by wound healing assay,and cell migration rates were calculated.The invasion of cells was determined by Transwell assay,and the invaded cells were counted.The expression of matrix metalloproteinases-2 (MMP-2),MMP-9,RhoA,and ROCK1 in cells was detected by Western blot.Results Compared with group C,cell migration rates and the number of invaded cells were significantly increased,and the expression of MMP-2,MMP-9,RhoA and ROCK1 was up-regulated in group L,and cell migration rates and the number of invaded cells were decreased,and the expression of MMP-2,MMP-9,RhoA and ROCK1 was down-regulated in group P.Compared with group L,cell migration rates and the number of invaded cells were significantly decreased,and the expression of MMP-2,MMP-9,RhoA and ROCK1 was down-regulated in group PL.Conclusion Inhibition of RhoA/ROCK1 signaling pathway is involved in the mechanism by which propofol decreases metastasis of human gastric cancer cells.

Effect of hydrogen gas on intestinal Rho/ROCK signaling pathway in septic mice / 中华麻醉学杂志

Objective To evaluate the effect of hydrogen gas (H2) on intestinal Ras homolog gene (Rho) /Rho-associated coiled coil-forming protein kinase (ROCK) signaling pathway in septic mice.Methods Sixty-four male ICR mice,weighing 20-25 g,aged 6 weeks,were randomly divided into 4 groups (n =16 each) using a random number table:sham operation group (group SH),H2 group (group H2),sepsis group (group S) and sepsis+H2 group (group S+H2).Sepsis was produced by cecal ligation and puncture (CLP).H2 and S+H2 groups inhaled 2％ H2 for 1 h starting from 1 and 6 h after CLP operation,respectively.Eight mice of each group were selected at 20 h after CLP operation,and gavaged with fluorescein-isothiocyanate-conjugated dextran (FITC-dextran),4 h later blood samples were obtained by cardiac puncture,and the concentration of FITC-dextran in serum was measured.The left 8 mice in each group were sacrificed at 24 h after CLP operation.After anesthesia,the sterile samples of blood,liver,spleen and kidney were obtained and cultured for bacterial growth to evaluate the condition of bacterial translocation.The intestinal tissues were obtained for examination of the epithelial ultrastructure (by transmission electron microscope),and of the pathological changes which were scored (by light microscope) and for determination of the expression of Rho,ROCK1 and ROCK2 (by Western blot).Results Compared with group SH,the serum concentration of FITC-dextran and pathological scores were significantly increased,the colony-forming units in bacterial culture plates of blood,liver,spleen and kidney were increased,and the expression of Rho,ROCK1 and ROCK2 was up-regulated in S and S+H2 groups,and no significant change was found in the parameters mentioned above in H2 group.Compared with group S,the serum concentration of FITC-dextran and pathological scores were significantly decreased,the colony-forming units in bacterial culture plates of blood,liver,spleen and kidney were decreased,and the expression of Rho,ROCK1 and ROCK2 was down-regulated,and the pathologic changes of intestines were mitigated in group S+H2.Conclusion The mechanism by which H2 alleviates the intestinal injury is related to inhibition of the activation of Rho/ROCK signaling pathway in septic mice.

The expressions of protein RhoA/RhoB ang Rock1/Rock2 in severe preeclampsia / 中国医师杂志

Objective To investigate expressions of Ras homologue protein family A (RhoA)/RhoB and Rho-associated coiled coil Rho forming protein Kinase 1 (Rock1)/Rock2 in placenta tissues of severe preeclampsia (sPE) to clarify the molecular mechanisms of sPE.Methods The locations and expressions of RhoA/RhoB and Rock1/Rock2 between two groups were detected with immunohistochemistry.Results RhoA,RhoB,Rock1,and Rock2 were mainly distributed in cytoplasms of syncytiotrophoblasts,cytotrophoblast,endothelial cells,and endometrial stromal cells.Rock1 and Rock2 were less expressed in nucleus.The expression levels of RhoA,RhoB,Rock1,and Rock2 in sPE group were significantly higher than control group (P ＜ 0.05).Conclusions The signal pathway that consists of upstream RhoA/RhoB and downstream Rock1/Rock2 is up-regulated in sPE.It demonstrates that signal molecules including RhoA,RhoB,Rockl,and Rock2 may involve in the sPE pathogenesis through affecting shallow placenta implantation in preeclampsia,ischemia,hypoxia and apoptosis of trophoblasts,and injury of vascular endothelial cells and artery vasospasm.

Effect of ART 1 gene silencing by RNA interference on the proliferation of mouse colon carcinoma cells and its possible mechanism / 肿瘤

Objective: To investigate the effect of ART 1 (arginine-specific adenosinediphosphateribosyltransferase 1) gene silencing by shRNA (short hairpin RNA) interference on the proliferation ability of mouse colon cancer CT26 cells, and to explore its possible mechanism. Methods: It was confirmed that ART1 expression existed in CT26 cells by immunofluorescence assay. Lentivirus of ART1-shRNA was infected into mouse colon carcinoma CT26 cells. The CT26 cells uninfected or infected with a negative NC-shRNA (control-shRNA) served as the controls. The expression of ART1 mRNA was detected by RTPCR, and the expressions of ART1, RhoA, c-myc, and c-fos proteins were examined by Western blotting. The cell proliferation in each group was measured by CCK8 (cell counting kit-8) assay. Results: It was determined that ART1 expression existed in the CT26 cells. Lentivirus of ART1-shRNA or NC-shRNA was infected into CT26 cells successfully, and the CT26 cell line with stable low-expression of ART1 was successfully established. Compared with CT26 cells infected with NC-shRNA lentivirus or those were un-infected, the expression of ART1 mRNA was significantly reduced in CT26 cells infected with ART1- shRNA lentivirus (P < 0.01), and the protein expression levels of ART1, RhoA, c-myc, and c-fos were all obviously decreased (P < 0.01). The inhibition rate of cell proliferation of CT26 cells infected with ART1-shRNA lentivirus was markedly increased compared with the control groups (P < 0.01). Conclusion: RNA interference targeting ART 1 gene can inhibit the proliferation ability of mouse colon carcinoma CT26 cells. This effect probably associates with the down-regulation of the expressions of RhoA and its downstream effectors c-myc and c-fos after silencing the expression of ART 1 gene. Copyright © 2012 by TUMOR.

Relationship between syncytiotrophoblast microvillous membrane shedding in severe preeclampsia and Rho/ROCK molecule expression / 解放军医学杂志

Objective To study the relationship between syncytiotrophoblast microvillous membrane (STBM) shedding in severe preeclampsia (sPE) and placenta Rho/ROCK molecule expression. Methods In this study, placenta tissue of 20 sPE lying-in women (sPE group) and 20 normal lying-in women (control group) were collected. The syncytiotrophoblast microvillous structure of the placenta tissue was scrutinized with transmission electron microscope (TEM). The microvillous numerical density (Nv), surface density (Sv), and volume density (Vv) of the cell surfaces were analyzed with stereology method. Western blotting and immunohistochemistry were used to detect the expression of Rho subfamily proteins (RhoA, RhoB, RhoC) and Rho kinases (ROCKI, ROCKII) in the placenta tissues. The correlation of the expression of Rho, ROCK proteins, and the shedding of STBM was examined with Spearman rank correlation analysis. Results The Nv, Sv, and Vv of the syncytiotrophoblast microvillous membranes in sPE group were significantly less than those of the control group (P<0.05). Compared with the control group, the expression levels of RhoA, RhoB, ROCKI, and ROCKII proteins in sPE group were noticeably elevated (P<0.05). In sPE group, the expressions of RhoB, ROCKI, and ROCKII proteins were positively correlated with the shedding of STBM (r=0.631, r=0.826, r=0.865, P<0.05). Conclusion The signaling pathways constituted by RhoB and its downstream molecules ROCKI/ROCKII, may play an important regulatory role in the upregulation of STBM shedding in sPE.

Effects of silenced Racl on invasion and migration of LoVo cells / 中华消化杂志

Objective To investigate the effects of silenced Racl on invasion and migration in LOVo cells.Methods The expression of Racl mRNA and protein in colorectal cancer cells(including LoVo SW480.SW620.SW1116,HT29)were detected by RT-PCR and Western blot,respectively.The changes of cytoskeleton were observed in LoVO cells after transfected with Racl-shRNA.then invasion and migration were recorded respectively in LoVo cells after transfected with Racl-N17 and Racl-L61.Results Racl mRNA and protein were overexpressed in all selected colorectal cancer cells.Deletion of Racl decreased the cross-linked actin network and pseudopodia,and inhibited the invasion and migration in LoVo cells.The migration experiment showed that the migrated cells were higher in Racl-shRNA[(75±5)cells].Racl-N17 [(93±5)cells]and Racl-L61[(267±7)cells]groups compared with control group[(214±8)cells,P＜0.01,＜O.01 and＜0.05,resprectively].The invasion experimental study revealed that the migrated cells were higher in Racl-shRNA[(35±5)cells],Racl-N17[(42±5)cells]and Racl-L61[(86±7)cells] groups compared with control group[(73±6)cells,P＜0.01,＜0.01 and＜0.05,resprectively].Condusion Deletoin of Racl can inhibit the invasion and migration in LDVo cells.

Expression and significance of RhoC gene in primary hepatocellular carcinoma / 中国普通外科杂志

ObjectiveTo investigate the expression of RhoC gene in primary hepatocellular carcinoma and to evaluate the relationship between RhoC gene expression and invasion and metastasis of primary hepatocellular carcinoma.MethodsThe mRNA expression of RhoC gene was examined by polymerase chain reation after reverse transcription (RT-PCR) in 25 cases of primary hepatocellular carcinoma (HCC) and adjacent non-cancerous tissuse. In addition, the mutation of RhoC gene was examined by polymerase chain reaction-single strand conformational polymorphism(PCR-SSCP)ResultsThe mRNA expression of RhoC in tumor tissue were higher than that in adjacent liver tissue,1.8?1.1 vs. 1.0?0.7( P