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OBJECTIVE To investigate the inhibitory effects of Ginkgo biloba extract （GBE） on renal inflammation in diabetic nephropathy （DN） model mice， and its potential mechanism. METHODS KK/Ay mice were fed with high fat and high sugar to induce DN model. They were divided into model group， positive control group [metformin 200 mg/（kg·d）]， GBE low-dose and high-dose groups [100， 200 mg/（kg·d）]， with 6 mice in each group. Six C57BL/6J mice were fed with a regular diet as the control group. Administration groups were given relevant liquid intragastrically， control group and model group were given constant volume of normal saline intragastrically， once a day， for 8 consecutive weeks. The body weight， fasting blood glucose， 24-hour food intake， 24-hour urine output， monocyte chemoattractant protein-1 （MCP-1）， interleukin-12 （IL-12）， IL-10， advanced glycation end products （AGEs）， blood urea nitrogen （BUN） and serum creatinine （Scr） of mice were measured， and the ratio of bilateral kidneys to body weight was also calculated. The pathological injury and fibrotic changes of the renal cortex were observed， and the expressions of macrophage polarization marker proteins [type M1： inducible nitric oxide synthase （iNOS）； type M2： arginase-1 （Arg-1）] and AGEs-the receptor of advanced glycation end products （RAGE）/Ras homolog gene pharm_chenjing@163.com family member A （RhoA）/Rho-associated coiled-coil forming protein kinase （ROCK） signaling pathway-related proteins were determined in renal cortex. RESULTS Compared with the model group， the symptoms such as renal cortical hyperplasia， vacuoles， infiltration of inflammatory cells， and renal cortical fibrosis had been improved in GBE low-dose and high-dose groups； body weight， serum level of IL-10， the expression of Arg-1 in the renal cortex were significantly higher than model group （P＜ 0.01）； fasting blood glucose， 24-hour food intake， 24-hour urine output， serum levels of MCP-1， IL-12， BUN， Scr and AGEs， the ratio of bilateral kidneys to body weight， renal injury score， the proportion of renal interstitial fibrosis， the protein expressions of iNOS， RAGE， RhoA and ROCK1 （except for GBE low-dose group） in renal cortex were significantly lower than model group （P＜0.01）. CONCLUSIONS GBE could improve kidney damage and alleviate inflammatory response in DN model mice， the mechanism of which may be related to inhibiting the AGEs-RAGE/RhoA/ROCK signaling pathway and regulating macrophage polarization.
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Objective @#To investigate the effect of isoprene cysteine carboxymethyltransferase (ICMT) gene on the migration and invasion of salivary adenoid cystic cancer cells (SACC) and the related mechanism, to provide experimental evidence for molecular targeted therapy of SACC.@*Methods@# Adenoid cystic cancer cells SACC-LM and SACC-83 were cultured in vitro, and siRNA was transfected into human SACC-LM and SACC-83 cells (experimental group) by transient transfection of a liposome vector. A blank control group and negative control group were set up respectively (transfected NC-siRNA). qRT-PCR was peformed to measure the mRNA expression of ICMT and RhoA in each group after transfection and to determine the silencing efficiency. The expression of ICMT, membrane RhoA, total RhoA, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and Rho associated with coiled helical binding protein kinase 1 (ROCK1) in each group was detected by Western blot. The proliferation abilityies of SACC cells was detected by CCK-8 assay. The migration and invasion ability of SACC cells were detected by comparing the relative healing area of cell scratch assay and the number of Transwell assay cells. @*Results@#After transfection of ICMT-siRNA into SACC-LM and SACC-83 cells, the expression of ICMT gene and protein in the experimental group was significantly decreased compared with the negative control group and blank control group (P<0.05), but there were no significant differences in the expression of RhoA gene and total protein among all groups (P>0.05). The expression of RhoA membrane proteins, ROCK1, MMP-2, MMP-9 in the experimental group was significantly decreased compared with that in the negative control group and blank control group (P<0.05). Cell proliferation ability was significantly decreased (P<0.05). The migration and invasion abilities were significantly decreased (P<0.05). @*Conclusion @#In vitro silencing of ICMT gene can effectively inhibit the migration and invasion of human SACC-LM and SACC-83 cells, and the mechanism may be related to RhoA-ROCK signaling pathway.
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AIM: To investigate the effects of hyperoside on traumatic brain injury (TBI) rats by regulating the Ras homolog gene family, member A (RhoA)/Rho-associated coiled coil-forming kinase (ROCK) signal pathway. METHODS: The TBI rat model was established by modified Feeney free fall hit method, and was randomly divided into model group, low-dose hyperoside (60 mg / kg) group, high-dose hyperoside (120 mg / kg) group, high-dose hyperoside (120 mg / kg) + no load group, and high-dose hyperoside (120 mg / kg) + RhoA overexpression group, with 10 rats in each group, another 10 healthy rats were set as sham operation group, after hyperoside and plasmid were grouped, the nerve injury was detected by modified neurological deficit score (mNSS) and dark avoidance test; Evans blue (EB) quantitative method was used to detect the permeability of blood brain barrier in rats; ultrastructural damage of blood-brain barrier was observed by transmission electron microscopy; the levels of tumor necrosis factor- α (TNF- α), interleukin-8 (IL-8), superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and brain tissue of rats were measured with the kit; and the expression of RhoA/ROCK pathway related proteins in rat brain was detected by Western blot. RESULTS: Compared with the sham operation group, the blood brain barrier structure of the model group rats was damaged, the step-through latency and SOD level decreased obviously (P0.05). CONCLUSION: Hyperoside can inhibit neuroinflammation and oxidative stress in TBI rats by down-regulating RhoA / ROCK signal pathway, thereby reducing the damage of blood brain barrier and repairing its neural function.
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Objective:To determine the protective effect of fasudil on acute lung injury in septic mice.Methods:Forty-five 4-6-week-old male C57BL mice were randomly(random number) assigned to three groups ( n=15 each group): control group, lipopolysaccharide (LPS) group and Fasudil intervention group (FAS+LPS). Acute lung injury model of septic mice was established with an intraperitoneal injection and intratracheal infusion of LPS. The mice in the FAS+LPS group were injected with fasudil hydrochloride intraperitoneally 30 min before intraperitoneal LPS injection and 1 h after intratracheal LPS infusion, respectively. All mice were sacrificed at 4 h after modeling, and lung tissues were collected. Hematoxylin-eosin staining was preformed to observe the morphological changes in the lung tissue. The wet /dry weight (W/D) ratio, malondialdehyde (MDA) content and the activity of myeloperoxidase (MPO) in the lung tissues were detected. Caspase-3 expression was examined by immunohistochemical (IHC) staining. Western blot was employed to detect the expression of RhoA, ROCK1, endothelial nitric oxide synthase (eNOS), and p-eNOS. Results:Inflammatory cell infiltration and erythrocyte exudation were significantly reduced, and the degree of interstitial oedema and derangement of alveolar structure appeared in a decreasing degree after FAS intervention. Compared with the LPS group, the W/D ratio, MDA content, MPO activity and the expression of Caspase-3 in the FAS+LPS group were significantly reduced (all P<0.01). Meanwhile, the expression of RhoA and ROCK1 of the LPS group were obviously higher than those in the control group ( P<0.05), and p-eNOS was obviously lower than that in the control group ( P<0.05). Furthermore, the expression of RhoA and ROCK1 of the FAS+LPS group were obviously lower than those in the LPS group, and p-eNOS was obviously higher than that in the LPS group. There was no significant difference on the expression of eNOS among the three groups. Conclusions:Fasudil can alleviate the degree of inflammatory cell infiltration, reduce apoptosis in lung tissue, inhibit the RhoA/ROCK1 signaling activity, and promote the phosphorylation expression of eNOS in septic mice.
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Aim To investigate the role of H2S pro¬duced by CSE in cerebral ischemia-reperfusion ( I/R) injury and its relationship with RhoA-ROCK2 signaling pathway.Methods Bilateral common carotid artery ligation was used to prepare a mouse cerebral ischemia- reperfusion injury model.Laser speckle method was used to detect cerebral blood flow, HE staining method was used to observe the pathological changes of brain hippocampus, and the activity of LDH, NSE, RhoA and ROCK,, H,S content and ROCK, protein expres¬sion were detected.Results The H,S synthase CSE substrate L-Cys ( 3(X) mg • kg-1) could significantly promote the recovery of cerebral blood flow in brain 1/ R mice, improve the pathological damage of hippocam¬pus , inhibit the increase of LDH activity in serum and NSE, RhoA and ROCK2 activity in brain tissues, and inhibit the decrease of serum H2S content and the in¬crease of ROCK2 protein expression in brain tissues.But the above effects of L-Cys could be significantly at¬tenuated by the CSE inhibitor PPG (50 mg • kg~ 1 ) ; the H2S donor NaHS (4.8 mg • kg"1 ) also had the same effect as L-Cys did.Conclusions H2S pro¬duced by CSE has a protective effect on mouse brain 1/ R injury, and its effect may be related to inhibiting RhoA-ROCK signaling pathway and increasing cerebral blood flow.
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【Objective】 To investigate the effects of naringenin on the polarization of high-glycemic RAW264.7 macrophages and its related mechanism. 【Methods】 The mother solution of NAR was prepared with dimethyl sulfoxide (DMSO), and the log-growth phase macrophage RAW264.7 was pre-tested. DMEM medium with different glucose concentrations (1, 2, 4, 5 and 6 g/L) was used for cultivation for 24 h. Before the experiment, DMEM was diluted into NAR mixture with different final concentrations, and the effect of NAR on RAW264.7 cell activity was detected by CCK-8 method; nitric oxide synthase (NOS) type classification determination of checkerboard induced nitric oxide synthase (iNOS) active filter was used to control the concentration of sugar and high-sugar stimulation. The control group were subdivided into normal control (NG) and osmotic pressure control (NG+M). The high-glucose stimulation group was divided into normal high glucose (HG), high glucose + naringenin (HG+NAR), high glucose + Fasudil (HG+F), and high glucose +C3 transferase (HG+C3). RAW264.7 was cultured for 24 h in each group; the expression levels of supernatant cytokines, namely, interleukin6 (il-6), tumor necrosis factor -α (TNF-α) and interleukin10 (IL-10), were detected by ELISA. Western blotting was used to determine the RhoA/ROCK pathway related proteins, iNOS and Arg-1 protein levels. Type (M1, M2) and proportion (M1/M2) of macrophages were analyzed by flow cytometry. 【Results】 Compared with those in NG group, in HG group RhoA/ROCK pathway-related proteins and iNOS expression were increased, while Arg-1 expression was decreased (P<0.05). The secretion of pro-inflammatory cytokines IL-6 and TNF-α was increased while anti-inflammatory cytokine IL-10 was decreased (P<0.05). The number of M1-type cells and M1/M2 ratio increased (P<0.05). Compared with HG group, RhoA/ROCK pathway related proteins and iNOS expression were decreased in HG+NAR group, HG+F group and HG+C3 group, while Arg-1 expression was increased, IL-6 and TNF- α secretion was decreased, IL-10 was increased, M2-type macrophages were increased, and M1/M2 was decreased (P<0.05). 【Conclusion】 NAR may promote the M2-type differentiation of macrophages stimulated by high glucose by down-regulating RhoA/ROCK signaling pathway.
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Atherosclerosis (AS) is a chronic inflammatory disease, the main causes of which include abnormal lipid metabolism, endothelial injury, physical and chemical injury, hemodynamic injury, genetic factors and so on. These causes can lead to inflammatory injury of blood vessels and local dysfunction. Bunao-Fuyuan decoction (BNFY) is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases, but its effect on AS is still unknown. The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells (VSMCs) on AS. At first, the expression of α-SMA protein in ox-LDL-induced VSMCs, which was detected by immunofluorescence staining and western blot. CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY. Meanwhile, the expression of proliferating protein Ki67 was detected by immunofluorescence staining. Western blot was also used to detect the expression of proliferation-related proteins CDK2, CyclinE1 and P27. Flow cytometry was used to detect the effect of BNFY on cell cycle. The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell. Western blot was used to detect the expression of adhesion factors ICAM1, VCAM1, muc1, VE-cadherin and RHOA/ROCK-related proteins in cells. We found that the expression of AS marker α-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs. The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY, and BNFY can inhibit cell cycle. Meanwhile, we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1, VCAM1, muc1 and VE-cadherin were inhibited too by BNFY. Finally, we found that BNFY inhibited the expression of RHOA, ROCK1, ROCK2, p-MLC proteins in the RHOA/ROCK signaling pathway. Therefore, we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.
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Objective To observe the effect of different hypoxic time on hydrogen sulfide (H2S), nitric oxide (NO) and Ras homolog gene family,member A/Rho associated coiled coil-forming kinase(RhoA-ROCK) pathway in rat cerebrovascular endothelial cells(EC),and investigate the effect of dermatogenous H2S on the RhoA-ROCK pathway. Methods Rat brain vascular EC was cultured by collagenase digestion. The EC was measured for H2S and NO after hypoxia for 1,2,4,8 and 24 h respectively. G-LISA was used to detect RhoA activity. Proteins expression changes were detected by Western blot. Results After 1 hour of hypoxia,the content of H2S decreased significantly, the NO content decreased significantly after hypoxia of 4 hours,the activity of RhoA increased significantly after hypoxia of 8 h. The expression of CSE protein decreased significantly after 4 h of hypoxia,the expression level of eNOS protein decreased significantly after 8 h of hypoxia,and the expression of ROCK1 and ROCK2 increased significantly at 8 h of hypoxia. Both endogenous and exogenous H2S inhibited RhoA activity. Conclusion During the hypoxic injury of rat cerebrovascular endothelial cells. The decrease of endogenous H2S occurred first, followed by NO,and the activation of RhoA-ROCK pathway occurred later,which may be secondary to the decrease of H2S.
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OBJECTIVE: To observe the effect of different strength of electroacupuncture (EA) stimulation on gastrointestinal motility and Ras homolog gene family member (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) signaling in diabetic gastroparesis (DGP) rats, so as to reveal the underlying mechanisms of EA for improving DGP. METHODS: Sixty SD rats were randomly and equally divided into blank control, DGP model, weak EA, medium EA, and strong EA groups (n＝12 rats in each). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mmol/kg, 2%) and high-sugar and high-fat fodder feeding for 8 weeks. EA (0.12, 0.24, 0.36 mA, 20 Hz/100 Hz) was applied to "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) for 20 min, once daily for 15 successive days. Blood glucose levels were measured weekly with blood glucose meter and blood glucose test paper. Fecal phenol red excretion method was used to display gastric emptying and small intestinal propulsion function. The expression of RhoA protein in the gastric antral smooth muscle tissue was detected by immunohistochemistry and Western blot (WB), separately, and that of ROCK, myosin phosphatase target subunit 1 (MYPT 1) and phosphorylated (p)-MYPT 1 proteins in gastric antrum detected by WB. RESULTS: Compared with the blank control group, the gastric emptying rate and small intestine propulsion rate of the model group were significantly decreased (P0.05). CONCLUSION: Electroacupuncture stimulation of ST 36-SP 6-ST 21 at 0.12, 0.24 and 0.36 mA can promote the gastrointestinal motility in DGP rats, which may be associated with its effects in enhancing RhoA/ROCK signaling in the gastric antral smooth muscle at different degrees.
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Objective To investigate the protective mechanism of MEK1/2 inhibitor PD98059 on ox-LDL induced injury of human umbilical vein endothelial cells (HUVEC),and its influence on the expression of LOX-1.Methods HUVEC damage models were established by using ox-LDL and were treated with PD98059 later,divided into the negative control group,the ox-LDL group,the positive control group and the PD98059+ox-LDL group.The effect of inhibition of MEK1/2 on ox-LDL induced HUVEC damage was measured.Results Compared with the negative control group,the levels in the ox-LDL group of LOX-1,pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were increased significantly,the proliferations of HUVEC and the productions of NO were decreased (P＜0.05).Compared with the ox-LDL group,the levels in the positive control group and the PD98059+ox-LDL group of pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were decreased,the proliferation of HUVEC and the production of NO were increased (P＜0.05).Conclusion PD98059 inhibit the MEK1/2 signaling pathway to suppress the ox-LDL induced damage of HUVEC by decreasing the expression of LOX-1.
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Objective:To study the effect of the prescription Tangshenning on the proliferation of the high glucoseinduced cells of near-end kidney tubules.Method:To prepare durg-contained serum from mice to enter into in vitor reaction system,the cells were randomly divided into 4 groups:the normal group,the model group,the,the irbesartan group,the tangshenning group and then detect the effects of all serum sections on the proliferation of high glucoseinduced epithelial cells of kidney tubules by the MTT colorimetric method,and the expression of RhoA,ROCK 1,Ecadherin and α-SMA in each group were detected by Western blotting.Result:The high glucose group of renal tubular epithelial cells form from the flat irregular polygon into long fusiform;After adding the drug-containing serum corresponding intervention into the cells into a flat in irregular polygon.The high glucose group of renal tubular epithelial cells show obvious proliferation condition,In the condition of 24 h,48 h,The high glucose group proliferation is better than the normal group (P＜0.01),in 60 h,The high glucose group proliferation is better than the normal group (P＜0.05);In 24 h,compared with the irbesartan,Tangshenning has better effect of inhibiting the cell proliferation(P＜0.05);In 48 h,Tangshenning has the better effect than irbesartan and Y27632 (P＜0.01);In 60 h,Tangshenning has the better effect than irbesartan and Y27632 (P＜0.05);Western blotting:Western blotting analysis showed that compared with the normal group,the expression of RhoA protein in high glucose group and Y27632 decreased (P＜0.01);The high glucose group and Tangshenning have significant difference (P＜0.01);Y27632 and Tangshenning have difference (P＜0.05).compared with the normal group,the expression of ROCK1 protein in high glucose group decreased (P＜0.01);The high glucose group,Tangshenning,the irbesartan and Y27632 have difference (P＜0.05).Compared with the normal group,the expression of a-SMA protein in high glucose group decreased (P＜0.01);The high glucose group,Tangshenning,the irbesartan and Y27632 have difference (P＜0.05).Compared with the normal group,the expression of E-Cadherin protein in high glucose group increased (P＜0.05).Y27632 and Tangshenning have difference (P＜0.05).The high glucose group,Tangshenning,the irbesartan and Y27632 have difference (P＜0.05).Conclusion:The prescription Tangshenning is able to inhibit the proliferation of high glucose-induced epithelial cells of kidney tubules and and can reverse renal tubularepithelial cell transdifferentiation via regulating RhoA/ROCK signaling pathway,and restrain renal interstitial fibrosis,thereby delaying the pathogenesis of diabetic kidney disease.
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To study the effect of the Tangshenping containing serum on the proliferation of the high glucose-induced epithelial cells of renal tubules.To prepare durg-contained serum from rats to enter into in vitor reaction system,the cellscultured via 10％ FBS-RPMI 1640 were randomly divided into 7groups:the normal group,themodel group,the Y27632 group,theirbesartangroup,the small dose Tangshenping group,the medium dose Tangshenping group and the high dose Tangshenping group and The cells were cultured in 3000 cells/well and grown in 96-well plates.Each group had 8 wells,then detect the effects of all serum sections on the proliferation of high glucose-induced epithelial cells of kidney tubules by the MTT colorimetric method after cultured for 12 h,24 h,48 h,and 60 h.Based on the results above,cell protein were extracted from each group at 24 h,and the expression of RhoA,ROCK1,α-SMA and E-cadherin in each group were detected by Western blotting.After high glucose stimulation,the shape of cell was shuttle-like or irregular triangle,the way it grew was radial;after the intervention of the corresponding serum,the shape of the cell was fiat and irregular polygonal.Started with 12h,compared with the normal group,OD value of other groups increased;at the 24h、48hand 60h,compared with the normal group,OD value of high glucose groupincreased significantly (P＜0.01);compared with the high glucose group,OD value of treatment groups decreased (P＜0.05);and 48 h,compared with the Y27632group,irbesartan groupand Tangshenping high dose group,OD value of Tangshenping low and medium dose groups decreased (P＜0.05);60 h,compared with Y27632 group,OD value of Tangshenping medium dose groups decreased;compared with irbesartan group,OD value of o Tangshenpinggroupsdecreased (P＜0.05);compared with Tangshenping high dose group,OD value of Tangshenpinglow groupsdecreased (P＜0.05) Western blotting analysis showed that compared with normal group,the expression of E-Cadherin protein in high glucose group reduced,and the expression of RhoA,ROCK1 and α-SMA protein increased;compared with high glucose group,the expression of E-Cadherin protein in each treating group increased,and the Tangshenping large dose group wassignificantly different (P＜0.01);the expression of RhoA,ROCK1 and or-SMA protein reduced,Tangshenping,the large dose group was significantlydifferent (P＜0.01);Compared with the Y27632 group,the expression of E-cadherin,ROCK1 and α-SMA protein in Tangshenping large dose group had no significant difference,while the expression of RhoAproteinreduced (P ＜0.01).Compared with theirbesartan group,the expression of E-cadherin,RhoA,ROCK1 and α-SMA protein in Tangshenping large dose group had no significant difference (P＞0.05).The Tangshenping containing serum is abletoinhibit the proliferation of high glucose-induced epithelial cells of kidney tubules,and can reverse renal tubular-epithelial cell transdifferentiation via regulating RhoA/ROCK signaling pathway,and restrain renal interstitial fibrosis,thereby delaying the pathogenesis of diabetic kidney disease.
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This study was aimed to explore the renoprotective effects of Tang-Shen-Ping (TSP) on RhoA/ROCK signaling pathway in KKAy mice with diabetic kidney disease (DKD).A total of 60 female 10-week SPF degree KKAy mice,which were fed with KK special food for 10 weeks,were made into DKD model.Mice were randomly divided in the model group,irbesartan group,low-,medium-and high-dose TSP group (0.525 g· kg-1,1.05 g· kg-1,and 2.1 g· kg-1).Ten female C57BL/6J mice were used as the normal control group.Mice of each group were intragastrically administered with corresponding medicine,respectively,while mice of the control group and the model group were given deionized water of the equal volume.The body weight was measured and the 24-hour urine protein quantification was detected every 4 weeks.At the end of the 26th week,all mice were sacrificed and the biochemical indicators,such as fasting blood glucose (FBG),serum blood urea nitrogen (BUN),serum creatinine (Scr),and triglyceride (TG) were measured.HE staining,Mallory staining and PAS staining were used to observe the pathological morphology of kidney tissues.Immunohistochemistry (IHC) and in situ hybridization (ISH) were used in the detection of transforming growth factor-β1 (TGF-β1),Ras homolog gene family member A (RhoA),Rho-associated coiled-coil-containing protein kinase 1 (ROCK1),α-smooth muscle actin (α-SMA),E-Cadherin (E-Cad) mRNA and protein expression.The results showed that compared with the model group,there were significant differences on body weight,the ratio of kidney weight to body weight,and urinary protein in the middle-and high-dose TSP group (P ＜ 0.01);the renal pathological damage was obviously decreased;contents of FBG,BUN,Scr and TG decreased (P ＜ 0.01);mRNA and protein expression of E-Cadherin increased;mRNA and protein expression of TGF-β1,RhoA,ROCK1 and α-SMA decreased with significant difference in the middle-and high-dosc TSP group (P ＜ 0.01).It was concluded that the renoprotective effects and epithelial-mesenchymal transdifferentiation (EMT) of renal tubular epithelial cells of TSP on DKD KKAy mice may be related to the regulation of RhoA/ROCK signaling pathway.
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AIM To explore the effects of Sini Decoction (Aconiti lateralis Radix Praeparata,Zingiberis Rhizoma,Glycyrrhizae Radix et Rhizoma) on rat myocardial fibrosis induced by isoproterenol.METHODS Forty SD rats were randomly divided into blank,model,captopril [100 mg/(kg · d)] and Sini Decoction [3.8 g/(kg · d)] groups,with ten rats in each group.Except for the blank group,the rest rats were given subcutaneous injection of isoproterenol [5 rg/(kg · d)] to prepare myocardial fibrosis model,and successive administration lasted for four weeks.At 20 h after the last administration,hemodynamics changes of heart were detected;heart weight indexes were calculated;the changes of myocardial pathological morphology were observed by HE and Masson staining;NO level in serum was measured by nitrale reduetase;SOD activity in serum was measured by xanthinoxidase method;MDA level in serum was measured by thiobarbituric acid method;the protein expressions of RhoA,ROCK1 and eNOS in myocardial tissue were detected by Western blot method.RESULTS Compared with the model group,the cardiac function of rats was significantly improved;the myocardial collagen content was significantly decreased;the MDA level in serum was obviously decreased;the NO level and SOD activity were significantly increased;the protein expressions of RhoA and ROCK1 in myocardium tissue were significantly reduced,and the protein expression of eNOS was markedly increased in the Sini Decoction group.CONCLUSION Sini Decoction can improve the isoproterenol-induced myocardial fibrosis in rats,and its mechanism may be related to inhibiting the RhoA/ ROCK signaling pathway,in turn,raising the expression of eNOS,which leads to reduced oxidative stress.
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<p><b>Objective</b>To investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells.</p><p><b>METHODS</b>Human prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry.</p><p><b>RESULTS</b>Fasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former (［37.26±1.17］% and ［32.38±2.73］%) than in the latter (［78.12±4.16］% and ［69.47±6.71］%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil (［31.88±2.49］% and ［28.65±2.99］%) as compared with the negative controls (［7.51±2.28］% and ［7.13±1.61］%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ).</p><p><b>CONCLUSIONS</b>The RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity.</p>
Subject(s)
Humans , Male , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Prostatic Neoplasms , Drug Therapy , Pathology , Signal Transduction , rho-Associated KinasesABSTRACT
Objective To investigate mechanisms underlying the regulation of the permeability of vascular endothelial cells by the Treponema pallidum membrane protein Tpp47.Methods Human umbilical vascular endothelial cell (HUVEC) monolayers were established as a model,and were directly cultured with the presence of recombinant Tpp47 protein (rTpp47-treated group),or boiled and inactivated rTpp47 (negative control group).Some HUVEC monolayers,which were pretreated with the RhoA/ROCK signal pathway inhibitor Y-27632 for 30 minutes and then cultured with the presence of rTpp47,served as the pretreatment group.After 1-and 4-hour additional culture,enzymelinked immunosorbent assay (ELISA) was performed to estimate the permeability of these cell monolayers to horseradish peroxidase (HRP).After 12 hours of culture,rhodamine-phalloidin was used to stain cytoskeletal proteins,and confocal laser scanning microscopy was performed to observe the arrangement of the cytoskeletal protein F-actin.Western-blot analysis was conducted to measure the expressions of RhoA in HUVECs treated with rTpp47 or inactivated rTpp47.Results The supernatant level of HRP (expressed as the absorbance value at 450 nm) was significantly higher in the rTpp47-treated group than in the negative control group (0.81 ± 0.10 vs.0.39 ± 0.09,P ＜ 0.05),but no significant difference was observed between the pretreatment group (0.51 ± 0.10) and rTpp47-treated group or negative control group (both P ＞ 0.05) after 1-hour culture.Similarly,the rTpp47-treated group showed significantly increased levels of HRP compared with the pretreatment group and negative control group (2.31-± 0.14 vs.1.21 ± 0.12 and 0.73 ± 0.12,both P ＜ 0.05),while there was no significant difference between the pretreatment group and negative control group after 4-hour culture.The expression of RhoA in HUVECs treated with rTpp47 was significantly higher than that in those treated with inactivated rTpp47.Confocal laser scanning microscopy showed that rTpp47 treatment led to the rearrangement of F-actin in HUVECs followed by the formation of stress fibers in cytoplasm,while Y-27632 could partly inhibit the rearrangement of F-actin.Conclusion The recombinant Treponema pallidum membrane protein Tpp47 can regulate the permeability of vascular endothelial cells through the RhoA/ROCK signal pathway.
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Objective Clinical treatment can delay the development of renal interstitial fibrosis , but it can not reverse renal dysfuntion.The article was to discuss the influence of recombinant human erythropoietin ( rHuEPO ) on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism . Methods The vitro cultured HK-2 cells were randomized into 7 groups:the blank control group , rHuEPO control group ( addition of 20U/mL rHuEPO), albumin stimulation group (addition of 5mg/mL albumin), 5mg/mL rHuEPO intervention group (5mg/mL albumin +5U/mL rHuEPO), 10 U/mL rHuEPO intervention group (5mg/mL albumin +10 U/mL rHuEPO), 20U/mL rHuEPO intervention group (5mg/mL albumin +20U/mL rHuEPO), and Rho inhibi-taion group (addition of 5mg/mL albumin 30min after 10μmol/L Y27632), 24 h acting time for each group.We observed the changes of cell morphology in each group .Reverse transcription polymerase chain reaction ( RT-PCR) was used to evaluate the mRNA levels of RhoA, ROCK1 and IL-6 , and ELISA was applied to measure the levels of supernatant TNF-αand IL-6 protein. Results The form of pebbles or paving stone was observed in blank control group and rHuEPO intervention groups , a long and thin spindle change with the appearance of fibre cells in albumin stimulation group , the transformation to pebbles in 5, 10, 20 mg/mL rHuEPO intervention groups , the form of oval and slightly increased intercellular space in Rho inhibitaion group .Compared with the blank control group , the expressions of RhoA mRNA, ROCK1 mRNA and IL-6 mRNA significantly increased in the albumin stimulation group (P<0.05), while significantly reduced in 5, 10, 20 mg/mL rHuEPO intervention groups (P<0.05), which was in negative relation with the rHuEPO concentrations .Compared with the albumin stimulation group , the expressions of ROCK 1 mRNA and IL-6 mRNA reduced in Rho inhibtation group (P<0.05), while there was no significant difference as to the expression of RhoA mRNA .ELISA results showed:compared with blank control group , the expressions of supernatant TNF-α([452.32 ±33.23] ng/L vs [1347.54 ±41.52] ng/L), IL-6 protein([884.62 ±0.73] pg/L vs [95.12 ±0.32]pg/LP<0.05) increased significantly.Compared with albumin stim-ulation group, the expressions of TNF-αin 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced signifi-cantly([1003.32 ±3.42] ng/L, [821.32 ±21.32] ng/L, [590.15 ±7.68] ng/L, [488.13 ±65.03] ng/L vs [1 347.54 ± 41.52]ng/L,P<0.05), while the expressions of IL-6 mRNA reduced accordingly in 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced significantly ([656.68 ±0.55] pg/L, [422.35 ±0.22] pg/L, [217.32 ±0.35] pg/L, [309.49 ±0.21] pg/L vs [884.62 ±0.73]pg/L,P<0.05).Moreover, there was significant statistical difference among 5, 10, 20 mg/mL rHuEPO intervention groups(P<0.05). Conclusion RHuEPO can inhibit the transdifferentiation process of HK-2 cells in-duced by albumin by suppressing inflammation factors , and the mechanism may be involved in RhoA/ROCK signaling pathway .
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Objective To explore the effect of intermittent hypoxia (IH) on RhoA/ROCK pathway in lung and on the muscularization in pulmonary vascular in rat model. Methods Wistar rats (n=40) were randomly divided into two groups:the normal oxygen control group (n=20) and the IH group ( n=20). For 4 weeks, rats in control group and IH group were ex?posed to intermittent normal oxygen (21%O2) or IH (5%-21%O2) respectively. Then, mRNA transcription and protein trans?lation levels of RhoA/ROCK were examined by Real-time PCR and Western blot. Expression of proliferation cell nuclear an?tigen (PCNA) andα-smooth muscle actin (SM-α-actin ) of lung and pulmonary artery were detected by immunohistochemis?try. Results RhoA mRNA transcription level(0.463 ± 0.067 vs 0.182 ± 0.040), ROCK mRNA transcription level(0.384 ± 0.062 vs 0.192 ± 0.052), RhoA protein expression level(0.827 ± 0.065 vs 0.424 ± 0.075)and ROCK protein expression level (0.488±0.088 vs 0.336±0.102)were higher in IH group than those in control group(P<0.05);Levels of PCNA in lung tissue [(54.67±1.80)%vs (9.14±0.91)%], PCNA in pulmonary artery [(49.40±1.21)%vs (8.38±1.13)%], SM-α-actin in lung tis?sue [(42.66±1.63)%vs (35.44±1.41)%] and SM-α-actin in pulmonary artery [(62.62±2.53)%vs (45.54±2.58)%] were also higher in IH group than those in control group(P<0.05). Conclusion Rho/ROCK pathway may play an important role in developing pulmonary hypertension (PH) associated with IH;and IH can promote the muscularization in pulmonary vascular to accelerate PH.
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Objective To study the effects of erythropoietin (rhEPO) in high glucose induced proliferation and apopto?sis of human kidney proximal tubular epithelial (HK-2) cells, and the possible mechanism thereof. Methods HK-2 cells cultured in vitro were divided into several groups randomly:blank control group, high glucose group, mannitol group, rhEPO control group, different concentrations of rhEPO treatment groups (5, 10, 20 U/mL) and Rho kinase group. The reverse tran?scription polymerase chain reaction (RT-PCR) was used to evaluate the mRNA levels of RhoA and ROCK after 24 hours. Tetrazolium salt method (MTT) was used to determine the cell proliferation. Cell apoptosis was detected by flow cytometry. Results Compared with blank control group the expression levels of RhoA and ROCK1 mRNA were significantly in?creased in high glucose group (P < 0.05). RhoA, ROCK1 mRNA expressions significantly decreased in rhEPO group than those of high glucose group (P<0.05). There was a positive correlation between the expression levels of RhoA mRNA and ROCK1 mRNA in high glucose group and rhEPO group. MTT method showed that rhEPO significantly promoted the prolifer?ation of HK-2 cells (P<0.05). Flow cytometry analysis showed that high glucose induced apoptosis in HK-2 cells, which was significantly inhibited in rhEPO group and Rho kinase group as compared to that of high glucose group in a concentra?tion dependent manner (P<0.05). Conclusion rhEPO can promote HK-2 cell proliferation and inhibit apoptosis, which may be related to RhoA/ROCK signaling pathway.
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To study the function and mechanism of the NgR-Rhoa- Rock signal pathways which exists in the retinal ganglion cells apoptosis in diabetes mellitus (DM) rats. ● METHODS: Some healthy SD rats were operated by means of single intraperitoneal injection of 1%streptozotocin based on the standard of 50mg/ kg wight, after that the blood sugar value was greater than 16. 7mmol/ L as DM model, then randomly divided into 3 groups, each group was 10 rats. ln addition to take 10 healthy SD rats as control group. Four groups of rats were bilaterally eyeball intravitreal injection in turn with NgR-siRNA virus 10μ L (siRNA group), NgR-siRNA virus diluted 10μ L ( DM group), NgR - siRNA virus - negative -control solution 10μ L (siRNA blank group), NgR- siRNA virus diluted 10μ L ( normal control group ), and fed normally. During that time, some life indexes like blood glucose, body mass, etc. were measured and recorded. After 12wk, the expression of NgR and Rhoa, HE staining, and TUNNEL staining were detected by Western blot analysis. ● RESULTS: Western blot analysis: compared with normal control group, the expression of NgR and Rhoa in DM group and siRNA blank group increased significantly (P 0. 05); compared with DM group and siRNA blank group, the expression of those proteins significantly lowered in siRNA group. HE staining: compared with normal control group, some extent ganglion cells arranged disorder, irregular shape, spacing not consistent were all found in three groups of model rats;compared with DM group and siRNA blank group, there was some improvement in siRNA group of ganglion cells about the order and shape size. TUNEL staining:compared with normal control group, there were retinal ganglion cells apoptosis in all of three groups of model rats. Compared with DM group and siRNA blank group, the number of retinal ganglion cells apoptotic cells was less, and the shape of cells had improved significantly in siRNA group. ●CONCLUSlON: ln the DM phase, the expression of NgR and Rhoa were up - regulation, the condition of diabetic retinal ganglion cell apoptosis was improved after that the NgR-Rhoa-Rock signal pathways had been inhibited.