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Background@#In consideration of characteristics and functions, extra-cellular signal-regulated protein kinase 5 (ERK5) signaling pathway could be a new target for spinal cord injury (SCI) treatment. Our study aimed to evaluate the roles of ERK5 signaling pathway in secondary damage of SCI.@*Methods@#We randomly divided 70 healthy Wistar rats into five groups: ten in the blank group, 15 in the sham surgery + BIX02188 (sham + B) group, 15 in the sham surgery + dimethyl sulfoxide (DMSO; sham + D) group, 15 in the SCI + BIX02188 (SCI + B) group, and 15 in the SCI + DMSO (SCI + D) group. BIX02188 is a specific inhibitor of the ERK5 signaling pathway. SCI was induced by the application of vascular clips (with the force of 30 g) to the dura on T10 level, while rats in the sham surgery group underwent only T9-T11 laminectomy. BIX02188 or DMSO was intra-thecally injected at 1, 6, and 12 h after surgery or SCI. Spinal cord samples were taken for testing at 24 h after surgery or SCI.@*Results@#Expression of phosphorylated-ERK5 (p-ERK5) significantly increased after SCI. Application of BIX02188 indeed inhibited ERK5 signaling pathway and reduced the degree of spinal cord tissue injury, neutrophil infiltration and proinflammatory cytokine expression, nuclear factor-κB (NF-κB) activation and apoptosis (measured by TdT-mediated 2′-deoxyuridine 5′-triphosphate nickend labeling, expression of Fas-ligand, BCL2-associated X [Bax], and B-cell lymphoma-2 [Bcl-2]). Double immunofluorescence revealed activation of ERK5 in neurons and microglia after SCI.@*Conclusion@#ERK5 signaling pathway was activated in spinal neurons and microglia, contributing to secondary injury of SCI. Moreover, inhibition of ERK5 signaling pathway could alleviate the degree of SCI, which might be related to its regulation of infiltration of inflammatory cells and release of inflammatory cytokines, expression of NF-κB and cell apoptosis.
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Objective Through observation of the expression and activity of extracellular regulated protein kinase 5 (ERK5) and its relationship with the learning and memory ability in rats with chronic fluorosis,to further study the pathogenesis of chronic fluorosis in nervous system.Methods Thirty SD rats were divided into 3 groups according to body weight by means of a random number table (10 rats in each group,half male and half female).The rats in control group were fed with free drinking tap water containing less than 0.5 mg/L fluoride (NaF);the rats in low fluoride group with 10.0 mg/L fluoride;the rat in high dose fluoride group with 50.0 mg/L fluoride.After 6months of experiment,rat brain tissue was took,mRNA expression level of ERK5 was detected by real-time fluorescence quantitative PCR (real-time PCR),protein expression level and activity of ERK5 were detected by Western blotting;the learning and memory ability of rats with chronic fluorosis were detected by Morris water maze test.Results The rat in groups exposed to fluoride exhibited different degrees of dental fluorosis and the fluoride content in urine of rats increased gradually with increase of fluoride doses (F =164.10,P ＜ 0.05).The protein levels of phosphor-ERK5 in the control group,low fluoride group and high fluoride group were 0.13 ± 0.03,0.29 ± 0.10and 0.43 ±0.17,respectively,the difference was statistically significant (F=11.96,P＜ 0.05),and low fluoride group and high fluoride group were higher than control group (all P ＜ 0.05).The total protein levels of ERK5 in control group,low fluoride group and high fluoride group were 0.32 ± 0.11,0.37 ± 0.13 and 0.49 ± 0.16,respectively,the difference was statistically significant (F =3.45,P ＜ 0.05),and high fluoride group was higher than control group (P ＜ 0.05).The expression of ERK5 mRNA in rat brains between groups was not significantly different (F =0.81,P ＞ 0.05).The second,third,and forth days of directional navigation experiment,the time of escape latency and the number of crossing the platform between groups were statistically significant (H =28.20,29.90,26.47,27.23,35.34,27.62,all P ＜ 0.01);the fifth day of space exploration experiment,the difference of the time of the first crossing platform and the number of crossing the platform between groups were statistically significant (H =31.41,30.80,all P ＜ 0.01);the protein level of phosphor-ERK5 in brain tissue of rats was negatively correlated with the number of the first crossing platform (r =-0.470,P ＜ 0.01),while positively related to escape latencies at the fifth day of the test (r =0.591,P ＜ 0.01).Conclusion The changes of ERK5 signaling pathway in rat brain tissue caused by chronic fluorosis are found,which are related to the decrease of leaming and memory ability of animals with chronic fluorosis.
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To investigate the effect and the mechanism of puerarin in attenuating PM2.5-induced human umbilical vein endothelial cells (EA.hy926) injury, the samples of fine particulate matter (PM2.5) were collected and made into suspension. Different concentrations of PM2.5 (0,20, 200, 400 mg•L⁻¹) were used to contaminate EA.hy926 cells for 24 h. The cells survival rate was detected by MTT assay; cells apoptosis of EA.hy926cells was detected by flow cytometry; the protein levels of p-ERK1/2, Bax and Bcl-2 were detected by Western blot; the contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), malonaldehyde (MDA), and the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH) were measured by ELISA. Puerarin at different concentrations (10, 50, 100 μmol•L⁻¹) or a specific inhibitor of ERK1/2 pathway PD98059 (20 μmol•L-1) was added into the EA.hy926 cells to observe the intervention effect and mechanism of puerarin. Compared with the control group, PM2.5 reduced the cells survival rate, up-regulatedp-ERK1/2 protein level and Bax/Bcl-2 ratio in a dose dependent manner to promote apoptosis; increased the contents of TNF-α, IL-6 and MDA, the activity of LDH, but decreased SOD activity in the EA.hy926 cells (P<0.05). Compared with PM2.5 group, puerarin increased the cells survival rate, down-regulated p-ERK1/2 protein level and Bax/Bcl-2 ratio in a dose dependent manner to inhibit the apoptosis; decreased the contents of TNF-α, IL-6 and MDA, the activity of LDH, but increased SOD activity in the EA.hy926 cells (P<0.05). The results indicated that puerarin could attenuate PM2.5-induced EA.hy926 cells injury via the inhibition of ERK1/2 pathway.
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This study aimed to clarify preliminarily the effects and mechanisms of Shenkang injection (SKI) promoting extracellular matrix(ECM)degradation via regulating extracellular-signal regulated protein kinase(ERK)1/2/matrix metalloproteinases(MMPs)signaling pathway in renal failure rats. Twenty rats were randomly divided into 4 groups：the Sham group,the Model group,the SKI group and the Enalapril maleate(EM)group. The model rats with renal failure were induced by intragastric administration of adenine and unilateral ureteral obstruction(UUO). After modeling, the rats in SKI group and EM group were intervened by intraperitoneal injection of SKI or intragastric administration of the EM suspension,while the rats in Sham group and Model group were administrated with distilled water respectively for 3 weeks. The 24 h urinary protein excretion(Upro)and urinary N-acety1-β-D-glucosaminidase(UNAG)in all rats were tested after drug administration. All rats were sacrificed after drug administration for 3 weeks,blood and kidney were collected,renal morphological characteristics were observed. Furthermore,serum biochemical indices and the protein expressions of collagen type IV(CIV),MMP-2,MMP-9,tissue inhibitors of metalloproteinase(TIMP)-1,ERK1/2 and phosphorylated-ERK1/2(p-ERK1/2)in the kidney were evaluated respectively. The results indicated that,after the intervention of SKI,serum creatinine(Scr),blood urea nitrogen(BUN),uric acid(UA),albumin(Alb),Upro,UNAG and renal morphological change in model rats were improved at different levels,respectively. Moreover,these actions were similar to EM. In addition to these,SKI adjusted the protein expressions of MMP-2,MMP-9 and TIMP-1,and down-regulated the protein expressions of p-ERK1/2 in the kidney. Moreover,these actions were different from EM. In conclusion,SKI promotes ECM degradation and delays the progression of renal failure possibly through regulating ERK1/2 signaling pathway activation in the kidney and intervening MMPs/TIMP-1 expressions in vivo.
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Abstrac:Aim To study the effect of hydroxysafflor yellow A ( HYSA ) on the proliferation of vascular smooth muscle cells ( VSMCs) and the related molecu-lar mechanism. Methods The inhibitory effects of hydroxysafflor yellow A on VSMC proliferation was de-tected using cell culture, MTT assay, Western blot and immunohistochemical staining. Results The results showed that HYSA inhibited cell proliferation induced by PDGF in a dose-dependent (5,10,20,40 μmol· L-1 ) manner, reduced proliferating cell nuclear anti-gen ( PCNA ) expression and blocked PDGFR-MEK-ERK1/2 signaling pathway activated by PDGF in VSMCs. Conclusion HYSA inhibits VSMCs prolifer-ation via reducing the expression of PCNA and blocking signal transduction of MEK-ERK1/2 in VSMCs.
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Objective To investigate the preventive effects of emilia sonchifolia on experimental hepatic steatosis in rats and its molecular mechanism.Methods Seventy Sprague-Dawley (SD) rats were randomly divided into five groups: normal control, model, high dose emilia sonchifolia, low dose emilia sonchifolia groups and high dose emilia sonchifolia + phosphorylated extracellular signal regulated protein kinase 1/2 (pERK1/2) inhibitor (PD98059) group (PD group). In normal control group, the rats were fed with normal diet, and in the other four groups, the rats were fed with high fat and low protein diet combined with 30% carbon tetrachloride (CCl4) peanut oil 2 mL/kg subcutaneous injection, once every 3 days for consecutive 3 weeks to establish animal models with hepatic steatosis. In emilia sonchifolia high and low dose groups, 5.0 g/kg and 2.5 g/kg doses of emilia sonchifolia were given respectively by gavage, once a day. In PD group, after administration of emilia sonchifolia high dose by gavage once a day, additionally PD98059 0.3 mg/kg was injected through a tail vein, once a week. After 3 weeks, all rats were switched to normal diet and treatment continued as before. At the end of the 5th week, liver tissues were taken for pathological analyses. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total cholesterol (TC), and triglyceride (TG) were determinated by automatic biochenical analyzer. The positive cell count and protein expressions of sterol-regulatory element binding protein 1 (SREBP-1), pERK1/2, toll like receptor 4 (TLR4) and high mobility group box-1 protein (HMGB1) were tested by immunohistochemistry, Western Blot and flow cytometry. The levels of superoxide dismutase (SOD) and malonaldehyde (MDA) in liver cell homogenate were detected by hydroxylamine and TBA method.Results Compared with the model group, the lobular inflammation in high and low dose emilia sonchifolia groups and PD group was attenuated (1.50±0.53, 1.80±0.43, 1.20±0.42 vs. 2.30±0.48), and ALT, AST, TC, TG, SREBP-1, and MDA were significantly decreased, the decrease in high dose emilia sonchifolia group being the most significant [ALT (U/L): 51.91±6.95 vs. 66.50±12.15, AST (U/L): 125.70±5.62 vs. 147.10±10.52, TC (mmol/L): 1.79±1.04 vs. 2.81±1.08, TG (mmol/L): 0.87±0.55 vs. 1.17±0.67, SREBP-1: (30.60±5.56)% vs. (53.10±5.02)%, MDA (nmol/mg): 5.20±0.87 vs. 10.61±5.45,P 0.05]. While the above index values in PD group were close to those in high dose emilia sonchifolia group, showing that PD98059 had no impact on emilia sonchifolia's action.Conclusions Emilia sonchifolia can alleviate hepatic injury and attenuate lobular inflammation in rat experimental hepatic steatosis. Its mechanism is possibly related to the reduction of oxidative stress reaction, and SREBP-1 may be as a mediator involved in the action.
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@#Objective To observe the effect of electroacupuncture on basic fibroblast growth factor (bFGF), Desmin and extracellular signal-regulated kinase (ERK1/2) expression of rabbits with acute lumbar muscle contusion, and discuss the possible mechanism of muscle tissue regeneration and repair. Methods 40 male New Zealand rabbits were randomly divided into blank group (BG, n=10), model group (MG, n=10), Weizhong (BL40) electroacupuncture group (WG, n=10) and local electroacupuncture group (LG, n=10). Lumbar muscle injury was established with blunt trauma, and the rabbits were assessed with Appearance Score. The WG and LG accepted electroacupuncture for 2 weeks after modeling. The lumbar muscle of each animal was stained with HE to observe pathological changes, and immunohistochemical staining was used to observe bFGF expression, and Western blotting was used to detect the expression of Desmin and ERK1/2 protein.Results Appearance Score was more in the MG, WG and LG than in the BG (P<0.01). Histological score ranked from more to less as MG,LG, WG and BG (P<0.05). The expression of bFGF ranked as LG, WG, MG and BG (P<0.01), Desmin ranked as WG=LG and BG=MG (P<0.05), ERK1/2 as LG=WG, MG and BG (P<0.05). Conclusion Electroacupuncture could promote the regeneration and repair of tissue after acute lumbar muscle contusion, which may be related to the up-regulation of bFGF/ERK signaling pathway.
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Objective To explore the effects and mechanisms of prostaglandin E2 (PGE2) receptor 1 antagonist (SC-19220) on proliferation,prostaglandin synthase and extracellular regulated protein kinases (ERK) signal pathway induced by transforming growth factor β1(TGF-β1) in glomerular mesangial cells.Methods Mouse glomerular mesangial cells (GMCs) were divided into 5 groups:control group,TGF-β1 (10 μg/L) group,TGF-β1 (10 μg/L) plus SC-19220 group (0.1,0.5,1.0 μmol/L).The proliferation of GMCs was measured by CCK-8.The PGE2 in supernatant was measured by ELISA.The expression of connective tissue growth factor (CTGF),laminin (LN),cyclooxygenase 2(COX2),membrane-bound prostaglandin E2 synthase 1 (mPGES1) protein and mRNA was examined by Westem blotting and real-time quantitative PCR,ERK1/2 or phospho-ERK1/2 was measured by Western blotting as well.Results TGF-β1 induced the proliferation of GMCs and increased the secretion of PGE2.Besides,TGF-β1 significantly up-regulated the expression of CTGF,LN,COX2 and mPGES1 mRNA and protein (P ＜ 0.05),and increased the expression of phospho-ERK1/2 protein (P ＜ 0.05).However,SC-19220 significantly attenuated the changes of above-mentioned parameters and their activities (P ＜ 0.05).All the effects of SC-19220 were in dose-dependent manner.Conclusions SC19220 may reduce TGF-β1-induced cell damage by suppressing the activity of ERK1/2,and feedback inhibition of COX2,mPGES1 and PGE2,thus decreases the expression of LN and CTGF.
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Objective: To investigate the effect of ECHS1 (enoyl-coenzyme A hydratase, short chain 1) on the proliferation of hepatocellular carcinoma HepG2 cells. Methods: The recombinant plasmid pGPU6/GFP/siRNA-ECHS1 for ECHS1 gene interference was transfected into HepG2 cells. The HepG2 cells with stable ECHS1 gene interference were screened by puromycin. The expression level of ECHS1 protein in HepG2 cells was detected by Western blotting. The proliferative ability of HepG2 cells after transfection with pGPU6/GFP/siRNA-ECHS1 was detected by CCK-8 (cell counting kit-8) assay and BrdU (5-bromo-2'-deoxyuridine) assay. The expression levels of ERK (extracellular signal regulated kinase), p-ERK (phosphorylated-ERK), cyclin D3 and cyclin D1 were detected by Western blotting. Results: The HepG2 cells with stable ECHS1 gene interference were successfully established. The expression level of ECHS1 protein in HepG2 cells after transfection with pGPU6/GFP/siRNA-ECHS1 was significantly lower than that in the blank control cells (HepG2 cells without transfection) and the negative control cells (HepG2 cells transfected with pGPU6 vector) (P < 0.05). As compared with the negative control cells, the proliferative ability was significantly inhibited (P < 0.05) and the expression levels of p-ERK, cyclin D3 and cyclin D1 proteins were lower in HepG2 cells after transfection with pGPU6/GFP/siRNA-ECHS1 (P < 0.05). Conclusion: ECHS1 may promote the proliferative ability of HepG2 cells via enhancing the expression levels of p-ERK, cyclin D3 and cyclin D1. Copyright © 2013 by TUMOR.
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Objective To investigate the effect of quetiapine on the behavior and expression of pERK1/2 in chronic unpredictable stress(CUS) model rats.Methods 32 adult male Sprague Dawley rats were randomly divided into four groups (n =8 for each group):control group,CUS group,CUS + QUE (5 mg/kg,L) group and CUS + QUE(10 mg/kg,M)group.The rats in control group were left undisturbed in their home cage for 28 days and the other groups were exposed to 28 consecutive days of CUS,then the rats in control group and CUS group were treated with 1％ DMSO in saline (5 ml/kg,intraperitoneal injection),the rats in CUS + QUE (L)group and CUS + QUE(M) group respectively treated with quetiapine (5 mg/kg)or quetiapine(10 mg/kg) for consecutive 7 days.The weight data of each group were recorded,and the behavioral changes in these rats were analyzed by open field test and forced swimming test;and the expression of pERK1/2 was measured by Western blot.Results (1)Compared with control group,quetiapine (10 mg/kg) ameliorated the inhibition of body weight gain that induced by chronic unpredictable stress (P ＜ 0.05),but quetiapine (5 mg/kg) did not have this effect.(2)Open field and Forced swimming test showed significant difference (P ＜ 0.05) of horizontal motion distance (F =17.846),the number of central region entering(F=4.720) and the immobility time(F=26.090) in each group.And these tests showed that horizontal motion distance and the number of central region entering in CUS group ((6696.30 ±1061.19)mm,(19.63 ±9.15)times) were significantly lower than that of control group ((10824.61 ± 1399.37) mm,(37.75 ± 13.02) times) and CUS + QUE (M) group ((9637.51 ± 1630.16) mm,(32.38 ± 6.23)),while the immobility time (110.73 ± 15.98)s were significantly higher than that of control group((66.13 ± 5.18)s)and CUS + QUE (M) group((73.40 ± 11.99) s,P ＜ 0.05).But there was no significant difference between that of CUS group and CUS + QUE(L) group(P＞0.05).(3)The expression of pERK1/2 in CUS group showed significant decrease when compared with control group or CUS + QUE (M) group,but showed no significant difference with CUS + QUE(L) group(F=6.641,P＜ 0.01).Conclusion Quetiapine can ameliorate depressive-like behaviors induced by chronic unpredictable stress,and this effect may be carried out by up-regulation the expression of pERK1/2 in the hippocampus.
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Objective To investigate dynamic changes of extracellular signal regulated protein kinase (ERK) 1/2 in lung fibroblast of newborn rats with chronic lung disease (CLD) caused by hyperoxia.Methods Full-term newborn rats were randomly divided into two groups:air-exposed group and hyperoxia - exposed with 90％ oxygen group.Rats were sacrificed separately 3 d,7 d and 14 days after exposure to air or 90％ oxygen. Then lung fibroblasts of rats were isolated and primarily cultured. By using Immunocystochemistry,Western-blot and RT-PCR methods,the levels of ERK1/2 protein and expressions of ERK1/2 mRNA were measured. Results The levels of p-ERK1/2 protein in lung fibroblast in the hyperoxia group were significant higher on the 7th day and 14th day after exposure to 90％ oxygen compared with those in the air-exposed group (P ＜0.01 ).And the levels of total ERK1/2 protein and expressions of ERK1/2 mRNA did not change noticeably and were not significantly different between two groups (P ＞0.05 ).Conclusions The activation of phosphorated ERK1/2 may lead to lung fibrosis caused by hyperoxia in newborn rats.
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ObjectiveTo investigate the effects of ziprasidone on the behavior and the expression of pERK1/2 in posttraumatic stress disorder(PTSD) model rats.Methods 24 adult male SD rats weighing (200 ±20) g were randomly divided into four groups (n =6):control group,single prolonged stress and foot shock (SPS&S) group,ziprasidone group and ziprasidone + U0126 group.The fear response to environment,high alertness,and anxiety & depression behavior of rats were tested by the open field,elevated plus-maze,and the expression of pERK1/2 was measured by Western blot.ResultsIn open field test(OFT),the SPS&S group( (76.23 ± 54.76) cm for horizontal motion distance,(4.60 ± 1.14) for the number of entering central region) showed significant difference compared with control group ( (343.77 ± 74.22 ) cm,( 12.40 ± 3.36 ) ) or ziprasidone group ( ( 274.98± 83.56) cm,( 12.00 ± 2.92) ) (P ＜ 0.01 ),but showed no significant difference with ziprasidone + U0126 group ( ( 138.14 ± 41.98) cm,(5.00 ± 1.58) ) (P ＞ 0.05 ).The results of elevated plus maze (EPM) were in accordance with the results of OFT.The expression of pERK1/2 in SPS&S group and ziprasidone + U0126 group showed significant decrease when compared with control group or ziprasidone group (P ＜ 0.01 ).ConclusionZiprasidone can obviously improve fear response to environment,high alterness and anxiety & depression behavior of rats,and these effects of ziprasidone may be carried out by up-regulation the expression of pERK1/2.
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Objective To observe the effects of Tongluo reeipe on extracellular signal-regulated protein kinase (ERK1/2) pathway in the kidneys of streptozotocin-induced rat models of type 1 diabetes mellitus, and to explore the renoprotective mechanism of Tongluo reeipe. Methods Adult male Sprague-Dawley rats were randomly divided into 2 groups: control group (A, n=10) and experimental group. Rats in the experimental group were induced by intraperitoneal injection of streptozotocin (60 mg/kg body mass), and were randomly subdivided into group B (diabetic control, n=10), group C (diabetic rats treated with low dose of Tongluo recipe,0.5g/[kg · d], n=10), group D (diabetic rats treated with medium dose of Tongluo recipe, 1.0g/[kg · d], n=10), group E (diabetic rats treated with high dose of Tongluo recipe, 2.0g/[kg · d], n=10) and group F (diabetic rats treated with losartan, 30 mg/[kg · d],n=10). At the end of the 12th week rats were sacrificed, and the serum glucose, triacylglycerol(TG), total cholesterol(Chol), serum creatinine(Scr), urea nitrogen(BUN) in blood, 24-hour urine protein(Upro24), microalbuminuria (MAU), and creatinine in urine (UCR) were examined. The kidney mass/body mass (KM/BM) and creatinine clearance rate (Ccr) were calculated. The left kídneys were collected, weighed and subjected to H-E staining and PAS staining to observe the pathological changes; serum and renal tissue angiotensin II (Ang II) levels were determined by ELISA; the expressíon of phosphorylated ERK1/2 (p-ERK1/2) was detected by immunohistochemical staining and vascular endothelial growth factor (VEGF) protein expression was determined by Western blotting analysis. Results Compared with group B, group D had a slighter mesangial proliferation under light microscope. Besides, rats in group D had significantly decreased Ccr, BUN, MAU, and Upro24 in the serum, and decreased Ang II in the serum and renal tissue, and decreased KM/BM, VEGF and p-ERK1/2 levels in renal tissue compared with rats in group B(P<0.05). The medium dose of Tongluo recipe and losartan showed similar effects in improving the renal function and inhibiting Ang II, VEGF, and p-ERK1/2 expression of diabetic rats. Conclusion The renoprotective effect of Tongluo recipe may be related to inhibition of ERK1/2 cascade pathway by decreasing the expression of Ang II and VEGF.
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Objective To observe the effects of Tongluo recipe on extracellular signal-regulated protein kinase (ERK1/2) pathway in the kidneys of streptozotocin-induced rat models of type 1 diabetes mellitus, andto explore the renoprotective mechanism of Tongluo recipe. Methods Adult male Sprague-Dawley rats were randomly divided into 2 groups: control group (A, n = 10) and experimental group. Rats in the experimental group were induced by intraperitoneal injection of streptozotocin (60 mg/kg body mass), and were randomly subdivided into group B (diabetic control, n = 10), group C (diabetic rats treated with low dose of Tongluo recipe, 0. 5 g/[kg • d], n = 10), group D (diabetic rats treated with medium dose of Tongluo recipe, 1. 0 g/[kg • d], n = 10), group E (diabetic rats treated with high dose of Tongluo recipe,2. 0 g/[kg • d], n = 10) and group F (diabetic rats treated with losartan, 30 mg/[kg • d],n=10). At the end of the 12th week rats were sacrificed, and the serum glucose, triacylglycerol(TG), total cholesterol(Choi), serum creatinine(Scr), urea nitrogen(BUN) in blood, 24-hour urine protein(Upro24), microalbuminuria (MAU), and creatinine in urine(UCR) were examined. The kidney mass/body mass (KM/BM) and creatinine clearance rate (Ccr) were calculated. The left kidneyswere collected, weighed and subjected to H-E staining and PAS staining to observe the pathological changes; serum and renal tissue angiotensin II (Ang II) levels were determined by ELISA; the expression of phosphorylated ERK1/2(p-ERK1/2) was detectedby immunohistochemical staining and vascular endothelial growth factor (VEGF) protein expression was determined by Western blotting analysis. Results Compared with group B, group D had a slighter mesangial proliferation under light microscope. Besides, rats in group D had significantly decreased Ccr, BUN, MAU, and Upro24 in the serum, and decreased Ang II in the serum and renal tissue, and decreased KM/BM, VEGF and p-ERK1/2 levels in renal tissue compared with rats in group B(P<0. 05). The medium dose of Tongluo recipe and losartan showed similar effects in improving the renal function and inhibiting Ang II, VEGF, and p-ERK1/2 expression of diabetic rats. Conclusion The renoprotective effect of Tongluo recipe may be related to inhibition of ERK1/2 cascade pathway by decreasing the expression of Ang II and VEGF.
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Objective To investigate the expression of extraeellular signal-regulated protein kinase (ERK1/2)pathway in rat brains with fluorosis and the effects of fluoride on learning and memory of the rats,and to reveal the mechanisms of damaged nervous system resulted from the toxicity of the ion.Methods Seventy-two SD rats were divided into 3 groups and 24 rats were in each group.Three groups were fed respectively with different concentrations of fluoride(NaF)for 6 months to establish rat models with fluorosis.Controls were fed with tap water (NaF＜0.5 mg/L):lower and higher concentration group were fed with water containing NaF(5,50 ms/L).Animals are sacrificed after 6 months of treatment with fluoride and the dissected brains were kept for analysis.The protein levels of ERK1/2 in rat brains were detected by Western-blotting and the mRNA level by RT-PCR. The spatial learning and memorizing ability was measured by Morris water maze test. Results The ERK1/2 protein in control group,lower and higher concentration group was 0.944±0.10,1.253±0.02,1.953±0.07,the differece being statistieally sighificant between any two groups (P ＜ 0.05). The phospho-ERKl/2 protein in control group,lower and higher concentration group was 0.73±0.08,0.77±0.07,1.28±0.11,the differece being statistieally sighificant between any two groups(P ＜ 0.05);the activation rate of phospho-ERK1/2 in lower and higher concentration group [(68.4± 3.8)%,(64.1±3.2)%] was decreased compared to control group[ (82.3±10.7)%],the differece being significant(P ＜ 0.05). In the navigation trial,longer escape latencies of lower concentration group on the second, the third,the fifth and the sixth day were observed[ (46.0±8.0),(24.0±2.7),(8.9±5.3),(7.4±4.1 )s] compared to the control[ (39.3±6.9),(19.1±9.1 ),(8.3±3.4),(4.8±2.7)s],the differece being significant (P ＜ 0.05 or ＜ 0.01 );the similar results were also observed in the higher concentration group[ (36.9±16.8),(37.7±12.9), (19.7±7.6),(12.2±5.7 )s],and the escape latencies of the higher concentration group on the third,the fifth and the sixth day were longer than that in lower concentration group. In the probe test,the rats took more time to reach the first cross in lower and higher concentration group[(1.17±0.75),(4.18±1.10)s] than control group[ (5.89± 0.56 ) s ],the differece being significant (P ＜ 0.05 or ＜ 0.01 ) ;stayed shorter [ ( 17.05±4.25 ),(18.20±4.57 ) s ] than control [(25.37±5.65 )s ] in platform area (P ＜ 0.01 );the activation rates of ERK1/2 were directly correlated with the time taken to reach the first cross platform located in the probe test(r = 0.364,P ＜ 0.05) and the activation rates were also directly correlated with the escape latencies on the sixth day(r = 0.497,P ＜ 0.05). Conclusion Long-term exposure of excessive fluoride induces the change of expression and activating rate of the ERK1/2 in rat brains,leading to the decreased capacity of learning and memory.
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In order to investigate if there exists interaction between mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) protein, and how the interaction regulates tumor necrosis factor-? (TNF-?) transcription activity, the human p38 and extracellular-signal regulated protein kinase 2 (ERK2) genes were amplified from human flag-p38 and flag-ERK2 by polymerase chain reaction (PCR) and cloned into pcDNA3-HA. Protein expression of the plasmids was examined by Western blotting. Co-immunoprecipitation was used to identify if there exists interaction between MAPK and STAT3 proteins. If the interaction was approved to be true, report gene system was applied to find how the interaction affect transcriptional expression of TNF-?. After STAT3 pathway was inhibited by RNA interfering, the action on TNF-? activity was determined. The results of DNA sequencing and enzyme digestion showed that the cloned p38 and ERK2 genes were correct, to be 1 080 bp or so. p38 and ERK2 proteins were expressed in 293T cell to be approximately 40 ku. Co-immunoprecipitation data showed that p38 and ERK2 proteins integrated with STAT3 protein in vivo. TNF-? reporter gene activity results found that protein complex of p38-STAT3 and ERK2-STAT3 coordinately increased TNF-? activity. After STAT3 was interfered, the TNF-? activity markedly decreased. These data indicated that there exists interaction between p38 and STAT3 protein, ERK2 and STAT3 protein. The complex of the proteins can coordinately regulate TNF-? expression. After interfereing STAT3 pathway, the coordinated action on TNF-? transcription activity might be obviously reduced.
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Focal adhesion kinase(FAK)and extracellular signal-regulated protein kinase(ERK),which are thought to play major roles in transducing surface receptor-derived signals into nucleus,are pivotal signal transduction molecules in mammalian cells.FAK is activated through phosphorylation by extracellular stimuli.p-FAK activates the downstream of ERK which changes cell action.FAK-ERK signal conduction factors are involved in a variety of biological responses,such as cell proliferation,differentiation,stress,apoptosis and malignant change.The biologic characters of FAK-ERK and advances in the study on its roles in tissue damage are reviewed in this article,which may provide a more effective way for estimation of wound age in forensic medicine.
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Objective:To investigate the effect of extracellular signal-regulated protein kinase (ERK) on hepatic fibrosis autoreversibility and its mechanism. Methods: Animal model of hepatic fibrosis autoreversibility was established 6 weeks after 8-week CCl_(4 ) exposure. Then the activation, expression and distribution of ERK in the liver were investigated by Northern blot and immunohistochemistry during fibrosis reverse. RT-PCR and cDNA hybridization were used to detect the RNA and protein expression of ERK substrates. Results: The transcription of ERK was highly activated throughout the hepatic fibrosis (recovery.) ERK was located in the nucleus of hepatic stellate cells (HSCs) in fibrotic liver while it was mainly expressed in the hepatocyte cytoplasm of the lobule, sinusoid space, and perifibrous tissue as hepatic fibrosis resolved. Downstream molecules of ERK like S6 protein kinase (RSK1), c-fos, phospholipase A2, ion channels, and gastrointestinal hormone receptors were also found up-regulated during the whole course. Conclusion: ERK may be closely related to hepatic fibrosis reversibility and it may protect and promote proliferation of hepatocyte and induce apoptosis of HSC.
ABSTRACT
Objective To study expression intensity and distribution of phosphorylated forms of extracellular signal-regulated protein kinase1/2 (ERK1/2), stress-activated protein kinase (SAPK) and P38MAPK in normal skin and hypertrophic scars and their potential biological significance. Methods The morphological characteristics of hypertrophic scars in different periods after wound healing and normal skin were examined histopathologically. Protein expression of p-ERK1/2, p-SAPK and p-P38MAPK was also assessed with immunohistochemical technique. Results In normal skin, phosphorylated forms of ERK1/2, SAPK and P38MAPK were mainly located in epidermal basal cells, in which the positive cellular rates of all the three proteins were low. Along with the maturation of hypertrophic scars, protein contents of p-ERK1/2 and p-SAPK were progressively increased. In mature hypertrophic scars, positive signals of these two proteins were mostly distributed in keratinocytes and some fibroblasts. The positive cellular rate of p-P38MAPK ascended in active hypertrophic scar, then decreased to a level which was still higher than that of normal skin. Conclusion The formation of hypertrophic scars may be associated with the alteration in signaling pathways which results in the increment of protein contents of p-ERK1/2, p-SAPK and p-P38MAPK.