Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB le...Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.展开更多
Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac p...Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac protection.Our previous work found that sphingosine-1-phosphate(S1P)could ameliorate cardiac hypertrophy.In this study,we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.Methods:Eight-week-old male C57BL/6 mice were randomly divided into a sham,transverse aortic constriction(TAC)or a TAC+S1P treatment group.Results:We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease inα-smooth muscle actin(α-SMA)and collagen type I(COL I)expression compared with the TAC group.We also found that one of the key S1P enzymes,sphingosine kinase 2(SphK2),which was mainly distributed in cytoblasts,was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro.In addition,our in vitro results showed that S1P treatment activated extracellular regulated protein kinases(ERK)phosphorylation mainly through the S1P receptor 2(S1PR2)and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.Conclusion:These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart.This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.展开更多
Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mech...Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.展开更多
目的探索CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响。方法选取30只SPF级SD雄性大鼠,依据随机数字表法分为健康组、模型组、CX3CR1抑制组,每组10只。除健康组外,其余各组均建立创伤性骨髓炎模型。其中健...目的探索CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响。方法选取30只SPF级SD雄性大鼠,依据随机数字表法分为健康组、模型组、CX3CR1抑制组,每组10只。除健康组外,其余各组均建立创伤性骨髓炎模型。其中健康组、模型组大鼠均每日常规腹腔注射生理盐水,CX3CR1干预组向残腔内注射CX3CR1中和抗体进行处理。采用ELISA法检测血清中IL-6、IL-10、IL-1β、TGF-β水平,应用改良X线Norden评分检测骨骼肌微纤维,HE染色观察病理变化,免疫印迹及PCR检测股骨组织中细胞外信号调节蛋白激酶(Extracellular regulated protein kinase,ERK1/2)、丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)蛋白及mRNA表达。结果与健康组比较,模型组TGF-β、IL-1β、IL-10、IL-6等炎症因子含量均升高(P<0.05);与模型组比较,CX3CR1抑制组炎症因子含量降低(P<0.05)。与健康组比较,模型组随时间推移X线Norden评分升高(P<0.05);与模型组比较,CX3CR1抑制组X线Norden评分降低(P<0.05)。HE染色显示,健康组骨质完好;模型组可见大量炎性细胞浸润、灶性脓肿及坏死灶;CX3CR1抑制组大鼠的骨质明显改善,炎症反应降低。与健康组比较,模型组ERK1/2、MAPK蛋白及mRNA表达升高(P<0.05);与模型组比较,CX3CR1抑制组ERK1/2、MAPK蛋白及mRNA表达降低(P<0.05)。结论抑制CX3CR1可改善创伤性骨髓炎大鼠的疾病反应,可能与降低炎症反应、ERK/MAPK信号通路以及改善骨骼肌微纤维相关。展开更多
目的观察北五味子多糖(polysaccharide of Schisandra chinensis,SCP)对血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱导的心肌成纤维细胞(CFb)增殖作用,并探讨其作用机制.方法以培养的新生Wistar大鼠乳鼠CFb为实验模型,实验分为对照组、模型组...目的观察北五味子多糖(polysaccharide of Schisandra chinensis,SCP)对血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱导的心肌成纤维细胞(CFb)增殖作用,并探讨其作用机制.方法以培养的新生Wistar大鼠乳鼠CFb为实验模型,实验分为对照组、模型组、3个药物剂量组.采用胰酶消化、差速贴壁法培养CFb;四氮唑盐(MTT)比色法检测CFb增殖;羟脯氨酸(Hyp)法测定胶原含量;分光光度计测定CFb中超氧化物歧化酶(SOD)活力、丙二醛(MDA)水平;硝酸还原酶法和分光光度法分别测定不同干预条件下CFb培养液中一氧化氮合酶(NOS)活性及一氧化氮(NO)水平;免疫组化技术检测CFb中细胞外调节蛋白激酶(ERK1/2)蛋白表达.结果SCP能够显著抑制AngⅡ诱导的CFb增殖(P<0.05或P<0.01),降低Hyp含量,提高SOD活力,降低MDA水平(P<0.05,P<0.01),提高NOS活性及NO水平(P<0.05,P<0.01),减低ERK1/2蛋白表达(P<0.05,P<0.01).结论SCP可通过抑制ERK1/2信号通路提高NOS活性,并升高NO水平,增强抗AngⅡ诱导的CFb增殖能力.展开更多
基金supported by Research Start-up Funding of Shenzhen Traditional Chinese Medicine Hospital,No.2021-07(to FB)Sanming Project of Medicine in Shenzhen,No.SZZYSM 202111011(to XDQ and FB)+1 种基金Key Discipline Established by Zhejiang Province,Jiaxing City Jointly-Pain Medicine,No.2019-ss-ttyx(to LSX)Jiaxing Key Laboratory of Neurology and Pain Medicine,No.[2014]81(to LSX)。
文摘Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.
基金supported by the National Natural Science Foundation of China(No.81873505).
文摘Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac protection.Our previous work found that sphingosine-1-phosphate(S1P)could ameliorate cardiac hypertrophy.In this study,we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.Methods:Eight-week-old male C57BL/6 mice were randomly divided into a sham,transverse aortic constriction(TAC)or a TAC+S1P treatment group.Results:We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease inα-smooth muscle actin(α-SMA)and collagen type I(COL I)expression compared with the TAC group.We also found that one of the key S1P enzymes,sphingosine kinase 2(SphK2),which was mainly distributed in cytoblasts,was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro.In addition,our in vitro results showed that S1P treatment activated extracellular regulated protein kinases(ERK)phosphorylation mainly through the S1P receptor 2(S1PR2)and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.Conclusion:These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart.This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.
基金the National Natural Science Foundation of China (No. 30570627)
文摘Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.
文摘目的探索CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响。方法选取30只SPF级SD雄性大鼠,依据随机数字表法分为健康组、模型组、CX3CR1抑制组,每组10只。除健康组外,其余各组均建立创伤性骨髓炎模型。其中健康组、模型组大鼠均每日常规腹腔注射生理盐水,CX3CR1干预组向残腔内注射CX3CR1中和抗体进行处理。采用ELISA法检测血清中IL-6、IL-10、IL-1β、TGF-β水平,应用改良X线Norden评分检测骨骼肌微纤维,HE染色观察病理变化,免疫印迹及PCR检测股骨组织中细胞外信号调节蛋白激酶(Extracellular regulated protein kinase,ERK1/2)、丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)蛋白及mRNA表达。结果与健康组比较,模型组TGF-β、IL-1β、IL-10、IL-6等炎症因子含量均升高(P<0.05);与模型组比较,CX3CR1抑制组炎症因子含量降低(P<0.05)。与健康组比较,模型组随时间推移X线Norden评分升高(P<0.05);与模型组比较,CX3CR1抑制组X线Norden评分降低(P<0.05)。HE染色显示,健康组骨质完好;模型组可见大量炎性细胞浸润、灶性脓肿及坏死灶;CX3CR1抑制组大鼠的骨质明显改善,炎症反应降低。与健康组比较,模型组ERK1/2、MAPK蛋白及mRNA表达升高(P<0.05);与模型组比较,CX3CR1抑制组ERK1/2、MAPK蛋白及mRNA表达降低(P<0.05)。结论抑制CX3CR1可改善创伤性骨髓炎大鼠的疾病反应,可能与降低炎症反应、ERK/MAPK信号通路以及改善骨骼肌微纤维相关。
文摘目的观察北五味子多糖(polysaccharide of Schisandra chinensis,SCP)对血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱导的心肌成纤维细胞(CFb)增殖作用,并探讨其作用机制.方法以培养的新生Wistar大鼠乳鼠CFb为实验模型,实验分为对照组、模型组、3个药物剂量组.采用胰酶消化、差速贴壁法培养CFb;四氮唑盐(MTT)比色法检测CFb增殖;羟脯氨酸(Hyp)法测定胶原含量;分光光度计测定CFb中超氧化物歧化酶(SOD)活力、丙二醛(MDA)水平;硝酸还原酶法和分光光度法分别测定不同干预条件下CFb培养液中一氧化氮合酶(NOS)活性及一氧化氮(NO)水平;免疫组化技术检测CFb中细胞外调节蛋白激酶(ERK1/2)蛋白表达.结果SCP能够显著抑制AngⅡ诱导的CFb增殖(P<0.05或P<0.01),降低Hyp含量,提高SOD活力,降低MDA水平(P<0.05,P<0.01),提高NOS活性及NO水平(P<0.05,P<0.01),减低ERK1/2蛋白表达(P<0.05,P<0.01).结论SCP可通过抑制ERK1/2信号通路提高NOS活性,并升高NO水平,增强抗AngⅡ诱导的CFb增殖能力.
