Background:Magnesium cantharidate(MC)is a protein phosphatase 2A(PP2A)inhibitor antitumor drug.However,its antitumor mechanism in hepatocellular carcinoma cell(HCC)remains unclear.Methods:PP2A lentiviral vector over e...Background:Magnesium cantharidate(MC)is a protein phosphatase 2A(PP2A)inhibitor antitumor drug.However,its antitumor mechanism in hepatocellular carcinoma cell(HCC)remains unclear.Methods:PP2A lentiviral vector over expression strategy was utilized both in vivo and in vitro to explore the antitumor effect in MC and okadaic acid(OA).Tumor weight was detected in mice after MC and OA exposure.Cell proliferation,cell cycle,apoptosis rate,and western blotting were detected to explore the effects on MC and OA in human hepatocarcinoma SMMC-7721 cells.Results:In vivo results demonstrated that MC inhibited HCC progression while OA promoted tumor growth.In vitro results demonstrated that MC effectively inhibited the growth of SMMC-7721 cells by arresting the cell cycle at the G2/M phase with inhibiting Cdc25C and activating the phosphorylation of the Cdc2 protein.Flow cytometry results further showed that MC increased apoptosis.Furthermore,the expression of phosphorylated ERK1/2 was lower in the MC group but higher in the OA group.Molecular docking results showed that MC docked well with ERK1/2.Conclusions:MC inhibited HCC progression by suppressing the growth and activating the apoptosis of cancer cells and suppressing the expression of PP2A and ERK1/2.展开更多
Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fib...Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fibroproliferation is an essential mechanism in ARDS.Mitofusion2(Mfn2)overexpression plays a role in inhibiting cell proliferation.However,the role and potential mechanism of Mfn2 on the proliferation of fibroblasts is still unknown.In this study,we aimed at exploring the effect of Mfn2 on the human embryonic lung fibroblasts(HELF)and discussed its related mechanism.The HELF were treated with the Mfn2 overexpressing lentivirus(adv-Mfn2).The cell cycle was detected by flow cytometry.MTT,PCR and Western blotting were used to investigate the effect of Mfn2 on the proliferation of the HELF,collagen expression,the RAS-RAF-1-ERK1/2 pathway and the expression of cycle-related proteins(p21,p27,Rb,Raf-1,p-Raf-1,Erk1/2 and p-Erk1/2).The co-immunoprecipitation assay was used to explore the interaction between Mfn2 and Ras.The results showed that the overexpression of Mfn2 inhibited the proliferation of the HELF and induced the cell cycle arrest at the G0/G1 phase.Meanwhile,Mfn2 also inhibited the expression of collagen I,p-Erk and p-Raf-1.In addition,an interaction between Mfn2 and Ras existed in the HELF.This study suggests that the overexpression of Mfn2 can decrease the proliferation of HELF in ARDS,which was associated with the inhibition of the RAS-RAF-1-ERK1/2 pathway.The results may offer a potential therapeutic intervention for patients with ARDS.展开更多
Background This study aimed to determine the effects of tumor necrosis factor(TNF-a) on endothelial cytoskeleton morphology and permeability,and to detect the underlying signaling mechanisms involved in these response...Background This study aimed to determine the effects of tumor necrosis factor(TNF-a) on endothelial cytoskeleton morphology and permeability,and to detect the underlying signaling mechanisms involved in these responses. Methods Cultured endothelial cells(ECs) were exposed to TNF-a,and EC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies.Endothelial permeability was detected by measuring the flux of HRP-albumin across the EC monolayers.To explore the signaling pathways behind TNF-a-induced EC alteration, ECs were treated with either the RhoGTPase inhibitor Y27632 or the MAPK inhibitors PD98059 and SB203580 before TNF-a administration.To further elucidate possible involvement of the RhoA and ERK pathways in TNF-induced EC changes,retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA,namely T19NRhoA and Q63LRhoA,were pre-infect-ed into ECs prior to TNF-a exposure.Results TNF-a induced F-actin cytoskeleton rearrangement,as well as EC hyperpermeability in a dose and time-dependent manner.The effects were attenuated in cells pretreated with Y27632 or PD98059,respectively.EC pre-infection with T19NRhoA also alleviated the effects of TNF-a.Furthermore,retrovirus-mediated administration of activated forms of Q63LRhoA alone induced rearrangement of F-actin and hyperpermeability as well as induced the activation of pERK.Conclusions These results indicate that RhoA-ERK/MAPK signal pathway play important roles in the mediation of TNF-a induced EC barrier dysfunction associated with morphological changes of the Factin.展开更多
Objective:To study the effect of estrogen on anovulatory dysfunctional uterine bleeding(ADUB).Methods:Primary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolylic activity of gelalinase was d...Objective:To study the effect of estrogen on anovulatory dysfunctional uterine bleeding(ADUB).Methods:Primary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolylic activity of gelalinase was determined by gelatin zymography analysis.Cellular mRNA and protein synthesis was blocked respectively to determine whether the increased expression of MMP-2/9 was induced by estrogen.The expression of VEGF was blocked by siRNA.After treatment with various factors.MMP-9,VEGF,total Erk and phosphorylated Erk expression in primary uterine epithelial cells was detected by Western blotting analysis.Cell MMP-2/9mRNA levels was measured by real-time RT-PCR.Results:The activity and expression of MMP2/9 was inereased in the endometrium of patients with ADUB.Estrogen could up-regulate the expression of VEGF and activate Erk 1/2-Elk1 signal path.After interference by siRNA,ERK1/2 pathway was blocked in cells,and the expression of MMP-2/9 was down-regulated.ERK1/2 specific blocker U0126 blocked ERK phosphorylation,and it could down-regulate the expression of MMP-2/9.Conclusions:The results showed that the estrogen can increase the expression of VEGF,and thus activate ERK1/2 pathway to induce MMP-2/9 expression.展开更多
Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells v...Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells via the estrogen receptor β （ERβ） and the cAMP-extracellular signal-regulated kinase （ERK1/2） pathway. Low levels （10-10-10-8 mol L-1） of 17β-estradiol increased cell number, but high levels （10-7-10-6 mol L-1） decreased it （P〈0.05）. Sertoli cell number began to recover for an additional 24 h in the medium without 17β-estradiol （10-6 mol L-l） （P〉0.05）. The effects of 17β-estradiol （10-9 mol L-1） peaked at the first 24 h （P〈0.05）. 17β-estradiol activated ERK1/2 from 5 min to 24 h, but the activiy of ERK1/2 began to decrease after 4 h. Both PD98059 and U0126, two ERK inhibitors, blocked cell division （P〈0.05）. 17β-estradiol （10-10-10-6 mol L-1） dose-dependently increased cAMP production （P 〈 0.05）, and both 17β-estradiol （10-9 mol L-1） and forskolin, which increases cAMP levels, induced cell proliferation and activated ERK1/2 （P〈 0.05）. Rp-cAMP, an antagonist of cAMP, blocked this 17β-estradiol activity （P〈 0.05）. Two estrogen receptor antagonists, ICI 182780 and ERβ antagonist （ERβAnt）, reduced Sertoli cell number, cAMP production and ERK1/2 activation （P〈 0.05）, but ERaAnt did not （P〉 0.05）. Therefore, 17β- estradiol mainly promotes pig Sertoli cell proliferation via ERβ to induce cAMP production and ERK activation to promote cell proliferation.展开更多
[Objectives] To investigate the role of Eriocalyxin B (EriB) in promoting colon cancer cell apoptosis through ERK1/2 pathway in vitro, and to provide a natural candidate drug for colon cancer treatment. [Methods] Colo...[Objectives] To investigate the role of Eriocalyxin B (EriB) in promoting colon cancer cell apoptosis through ERK1/2 pathway in vitro, and to provide a natural candidate drug for colon cancer treatment. [Methods] Colon cancer cells treated with different concentrations of EriB were detected by CCK-8 assay;cell scratch assay and crystal violet staining were used to detect the invasion and migration of colon cancer cell;cell apoptosis was detected by Annexin V/PI double staining, and cell cycle was detected by PI staining;Western Blotting was used to detect epithelial-mesenchymal transition and apoptosis-related proteins in colon cancer cells treated with EriB. [Results] After EriB treatment, the proliferation, migration and apoptosis of colon cancer cells were significantly inhibited, and the ratio of P-ERK1/2 to ERK was significantly decreased. [Conclusions] EriB can effectively inhibit the proliferation of colon cancer cells and promote the apoptosis of colon cancer cells through ERK1/2 pathway.展开更多
Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-asso...Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.展开更多
基金The research was financially supported by the National Natural Science Foundation of China(no.81760746)Science and Technology Department of Zunyi city of Guizhou province of China([2020]7)Guizhou Provincial Science&Technology Program(ZK[2022]615).
文摘Background:Magnesium cantharidate(MC)is a protein phosphatase 2A(PP2A)inhibitor antitumor drug.However,its antitumor mechanism in hepatocellular carcinoma cell(HCC)remains unclear.Methods:PP2A lentiviral vector over expression strategy was utilized both in vivo and in vitro to explore the antitumor effect in MC and okadaic acid(OA).Tumor weight was detected in mice after MC and OA exposure.Cell proliferation,cell cycle,apoptosis rate,and western blotting were detected to explore the effects on MC and OA in human hepatocarcinoma SMMC-7721 cells.Results:In vivo results demonstrated that MC inhibited HCC progression while OA promoted tumor growth.In vitro results demonstrated that MC effectively inhibited the growth of SMMC-7721 cells by arresting the cell cycle at the G2/M phase with inhibiting Cdc25C and activating the phosphorylation of the Cdc2 protein.Flow cytometry results further showed that MC increased apoptosis.Furthermore,the expression of phosphorylated ERK1/2 was lower in the MC group but higher in the OA group.Molecular docking results showed that MC docked well with ERK1/2.Conclusions:MC inhibited HCC progression by suppressing the growth and activating the apoptosis of cancer cells and suppressing the expression of PP2A and ERK1/2.
基金This project was supported by Wuhan Medical Science Foundation of China(No.WX17B07,No.WX19A09,and No.WJ2019H324).
文摘Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fibroproliferation is an essential mechanism in ARDS.Mitofusion2(Mfn2)overexpression plays a role in inhibiting cell proliferation.However,the role and potential mechanism of Mfn2 on the proliferation of fibroblasts is still unknown.In this study,we aimed at exploring the effect of Mfn2 on the human embryonic lung fibroblasts(HELF)and discussed its related mechanism.The HELF were treated with the Mfn2 overexpressing lentivirus(adv-Mfn2).The cell cycle was detected by flow cytometry.MTT,PCR and Western blotting were used to investigate the effect of Mfn2 on the proliferation of the HELF,collagen expression,the RAS-RAF-1-ERK1/2 pathway and the expression of cycle-related proteins(p21,p27,Rb,Raf-1,p-Raf-1,Erk1/2 and p-Erk1/2).The co-immunoprecipitation assay was used to explore the interaction between Mfn2 and Ras.The results showed that the overexpression of Mfn2 inhibited the proliferation of the HELF and induced the cell cycle arrest at the G0/G1 phase.Meanwhile,Mfn2 also inhibited the expression of collagen I,p-Erk and p-Raf-1.In addition,an interaction between Mfn2 and Ras existed in the HELF.This study suggests that the overexpression of Mfn2 can decrease the proliferation of HELF in ARDS,which was associated with the inhibition of the RAS-RAF-1-ERK1/2 pathway.The results may offer a potential therapeutic intervention for patients with ARDS.
文摘Background This study aimed to determine the effects of tumor necrosis factor(TNF-a) on endothelial cytoskeleton morphology and permeability,and to detect the underlying signaling mechanisms involved in these responses. Methods Cultured endothelial cells(ECs) were exposed to TNF-a,and EC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies.Endothelial permeability was detected by measuring the flux of HRP-albumin across the EC monolayers.To explore the signaling pathways behind TNF-a-induced EC alteration, ECs were treated with either the RhoGTPase inhibitor Y27632 or the MAPK inhibitors PD98059 and SB203580 before TNF-a administration.To further elucidate possible involvement of the RhoA and ERK pathways in TNF-induced EC changes,retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA,namely T19NRhoA and Q63LRhoA,were pre-infect-ed into ECs prior to TNF-a exposure.Results TNF-a induced F-actin cytoskeleton rearrangement,as well as EC hyperpermeability in a dose and time-dependent manner.The effects were attenuated in cells pretreated with Y27632 or PD98059,respectively.EC pre-infection with T19NRhoA also alleviated the effects of TNF-a.Furthermore,retrovirus-mediated administration of activated forms of Q63LRhoA alone induced rearrangement of F-actin and hyperpermeability as well as induced the activation of pERK.Conclusions These results indicate that RhoA-ERK/MAPK signal pathway play important roles in the mediation of TNF-a induced EC barrier dysfunction associated with morphological changes of the Factin.
基金supported by Natural Science Foundation of Hainan Province(No.812148)
文摘Objective:To study the effect of estrogen on anovulatory dysfunctional uterine bleeding(ADUB).Methods:Primary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolylic activity of gelalinase was determined by gelatin zymography analysis.Cellular mRNA and protein synthesis was blocked respectively to determine whether the increased expression of MMP-2/9 was induced by estrogen.The expression of VEGF was blocked by siRNA.After treatment with various factors.MMP-9,VEGF,total Erk and phosphorylated Erk expression in primary uterine epithelial cells was detected by Western blotting analysis.Cell MMP-2/9mRNA levels was measured by real-time RT-PCR.Results:The activity and expression of MMP2/9 was inereased in the endometrium of patients with ADUB.Estrogen could up-regulate the expression of VEGF and activate Erk 1/2-Elk1 signal path.After interference by siRNA,ERK1/2 pathway was blocked in cells,and the expression of MMP-2/9 was down-regulated.ERK1/2 specific blocker U0126 blocked ERK phosphorylation,and it could down-regulate the expression of MMP-2/9.Conclusions:The results showed that the estrogen can increase the expression of VEGF,and thus activate ERK1/2 pathway to induce MMP-2/9 expression.
基金supported by the National Natural Science Foundation of China(30270955)the Foundamental Research Funds for the Central Universities,China(XDJK2009B035)
文摘Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells via the estrogen receptor β （ERβ） and the cAMP-extracellular signal-regulated kinase （ERK1/2） pathway. Low levels （10-10-10-8 mol L-1） of 17β-estradiol increased cell number, but high levels （10-7-10-6 mol L-1） decreased it （P〈0.05）. Sertoli cell number began to recover for an additional 24 h in the medium without 17β-estradiol （10-6 mol L-l） （P〉0.05）. The effects of 17β-estradiol （10-9 mol L-1） peaked at the first 24 h （P〈0.05）. 17β-estradiol activated ERK1/2 from 5 min to 24 h, but the activiy of ERK1/2 began to decrease after 4 h. Both PD98059 and U0126, two ERK inhibitors, blocked cell division （P〈0.05）. 17β-estradiol （10-10-10-6 mol L-1） dose-dependently increased cAMP production （P 〈 0.05）, and both 17β-estradiol （10-9 mol L-1） and forskolin, which increases cAMP levels, induced cell proliferation and activated ERK1/2 （P〈 0.05）. Rp-cAMP, an antagonist of cAMP, blocked this 17β-estradiol activity （P〈 0.05）. Two estrogen receptor antagonists, ICI 182780 and ERβ antagonist （ERβAnt）, reduced Sertoli cell number, cAMP production and ERK1/2 activation （P〈 0.05）, but ERaAnt did not （P〉 0.05）. Therefore, 17β- estradiol mainly promotes pig Sertoli cell proliferation via ERβ to induce cAMP production and ERK activation to promote cell proliferation.
基金Supported by Natural Science Research Project of Colleges and Universities in Anhui Province(KJ2019A1110,KJ2020A0863)Innovation Team Research Fund Project of Anhui Medical College(WJH202007t,WJH202008t)2021 Anhui Provincial Production-Education Integration Training Base Project for Quality Engineering(2021cjrh020).
文摘[Objectives] To investigate the role of Eriocalyxin B (EriB) in promoting colon cancer cell apoptosis through ERK1/2 pathway in vitro, and to provide a natural candidate drug for colon cancer treatment. [Methods] Colon cancer cells treated with different concentrations of EriB were detected by CCK-8 assay;cell scratch assay and crystal violet staining were used to detect the invasion and migration of colon cancer cell;cell apoptosis was detected by Annexin V/PI double staining, and cell cycle was detected by PI staining;Western Blotting was used to detect epithelial-mesenchymal transition and apoptosis-related proteins in colon cancer cells treated with EriB. [Results] After EriB treatment, the proliferation, migration and apoptosis of colon cancer cells were significantly inhibited, and the ratio of P-ERK1/2 to ERK was significantly decreased. [Conclusions] EriB can effectively inhibit the proliferation of colon cancer cells and promote the apoptosis of colon cancer cells through ERK1/2 pathway.
基金supported by the National Natural Science Foundation of China,Nos.82071008(to BL)and 82004001(to XJ)Medical Science and Technology Program of Health Commission of Henan Province,No.LHGJ20210072(to RQ)Science and Technology Department of Henan Province,No.212102310307(to XJ)。
文摘Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.
