Background:Kirsten rat sarcoma(KRAS)and mutant KRAS^(G12D)have been implicated in human cancers,but it remains unclear whether their activation requires ubiquitination.This study aimed to investigate whether and how F...Background:Kirsten rat sarcoma(KRAS)and mutant KRAS^(G12D)have been implicated in human cancers,but it remains unclear whether their activation requires ubiquitination.This study aimed to investigate whether and how F-box and leucine-rich repeat 6(FBXL6)regulates KRAS and KRAS^(G12D)activity in hepatocellular carcinoma(HCC).Methods:We constructed transgenic mouse strains LC(LSL-Fbxl6^(KI/+);Alb-Cre,n=13),KC(LSL-Kras^(G12D/+);Alb-Cre,n=10)and KLC(LSL-Kras^(G12D/+);LSL-Fbxl6^(KI/+);Alb-Cre,n=12)mice,and then monitored HCC for 320 d.Multiomics approaches and pharmacological inhibitors were used to determine oncogenic signaling in the context of elevated FBXL6 and KRAS activation.Co-immunoprecipitation(Co-IP),Western blotting,ubiquitination assay,and RAS activity detection assay were employed to investigate the underlying molecular mechanism by which FBXL6 activates KRAS.The pathological relevance of the FBXL6/KRAS/extracellular signal-regulated kinase(ERK)/mammalian target of rapamycin(mTOR)/proteins of relevant evolutionary and lymphoid interest domain 2(PRELID2)axis was evaluated in 129 paired samples from HCC patients.Results:FBXL6 is highly expressed in HCC as well as other human cancers(P<0.001).Interestingly,FBXL6 drives HCC in transgenic mice.Mechanistically,elevated FBXL6 promotes the polyubiquitination of both wild-type KRAS and KRAS^(G12D)at lysine 128,leading to the activation of both KRAS and KRAS^(G12D)and promoting their binding to the serine/threonine-protein kinase RAF,which is followed by the activation of mitogen-activated protein kinase kinase(MEK)/ERK/mTOR signaling.The oncogenic activity of the MEK/ERK/mTOR axis relies on PRELID2,which induces reactive oxygen species(ROS)generation.Furthermore,hepatic FBXL6 upregulation facilitates KRAS^(G12D)to induce more severe hepatocarcinogenesis and lung metastasis via the MEK/ERK/mTOR/PRELID2/ROS axis.Dual inhibition of MEK and mTOR effectively suppresses tumor growth and metastasis in this subtype of cancer in vivo.In clinical samples,FBXL6 expression positively correlates with p-ERK(χ^(2)=85.067,P<0.001),p-mTOR(χ^(2)=66.919,P<0.001)and PRELID2(χ^(2)=20.891,P<0.001).The Kaplan-Meier survival analyses suggested that HCC patients with high FBXL6/p-ERK levels predicted worse overall survival(log-rank P<0.001).Conclusions:FBXL6 activates KRAS or KRAS^(G12D)via ubiquitination at the site K128,leading to activation of the ERK/mTOR/PRELID2/ROS axis and tumorigenesis.Dual inhibition of MEK and mTOR effectively protects against FBXL6-and KRAS^(G12D)-induced tumorigenesis,providing a potential therapeutic strategy to treat this aggressive subtype of liver cancer.展开更多
Objective： Mitogen-activated protein kinases （MAPKs） are correlated with a more malignant phenotype in many cancers. This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 ...Objective： Mitogen-activated protein kinases （MAPKs） are correlated with a more malignant phenotype in many cancers. This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 （MKP-1） and phosphorylated extracellular signal-regulated kinase 1/2 （p-ERKl/2）, as the key regulatory mechanism of the MAPKs, in lung squamous cell carcinoma （SCC）. Methods： We assessed the expressions of MKP-1 and p-ERK1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction （RT-PCR） analysis. Results： Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma, and it was negatively correlated with tumor differentiation （P〈0.01）. However, the expression of p-ERK1/2 or ERKl/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma, and it was positively correlated with tumor differentiation （P〈0.01）. Conclusions： Our data indicates the relevance of MKP-1 and p-ERK1/2 in SCC as a potential positive and negative prognostic factor. The imbalanced expression of MKP-1 and p-ERKl/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.展开更多
Extracellular signal-regulated protein kinase 5 （ERK5）, also known as big mitogen-activated protein kinase 1 （MAPK1）, is an important member of ERK family, which is a subfamily of the large MAPK family. ERK5 is ex...Extracellular signal-regulated protein kinase 5 （ERK5）, also known as big mitogen-activated protein kinase 1 （MAPK1）, is an important member of ERK family, which is a subfamily of the large MAPK family. ERK5 is expressed in many tissues, including the dorsal root ganglion （DRG） neurons and the spinal cord. In this review, we focus on elaborating ERK5-associated pathway in pathological pain, in which the ERK5/CREB （cyclic adenosine monophos- phate （cAMP）-response element-binding protein） pathway plays a crucial role in the transduction of pain signal and contributes to pain hypersensitivity. ERK5 activation in the spinal dorsal horn occurs mainly in microglia. The activation of ERK5 can be mediated by N-methyI-D-aspartate （NMDA） receptors. We also elaborate the relationship between ERK5 activation and nerve growth factor-tyrosine kinase A （NGF-TrkA）, and the connection between ERK5 activation and brain-derived neurotrophic factor （BDNF） in pathological pain in detail.展开更多
The human immunodeficiency virus type 1 （HIV-1） can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase （M...The human immunodeficiency virus type 1 （HIV-1） can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase （MAPK） signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase （ERK） pathway inhibitor, PD98059, the Jun N-terminal kinase （JNK） pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1NL4-3 luciferase reporter virus and HIV-1NL4-3 virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-INL4-3 luciferase reporter virus inhibition activity but no HIV-1NL4-3 virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.展开更多
Objective： To investigate the relationship between extracellular signal-regulated kinase （ERK） pathway, multidrug resistance gene （mdr-1）, ribonucleotide recluctase M1 （RRM1） gene and their roles in gemcitabine...Objective： To investigate the relationship between extracellular signal-regulated kinase （ERK） pathway, multidrug resistance gene （mdr-1）, ribonucleotide recluctase M1 （RRM1） gene and their roles in gemcitabine （GEM） chemoresistance in pancreatic cancer cell line SW1990. Methods： The GEM-resistance cell model was constructed by a stepwise method. Immunohistochemistry was used to measure the expression of ERK protein （ERK1/2） in the established cell strains in a semiquantitative way. The mRNA expression of mdr-1 and RRM1 were detected by RT-PCR. MTT assay was performed to determine the IC50 value. Results： The established GEM-resistant cell strains were able to gain stable growth and passage ability in the medium contained different concentration levels of GEM （0, 30, 60, 100, 150 and 200 nmol/L）. The expression of ERK protein, mdr-1 and RRM1 gene were elevated accompanied by the increase of GEM concentration. There was a highly positive correlation between mdr-1, RRM1 expression and GEM-resistanca level （r = 0.960, P = 0.002 and r = 0.966, P = 0.002）. The expression of ERK protein also correlated with the mdr-1 and RRM1 level （r = -0.943, P = 0.005 and r = -0.883, P = 0.02）. At the GEM-resistance level of 200 nmol/L, the grey scale value of ERK1/2 was 164.22 ±13.17, mdr-1/β-actin and RRM1/β-actin were 1.41 ±0.04 and 1.45 ± 0.18, respectively; after treated with ERK pathway inhibitor U0126, these values synchronously decreased to 186.85 ± 13.14, 0.2 3± 0.02 and 0.21 ± 0.03, respectively; inversely, the ERK1/2 grey scale value was 106.55 ± 16.45, mdr-l/β-actin and RRMl/β-actin were 1.50± 0.07 and 1.52 ± 0.12, respectively, which presented a tendency of synchronously increase after treated with ERK pathway activator EGF. The IC50 values in GEM-resistant cells of 0 nmol/L and 200 nmol/L levels were 4.104 and 10.20, respectively. After treated with U0126, these values decreased to 3.26 and 4.50, respectively; while treated with EGF, the IC50 values increased to 8.89 and 17.17, respectively. Conclusion： The ERK pathway may induce the GEM-chemoresistance in pancreatic cell line SW1990 through the participation in the regulation of the mdr-1 and RRM1 gene expression.展开更多
OBJECTIVE:To investigate the efficacy of Runjing(RJ)extract on oligoasthenoteratozoospermia(OAT)induced by ornidazole(ORN)in rats,and to study the underlying mechanism.METHODS:Twenty-four adult male Sprague-Dawley rat...OBJECTIVE:To investigate the efficacy of Runjing(RJ)extract on oligoasthenoteratozoospermia(OAT)induced by ornidazole(ORN)in rats,and to study the underlying mechanism.METHODS:Twenty-four adult male Sprague-Dawley rats were treated with normal saline(control),ORN(OAT model),ORN+4.725 g-dose)and ORN+18.9 g·kg^(-1)·d^(-1) RJ extract(low·kg^(-1)·d^(-1) RJ extract(high-dose)for 4 weeks.The rats were then euthanized and sperm and testis samples were collected for analysis.Sperm count,motility and morphology were calculated by sperm suspension from cauda epididymis.Testicular histopathological changes were analyzed by hematoxylin and eosin staining and Td T mediated d UTP nick end labelling.Moreover,the expression of vimentin and extracellular signal-regulated kinase(ERK)were examined through Western blot,and the distribution of vimentin was detected via immunohistochemistry.RESULTS:ORN successfully induces seminiferous epithelium injury,cellular apoptosis,and finally OAT(P<0.05).However,both low-dose and highdose RJ extract partially rescues the altered phenotypes(P<0.05).Moreover,the expressions of vimentin and ERK were significantly altered in ORN testes(all P<0.001),while RJ extract partially reversed these effects(P<0.01 or P<0.001).CONCLUSION:RJ extract can help maintain spermatogenesis through ERK signalling,and regulating vimentin expression.展开更多
Objective： To examine modulations caused by cyclooxygenase-2 （COX-2） inhibitors on altered microenvironments and overbalanced neurotransmitters in pilocarpine-induced epileptic status rats and to investigate possib...Objective： To examine modulations caused by cyclooxygenase-2 （COX-2） inhibitors on altered microenvironments and overbalanced neurotransmitters in pilocarpine-induced epileptic status rats and to investigate possible mechanisms. Methods： Celecoxib （a COX-2 inhibitor） was administered 45 min prior to pilocarpine administration. The effects of COX-2 inhibitors on mlPSCs （miniature GABAergic inhibitory postsynaptic currents） of CA3 pyramidal cells in the hippocampus were recorded. Expressions of COX-2, c-Fos, newly generated neurons, and activated microgliosis were analyzed by immunohistochemistry, and expressions of c^-subunit of y-amino butyric acid （GABAA） receptors and mitogen-activated protein kinase/extracellular signal-regulated protein kinase （MAPK/ERK） activity were detected by Western blotting. Results： Pretreatment with celecoxib showed protection against pilocarpine-induced seizures. Celecoxib prevented microglia activation in the hilus and inhibited the abnormal neurogenesis and astrogliosis in the hippocampus by inhibiting MAPK/ERK activity and c-Fos transcription. Celecoxib also up-regulated the expression of GABAA receptors. NS-398 （N-2-cyclohexyloxy-4-nitrophenyl-methanesulfonamide）, another COX-2 inhibitor, enhanced the frequency and decay time of mIPSCs. Conclusion： The COX-2 inhibitor celecoxib decreased neuronal excitability and prevented epileptogenesis in pilocarpine-induced status epilepticus rats. Celecoxib regulates synaptic reorganization by inhibiting astrogliosis and ectopic neurogenesis by attenuating MAPK/ERK signal activity, mediated by a GABAergic mechanism.展开更多
Accumulating evidence indicates that endostatin inhibits fibrosis. However, the mechanism is yet to be clarified. The aim of this study is to evaluate the effect of endostatin on platelet-derived growth factor-BB （PD...Accumulating evidence indicates that endostatin inhibits fibrosis. However, the mechanism is yet to be clarified. The aim of this study is to evaluate the effect of endostatin on platelet-derived growth factor-BB （PDGF-BB）- or transforming growth factor β1 （TGF-β1）-induced fibrosis in cultured human skin fibroblasts, and to further examine the molecular mechanisms involved. Human dermal flbroblasts were cultured in Dulbecco＇s modified Eagle＇s medium （DMEM） and serum-starved for 48 h before treatment. Cells were grouped as follows： ＂PDGF-BB＂, ＂PDGF-BB＋ endostatin＂, ＂TGF-β1＂, ＂TGF-β1＋endostatin＂, ＂endostatin＂, and ＂blank control＂. The fibroblasts were stimulated with either TGF-β1 or PDGF-BB for 72 h in order to set up the fibrosis model in vitro. The cells were co-cultured with either TGF-β1 or PDGF-BB and endostatin and were used to check the inhibiting effect of endostatin. A blank control group and an endostatin group were used as negative control groups. The biomarkers of fibrosis, including the expression of collagen I, hydrroxyproline, and α-smooth muscle actin （a-SMA）, were evaluated using an enzyme-linked immune- sorbent assay （ELISA） and Western blot. The expression of phosphorylated PDGF receptor β （p-PDGFRβ）, PDGFRβ, phosphorylated extracellular signal-regulated kinase （p-ERK）, and ERK was detected using Western blot and im- munofiuorescent staining was used to explore the mechanisms. Both PDGF-BB and TGF-β1 significantly up-regulated the expression of collagen I, hydroxyproline, and a-SMA. Endostatin significantly attenuated both the PDGF-BB- and TGF-β1-induced over-expression of collagen I, hydroxyproline, and a-SMA. PDGF-BB and TGF-β1 both promoted the expression of PDGFR, ERK, and p-ERK. Endostatin inhibited the expression of PDGFR and p-ERK but did not affect the expression of total ERK. Endostatin inhibited hypertrophic scar by modulating the PDGFRI3/ERK pathway. En- dostatin could be a promising multi-target drug in future fibrosis therapy.展开更多
OBJECTIVE: To study the anti-tumor effects of the extracts from Huangqi(Radix Astragali Mongolici)and Ezhu(Rhizoma Curcumae Phaeocaulis) on the growth of Lewis lung carcinoma(LLC) in a xenograft mouse model and to inv...OBJECTIVE: To study the anti-tumor effects of the extracts from Huangqi(Radix Astragali Mongolici)and Ezhu(Rhizoma Curcumae Phaeocaulis) on the growth of Lewis lung carcinoma(LLC) in a xenograft mouse model and to investigate the possible underlying mechanism.METHODS: LLC tumor-bearing C57 BL/6 mice were treated with normal saline, cisplatin(2 mg/kg intraperitoneally every other day), or Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis)(1∶1, 2∶1, or 3∶1 ratio;5, 8, or 11 g/kg crude drug intragastrically every day) for 15 d.Body weights and tumor volumes were measured every other day. Tumors were excised on day 15 and analyzed. Tumor microvessel density(MVD)was assessed by immunohistochemical staining of CD34;and expression of vascular endothelial cell growth factor(VEGF), the mitogen-activated protein kinases p38 mitogen-activated protein kinase(MAPK), extracellular signal-regulated kinases 1 and 2(ERK1/2), and Jun N-terminal kinase(JNK)and their phosphorylated forms were assessed by Western blotting.RESULTS: Treatment with cisplatin caused a significant loss of body weight compared with controls,whereas Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) extract combinations had no effect. Extracts from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) significantly decreased tumor weight and tumor MVD compared with controls,and at the 3∶1 treatment group had similar efficacy to cisplatin in reducing MVD. Tumors from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) treatments also showed decreased p38 MAPK, p-p38 MAPK, ERK1/2, p-ERK1/2,JNK, and p-JNK expression compared with the control group(all P < 0.01). VEGF protein expression was significantly reduced in the 2∶1 and 3∶1 treatment groups compared with the control group(P < 0.01).CONCLUSION: Extracts from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) hindered LLC growth in the xenograft mouse model, possibly via inhibition of the MAPK signaling pathway, VEGF production, and tumor angiogenesis.展开更多
基金supported by the National Natural Science Foundation of China(82370631)the Talent Foundations from Army Medical University(4174C6),the Chongqing Government(CQYC20220303727)to Xie CMthe National Natural Science Foundation of China(31900449)to Xiong HJ.
文摘Background:Kirsten rat sarcoma(KRAS)and mutant KRAS^(G12D)have been implicated in human cancers,but it remains unclear whether their activation requires ubiquitination.This study aimed to investigate whether and how F-box and leucine-rich repeat 6(FBXL6)regulates KRAS and KRAS^(G12D)activity in hepatocellular carcinoma(HCC).Methods:We constructed transgenic mouse strains LC(LSL-Fbxl6^(KI/+);Alb-Cre,n=13),KC(LSL-Kras^(G12D/+);Alb-Cre,n=10)and KLC(LSL-Kras^(G12D/+);LSL-Fbxl6^(KI/+);Alb-Cre,n=12)mice,and then monitored HCC for 320 d.Multiomics approaches and pharmacological inhibitors were used to determine oncogenic signaling in the context of elevated FBXL6 and KRAS activation.Co-immunoprecipitation(Co-IP),Western blotting,ubiquitination assay,and RAS activity detection assay were employed to investigate the underlying molecular mechanism by which FBXL6 activates KRAS.The pathological relevance of the FBXL6/KRAS/extracellular signal-regulated kinase(ERK)/mammalian target of rapamycin(mTOR)/proteins of relevant evolutionary and lymphoid interest domain 2(PRELID2)axis was evaluated in 129 paired samples from HCC patients.Results:FBXL6 is highly expressed in HCC as well as other human cancers(P<0.001).Interestingly,FBXL6 drives HCC in transgenic mice.Mechanistically,elevated FBXL6 promotes the polyubiquitination of both wild-type KRAS and KRAS^(G12D)at lysine 128,leading to the activation of both KRAS and KRAS^(G12D)and promoting their binding to the serine/threonine-protein kinase RAF,which is followed by the activation of mitogen-activated protein kinase kinase(MEK)/ERK/mTOR signaling.The oncogenic activity of the MEK/ERK/mTOR axis relies on PRELID2,which induces reactive oxygen species(ROS)generation.Furthermore,hepatic FBXL6 upregulation facilitates KRAS^(G12D)to induce more severe hepatocarcinogenesis and lung metastasis via the MEK/ERK/mTOR/PRELID2/ROS axis.Dual inhibition of MEK and mTOR effectively suppresses tumor growth and metastasis in this subtype of cancer in vivo.In clinical samples,FBXL6 expression positively correlates with p-ERK(χ^(2)=85.067,P<0.001),p-mTOR(χ^(2)=66.919,P<0.001)and PRELID2(χ^(2)=20.891,P<0.001).The Kaplan-Meier survival analyses suggested that HCC patients with high FBXL6/p-ERK levels predicted worse overall survival(log-rank P<0.001).Conclusions:FBXL6 activates KRAS or KRAS^(G12D)via ubiquitination at the site K128,leading to activation of the ERK/mTOR/PRELID2/ROS axis and tumorigenesis.Dual inhibition of MEK and mTOR effectively protects against FBXL6-and KRAS^(G12D)-induced tumorigenesis,providing a potential therapeutic strategy to treat this aggressive subtype of liver cancer.
基金supported by the National Natural Science Foundation of China (No. 30900654)the Science and Technology Department of Zhejiang Province (No. 2009R10031)the Health Bureau of Zhejiang Province (No. 2009QN010), China
文摘Objective： Mitogen-activated protein kinases （MAPKs） are correlated with a more malignant phenotype in many cancers. This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 （MKP-1） and phosphorylated extracellular signal-regulated kinase 1/2 （p-ERKl/2）, as the key regulatory mechanism of the MAPKs, in lung squamous cell carcinoma （SCC）. Methods： We assessed the expressions of MKP-1 and p-ERK1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction （RT-PCR） analysis. Results： Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma, and it was negatively correlated with tumor differentiation （P〈0.01）. However, the expression of p-ERK1/2 or ERKl/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma, and it was positively correlated with tumor differentiation （P〈0.01）. Conclusions： Our data indicates the relevance of MKP-1 and p-ERK1/2 in SCC as a potential positive and negative prognostic factor. The imbalanced expression of MKP-1 and p-ERKl/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.
基金supported by the Medical and Healthcare Project of Zhejiang Province(No.2015119381),China
文摘Extracellular signal-regulated protein kinase 5 （ERK5）, also known as big mitogen-activated protein kinase 1 （MAPK1）, is an important member of ERK family, which is a subfamily of the large MAPK family. ERK5 is expressed in many tissues, including the dorsal root ganglion （DRG） neurons and the spinal cord. In this review, we focus on elaborating ERK5-associated pathway in pathological pain, in which the ERK5/CREB （cyclic adenosine monophos- phate （cAMP）-response element-binding protein） pathway plays a crucial role in the transduction of pain signal and contributes to pain hypersensitivity. ERK5 activation in the spinal dorsal horn occurs mainly in microglia. The activation of ERK5 can be mediated by N-methyI-D-aspartate （NMDA） receptors. We also elaborate the relationship between ERK5 activation and nerve growth factor-tyrosine kinase A （NGF-TrkA）, and the connection between ERK5 activation and brain-derived neurotrophic factor （BDNF） in pathological pain in detail.
基金supported by the Key Projects in the National Science and Technology Pillar Program during the Eleventh Five-Year Plan Period of China (2008ZX10001-002)Major Science and Technology Innovation Cross Project of the Chinese Academy of Sciences (KSCX1-YW-10)
文摘The human immunodeficiency virus type 1 （HIV-1） can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase （MAPK） signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase （ERK） pathway inhibitor, PD98059, the Jun N-terminal kinase （JNK） pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1NL4-3 luciferase reporter virus and HIV-1NL4-3 virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-INL4-3 luciferase reporter virus inhibition activity but no HIV-1NL4-3 virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.
文摘Objective： To investigate the relationship between extracellular signal-regulated kinase （ERK） pathway, multidrug resistance gene （mdr-1）, ribonucleotide recluctase M1 （RRM1） gene and their roles in gemcitabine （GEM） chemoresistance in pancreatic cancer cell line SW1990. Methods： The GEM-resistance cell model was constructed by a stepwise method. Immunohistochemistry was used to measure the expression of ERK protein （ERK1/2） in the established cell strains in a semiquantitative way. The mRNA expression of mdr-1 and RRM1 were detected by RT-PCR. MTT assay was performed to determine the IC50 value. Results： The established GEM-resistant cell strains were able to gain stable growth and passage ability in the medium contained different concentration levels of GEM （0, 30, 60, 100, 150 and 200 nmol/L）. The expression of ERK protein, mdr-1 and RRM1 gene were elevated accompanied by the increase of GEM concentration. There was a highly positive correlation between mdr-1, RRM1 expression and GEM-resistanca level （r = 0.960, P = 0.002 and r = 0.966, P = 0.002）. The expression of ERK protein also correlated with the mdr-1 and RRM1 level （r = -0.943, P = 0.005 and r = -0.883, P = 0.02）. At the GEM-resistance level of 200 nmol/L, the grey scale value of ERK1/2 was 164.22 ±13.17, mdr-1/β-actin and RRM1/β-actin were 1.41 ±0.04 and 1.45 ± 0.18, respectively; after treated with ERK pathway inhibitor U0126, these values synchronously decreased to 186.85 ± 13.14, 0.2 3± 0.02 and 0.21 ± 0.03, respectively; inversely, the ERK1/2 grey scale value was 106.55 ± 16.45, mdr-l/β-actin and RRMl/β-actin were 1.50± 0.07 and 1.52 ± 0.12, respectively, which presented a tendency of synchronously increase after treated with ERK pathway activator EGF. The IC50 values in GEM-resistant cells of 0 nmol/L and 200 nmol/L levels were 4.104 and 10.20, respectively. After treated with U0126, these values decreased to 3.26 and 4.50, respectively; while treated with EGF, the IC50 values increased to 8.89 and 17.17, respectively. Conclusion： The ERK pathway may induce the GEM-chemoresistance in pancreatic cell line SW1990 through the participation in the regulation of the mdr-1 and RRM1 gene expression.
基金Supported by the Suzhou Science and Technology Development Plan:Based on the Theory of Collaterals Disease and Damp Disease Explore Promoting Dampness and Dredging Collateral Methods through P38mapk Signaling Pathways Regulate Inflammation Factor of ammatory MechanismⅢRe Type Prostatitis Anti-inflsearch(No.Sysd2018174)Zhangjiagang Science and Technology Plan:Based on the Mapk Pathway Research Runjing Decoction to Oxidative Stress Injury Caused by Weak SD Rats and the Protective Effects of Disease Mechanism Research,(No.ZKS1835)Youth Science and Technology Project of Zhangjiagang Health Plan:Based on the Theory of Essence Chamber to Explore Runjing Decoction Treating Idiopathic Less Weak Sperm Disease Clinical Research and the Analysis of Security(No.ZJGQNKJ201927)。
文摘OBJECTIVE:To investigate the efficacy of Runjing(RJ)extract on oligoasthenoteratozoospermia(OAT)induced by ornidazole(ORN)in rats,and to study the underlying mechanism.METHODS:Twenty-four adult male Sprague-Dawley rats were treated with normal saline(control),ORN(OAT model),ORN+4.725 g-dose)and ORN+18.9 g·kg^(-1)·d^(-1) RJ extract(low·kg^(-1)·d^(-1) RJ extract(high-dose)for 4 weeks.The rats were then euthanized and sperm and testis samples were collected for analysis.Sperm count,motility and morphology were calculated by sperm suspension from cauda epididymis.Testicular histopathological changes were analyzed by hematoxylin and eosin staining and Td T mediated d UTP nick end labelling.Moreover,the expression of vimentin and extracellular signal-regulated kinase(ERK)were examined through Western blot,and the distribution of vimentin was detected via immunohistochemistry.RESULTS:ORN successfully induces seminiferous epithelium injury,cellular apoptosis,and finally OAT(P<0.05).However,both low-dose and highdose RJ extract partially rescues the altered phenotypes(P<0.05).Moreover,the expressions of vimentin and ERK were significantly altered in ORN testes(all P<0.001),while RJ extract partially reversed these effects(P<0.01 or P<0.001).CONCLUSION:RJ extract can help maintain spermatogenesis through ERK signalling,and regulating vimentin expression.
文摘Objective： To examine modulations caused by cyclooxygenase-2 （COX-2） inhibitors on altered microenvironments and overbalanced neurotransmitters in pilocarpine-induced epileptic status rats and to investigate possible mechanisms. Methods： Celecoxib （a COX-2 inhibitor） was administered 45 min prior to pilocarpine administration. The effects of COX-2 inhibitors on mlPSCs （miniature GABAergic inhibitory postsynaptic currents） of CA3 pyramidal cells in the hippocampus were recorded. Expressions of COX-2, c-Fos, newly generated neurons, and activated microgliosis were analyzed by immunohistochemistry, and expressions of c^-subunit of y-amino butyric acid （GABAA） receptors and mitogen-activated protein kinase/extracellular signal-regulated protein kinase （MAPK/ERK） activity were detected by Western blotting. Results： Pretreatment with celecoxib showed protection against pilocarpine-induced seizures. Celecoxib prevented microglia activation in the hilus and inhibited the abnormal neurogenesis and astrogliosis in the hippocampus by inhibiting MAPK/ERK activity and c-Fos transcription. Celecoxib also up-regulated the expression of GABAA receptors. NS-398 （N-2-cyclohexyloxy-4-nitrophenyl-methanesulfonamide）, another COX-2 inhibitor, enhanced the frequency and decay time of mIPSCs. Conclusion： The COX-2 inhibitor celecoxib decreased neuronal excitability and prevented epileptogenesis in pilocarpine-induced status epilepticus rats. Celecoxib regulates synaptic reorganization by inhibiting astrogliosis and ectopic neurogenesis by attenuating MAPK/ERK signal activity, mediated by a GABAergic mechanism.
基金Project supported by the Zhejiang Provincial Natural Science Foundation of China(No.LY15H150004)the Teaching Department of the Zhejiang Province(No.Y201330073),China
文摘Accumulating evidence indicates that endostatin inhibits fibrosis. However, the mechanism is yet to be clarified. The aim of this study is to evaluate the effect of endostatin on platelet-derived growth factor-BB （PDGF-BB）- or transforming growth factor β1 （TGF-β1）-induced fibrosis in cultured human skin fibroblasts, and to further examine the molecular mechanisms involved. Human dermal flbroblasts were cultured in Dulbecco＇s modified Eagle＇s medium （DMEM） and serum-starved for 48 h before treatment. Cells were grouped as follows： ＂PDGF-BB＂, ＂PDGF-BB＋ endostatin＂, ＂TGF-β1＂, ＂TGF-β1＋endostatin＂, ＂endostatin＂, and ＂blank control＂. The fibroblasts were stimulated with either TGF-β1 or PDGF-BB for 72 h in order to set up the fibrosis model in vitro. The cells were co-cultured with either TGF-β1 or PDGF-BB and endostatin and were used to check the inhibiting effect of endostatin. A blank control group and an endostatin group were used as negative control groups. The biomarkers of fibrosis, including the expression of collagen I, hydrroxyproline, and α-smooth muscle actin （a-SMA）, were evaluated using an enzyme-linked immune- sorbent assay （ELISA） and Western blot. The expression of phosphorylated PDGF receptor β （p-PDGFRβ）, PDGFRβ, phosphorylated extracellular signal-regulated kinase （p-ERK）, and ERK was detected using Western blot and im- munofiuorescent staining was used to explore the mechanisms. Both PDGF-BB and TGF-β1 significantly up-regulated the expression of collagen I, hydroxyproline, and a-SMA. Endostatin significantly attenuated both the PDGF-BB- and TGF-β1-induced over-expression of collagen I, hydroxyproline, and a-SMA. PDGF-BB and TGF-β1 both promoted the expression of PDGFR, ERK, and p-ERK. Endostatin inhibited the expression of PDGFR and p-ERK but did not affect the expression of total ERK. Endostatin inhibited hypertrophic scar by modulating the PDGFRI3/ERK pathway. En- dostatin could be a promising multi-target drug in future fibrosis therapy.
基金Supported by the National Natural Science Foundation of China(No.81673810)
文摘OBJECTIVE: To study the anti-tumor effects of the extracts from Huangqi(Radix Astragali Mongolici)and Ezhu(Rhizoma Curcumae Phaeocaulis) on the growth of Lewis lung carcinoma(LLC) in a xenograft mouse model and to investigate the possible underlying mechanism.METHODS: LLC tumor-bearing C57 BL/6 mice were treated with normal saline, cisplatin(2 mg/kg intraperitoneally every other day), or Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis)(1∶1, 2∶1, or 3∶1 ratio;5, 8, or 11 g/kg crude drug intragastrically every day) for 15 d.Body weights and tumor volumes were measured every other day. Tumors were excised on day 15 and analyzed. Tumor microvessel density(MVD)was assessed by immunohistochemical staining of CD34;and expression of vascular endothelial cell growth factor(VEGF), the mitogen-activated protein kinases p38 mitogen-activated protein kinase(MAPK), extracellular signal-regulated kinases 1 and 2(ERK1/2), and Jun N-terminal kinase(JNK)and their phosphorylated forms were assessed by Western blotting.RESULTS: Treatment with cisplatin caused a significant loss of body weight compared with controls,whereas Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) extract combinations had no effect. Extracts from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) significantly decreased tumor weight and tumor MVD compared with controls,and at the 3∶1 treatment group had similar efficacy to cisplatin in reducing MVD. Tumors from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) treatments also showed decreased p38 MAPK, p-p38 MAPK, ERK1/2, p-ERK1/2,JNK, and p-JNK expression compared with the control group(all P < 0.01). VEGF protein expression was significantly reduced in the 2∶1 and 3∶1 treatment groups compared with the control group(P < 0.01).CONCLUSION: Extracts from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) hindered LLC growth in the xenograft mouse model, possibly via inhibition of the MAPK signaling pathway, VEGF production, and tumor angiogenesis.
