观察化瘀通络中药对糖尿病肾病大鼠肾脏炎症的干预作用,并探讨其机制。40只SD大鼠随机选取10只为正常组(C组),其余30只采用一次性腹腔注射链脲佐菌素(STZ,40 mg/kg)联合高糖高脂饲料喂养建立糖尿病模型。造模成功大鼠随机分为模型组(M组...观察化瘀通络中药对糖尿病肾病大鼠肾脏炎症的干预作用,并探讨其机制。40只SD大鼠随机选取10只为正常组(C组),其余30只采用一次性腹腔注射链脲佐菌素(STZ,40 mg/kg)联合高糖高脂饲料喂养建立糖尿病模型。造模成功大鼠随机分为模型组(M组)和化瘀通络中药组(Z组)。Z组予以中药干预。16周后,各组大鼠留尿检测24 h尿蛋白定量(24 h UTP);ELISA法检测血清中单核细胞趋化蛋白-1(MCP-1)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达;取肾组织,应用免疫荧光法检测巨噬细胞标志蛋白CD68、经典活化巨噬细胞标志蛋白诱导性一氧化氮合成酶(iNOS)的表达,应用Western blot法检测TAK1/JNK信号通路相关蛋白转化生长因子激活激酶-1(TAK1)、p-TAK1、c-Jun氨基末端激酶(JNK)和p-JNK的表达。实验结果显示,与C组比较,M组大鼠24 h UTP、MCP-1、IL-1β和TNF-α水平均明显升高(P<0.001);肾间质可见大量CD68和iNOS蛋白的沉积;p-TAK1和p-JNK蛋白的表达量明显增多(P<0.001)。与M组比较,Z组大鼠24 h UTP、MCP-1、IL-1β和TNF-α水平均显著降低(P<0.001,P<0.001,P<0.01,P<0.001);CD68和iNOS蛋白的沉积显著减少;p-TAK1和p-JNK蛋白的表达量均显著减少(P<0.05)。结果表明化瘀通络中药可降低糖尿病肾病大鼠蛋白尿,改善肾脏炎症反应,其机制可能与其抑制TAK1/JNK信号通路减少肾组织内巨噬细胞浸润、活化和炎症因子释放相关。展开更多
Interferon regulatory factor （IRF）3 is critical for the transcriptional induction of chemokines and cytokines during viral or bacterial invasion. The kinases Tank binding kinase （TBK）1 and Ikappa B kinase （IKK）...Interferon regulatory factor （IRF）3 is critical for the transcriptional induction of chemokines and cytokines during viral or bacterial invasion. The kinases Tank binding kinase （TBK）1 and Ikappa B kinase （IKK）ε can phosphorylate the C-terminal part of IRF3 and play important roles in IRF3 activation. In this study, we show that another kinase, c-Jun-NH2-terminal kinase （JNK）, phosphorylates IRF3 on its N-terminal serine 173 residue, and TAK1 can stimu- late IRF3 phosphorylation via JNK. JNK specific inhibitor SP600125 inhibits the N-terminal phosphorylation with- out affecting the C-terminal phosphorylation. In addition, IRF3-mediated gene expressions on lipopolysaccharide （LPS） or polyinosinic-cytidylic acid （polyI：C） treatment are severely impaired by SP600125, as well as for reporter gene assay of IRF3 activation. Knockdown of TAK1 further confirmed these observations. Interestingly, constitu- tive active IRF3（5D） can be inhibited by SP600125; JNK1 can synergize the action of IRF3（5D）, but not the S173A- IRF3（5D） mutant. More importantly, polyI：C failed to induce the phosphorylation of mutant S173A and SP600125 dramatically abrogated IRF3 phosphorylation and dimerization that was stimulated by polyhC. Thus, this study demonstrates that the TAK1-JNK cascade is required for IRF3 function, in addition to TBK1/IKKε, uncovering a new mechanism for mitogen-activated protein （MAP） kinase to regulate the innate immunity.展开更多
Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor...Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear.In this study,rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine(CPA)for five weeks.The mobility of rats was evaluated by forced swimming test,while their cognitive capabilities were evaluated by Y-maze test.Expression of sortilin,α-synuclein,p-JUN,and c-JUN proteins in the substantia nigra were detected by western blot analysis.In addition,immunofluorescence staining of sortilin andα-synuclein was performed to detect expression in the substantia nigra.The results showed that,compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine(5 mg/kg)+CPA co-treated rats,motor and memory abilities were reduced,surface expression of sortin andα-synuclein in dopaminergic neurons was reduced,and total sortilin and totalα-synuclein were increased in CPA-treated rats.MN9D cells were incubated with 500 nM CPA alone or in combination with 10μM SP600125(JNK inhibitor)for 48 hours.Quantitative real-time polymerase chain reaction analysis of sortilin andα-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone,but the combination of CPA and SP600125 could inhibit this expression.Predictions made using Jasper,PROMO,and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents.A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction.After sortilin expression was inhibited by sh-SORT1,expression of p-JUN and c-JUN was detected by western blot analysis.Long-term adenosine A1 receptor activation levels upregulatedα-synuclein expression at the post-transcriptional level by affecting sortilin expression.The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind toα-synuclein.Co-immunoprecipitation revealed an interaction between sortilin andα-synuclein in MN9D cells.Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression andα-synuclein accumulation,and dramatically improved host cognition and kineticism.This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan(approval No.AUP#20070090)in March 2007 and the Animals Ethics Committee of University of South China(approval No.LL0387-USC)in June 2017.展开更多
We investigated the liver protective activity of dandelion polyphenols(DP)against acetaminophen(APAP;Paracetamol)-induced hepatotoxicity.Mice were acclimated for 1 week and randomly divided into the following groups(n...We investigated the liver protective activity of dandelion polyphenols(DP)against acetaminophen(APAP;Paracetamol)-induced hepatotoxicity.Mice were acclimated for 1 week and randomly divided into the following groups(n=9 per group):Control,APAP,APAP+DP(100 mg·kg^–1),APAP+DP(200 mg·kg^–1),and APAP+DP(400 mg·kg^–1)groups.Mice were pretreated with DP(100,200,and 400 mg·kg^–1)by oral gavage for 7 d before being treated with 350 mg·kg^–1 APAP for 24 h to induced hepatotoxicity.Severe liver injury was observed,and hepatotoxicity was analyzed after 24 h by evaluation of biochemical markers,protein expressions levels,and liver histopathology.Pretreatment with DP was able to restore serum liver characteristics(aspartate transaminase,AST;alanine aminotransferase,ALT;alkaline phosphatase,AKP),improve redox imbalance(superoxide dismutase,SOD;glutathione,GSH;malondialdehyde,MDA),and decrease inflammatory factors(tumor necrosis factor-α,TNF-α;interleukin-1β,IL-1β).Pretreatment with DP also significantly inhibited the expression levels of nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2).Furthermore,DP pretreatment could inhibit the apoptosis of liver cells caused by APAP through up-regulation of Bcl-2 and down-regulation of Bax and caspase-9 protein.DP also down-regulated p-JNK protein expression levels to inhibit APAP-induced mitochondrial oxidative stress and up-regulated the expression of Nrf-2 and its target gene HO-1.The histopathological staining demonstrated that DP pretreatment could inhibit APAP-induced hepatocyte infiltration,congestion,and necrosis.Our results demonstrate that DP pretreatment could protect against APAP-induced hepatic injury by activating the Nrf-2/HO-1 pathway and inhibition of the intrinsic apoptosis pathway.展开更多
Background:Arteriosclerosis obliterans(ASO)is a major cause of adult limb loss worldwide.Autophagy of vascular endothelial cell(VEC)contributes to the ASO progression.However,the molecular mechanism that controls VEC ...Background:Arteriosclerosis obliterans(ASO)is a major cause of adult limb loss worldwide.Autophagy of vascular endothelial cell(VEC)contributes to the ASO progression.However,the molecular mechanism that controls VEC autophagy remains unclear.In this study,we aimed to explore the role of the GRB2 associated binding protein 1(GAB1)in regulating VEC autophagy.Methods:In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression.Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima.Gain-and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1.Results:The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor(0.80 vs.0.20,t=6.43,P<0.05).The expression level of GAB1 mRNA(1.00 vs.0.24,t=7.41,P<0.05)and protein(0.72 vs.0.21,t=5.97,P<0.05)was significantly decreased in ASO group as compared with the control group.Loss of GAB1 led to a remarkable decrease in LC3II(1.19 vs.0.68,t=5.99,P<0.05),whereas overexpression of GAB1 significantly led to a decrease in LC3II level(0.41 vs.0.93,t=7.12,P<0.05).Phosphorylation levels of JNK and p38 were significantly associated with gain-and loss-of-function of GAB1 protein.Conclusion:Loss of GAB1 promotes VEC autophagy which is associated with ASO.GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.展开更多
HepatitisBvirus(HBV)infection causes acuteand chronic liver diseases,but is not directly cytopathic.Liver injury results fromrepeated attempts of the cellular immune response system to control the viral infection.Here...HepatitisBvirus(HBV)infection causes acuteand chronic liver diseases,but is not directly cytopathic.Liver injury results fromrepeated attempts of the cellular immune response system to control the viral infection.Here,we investigate the roles of cellular factors and signaling pathways involved in the regulation of HBV replication to reveal the mechanism underlying HBV infection and pathogenesis.Weshowthat collagen triple helix repeat containing 1(CTHRC1)expression is elevated in HBV-infected patients andin HBV-transfected cells through epigenetic modification and transcriptional regulation.CTHRC1 facilitates HBV replication in cultured cells and BALB/c mice by activating the PKCa/ERK/JNK/c-Jun cascade to repress the IFN/JAK/STAT pathway.HBV-activated CTHRC1 downregulates the activityof typeI interferon(IFN),theproductionof IFN-stimulatedgenes(ISGs),andthephosphorylationofsignal transducerandactivator of transcription 1/2(STAT1/2),whereas it upregulates the phosphorylation and ubiquitination of type I IFN receptors(IFNARa/b).Thus,our results showthat HBV uses a novelmechanismto hijack cellular factors and signal cascades in order to evade host antiviral immunity and maintain persistent infection.We also demonstrate that CTHRC1 has a novel role in viral infection.展开更多
文摘观察化瘀通络中药对糖尿病肾病大鼠肾脏炎症的干预作用,并探讨其机制。40只SD大鼠随机选取10只为正常组(C组),其余30只采用一次性腹腔注射链脲佐菌素(STZ,40 mg/kg)联合高糖高脂饲料喂养建立糖尿病模型。造模成功大鼠随机分为模型组(M组)和化瘀通络中药组(Z组)。Z组予以中药干预。16周后,各组大鼠留尿检测24 h尿蛋白定量(24 h UTP);ELISA法检测血清中单核细胞趋化蛋白-1(MCP-1)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达;取肾组织,应用免疫荧光法检测巨噬细胞标志蛋白CD68、经典活化巨噬细胞标志蛋白诱导性一氧化氮合成酶(iNOS)的表达,应用Western blot法检测TAK1/JNK信号通路相关蛋白转化生长因子激活激酶-1(TAK1)、p-TAK1、c-Jun氨基末端激酶(JNK)和p-JNK的表达。实验结果显示,与C组比较,M组大鼠24 h UTP、MCP-1、IL-1β和TNF-α水平均明显升高(P<0.001);肾间质可见大量CD68和iNOS蛋白的沉积;p-TAK1和p-JNK蛋白的表达量明显增多(P<0.001)。与M组比较,Z组大鼠24 h UTP、MCP-1、IL-1β和TNF-α水平均显著降低(P<0.001,P<0.001,P<0.01,P<0.001);CD68和iNOS蛋白的沉积显著减少;p-TAK1和p-JNK蛋白的表达量均显著减少(P<0.05)。结果表明化瘀通络中药可降低糖尿病肾病大鼠蛋白尿,改善肾脏炎症反应,其机制可能与其抑制TAK1/JNK信号通路减少肾组织内巨噬细胞浸润、活化和炎症因子释放相关。
文摘Interferon regulatory factor （IRF）3 is critical for the transcriptional induction of chemokines and cytokines during viral or bacterial invasion. The kinases Tank binding kinase （TBK）1 and Ikappa B kinase （IKK）ε can phosphorylate the C-terminal part of IRF3 and play important roles in IRF3 activation. In this study, we show that another kinase, c-Jun-NH2-terminal kinase （JNK）, phosphorylates IRF3 on its N-terminal serine 173 residue, and TAK1 can stimu- late IRF3 phosphorylation via JNK. JNK specific inhibitor SP600125 inhibits the N-terminal phosphorylation with- out affecting the C-terminal phosphorylation. In addition, IRF3-mediated gene expressions on lipopolysaccharide （LPS） or polyinosinic-cytidylic acid （polyI：C） treatment are severely impaired by SP600125, as well as for reporter gene assay of IRF3 activation. Knockdown of TAK1 further confirmed these observations. Interestingly, constitu- tive active IRF3（5D） can be inhibited by SP600125; JNK1 can synergize the action of IRF3（5D）, but not the S173A- IRF3（5D） mutant. More importantly, polyI：C failed to induce the phosphorylation of mutant S173A and SP600125 dramatically abrogated IRF3 phosphorylation and dimerization that was stimulated by polyhC. Thus, this study demonstrates that the TAK1-JNK cascade is required for IRF3 function, in addition to TBK1/IKKε, uncovering a new mechanism for mitogen-activated protein （MAP） kinase to regulate the innate immunity.
基金supported by the National Natural Sciences Foundation of China,No.81770460(to YCL)the Postdoctoral Research Fellowship of the Saskatchewan Health Research Foundation,No.SHRF,4144(to YCL)+2 种基金the third level of the Chuanshan Talent project of the University of South China,No.2017CST20(to YCL)the Aid Program,No.2017KJ268 and the Key Lab for Clinical Anatomy&Reproductive Medicine,No.2017KJ182 from the Science and Technology Bureau of Hengyang City,China(to YCL and XC)the Postgraduate Student Research Innovation Projects of Hunan Province,China,No.CX2018B62(to ABG)
文摘Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear.In this study,rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine(CPA)for five weeks.The mobility of rats was evaluated by forced swimming test,while their cognitive capabilities were evaluated by Y-maze test.Expression of sortilin,α-synuclein,p-JUN,and c-JUN proteins in the substantia nigra were detected by western blot analysis.In addition,immunofluorescence staining of sortilin andα-synuclein was performed to detect expression in the substantia nigra.The results showed that,compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine(5 mg/kg)+CPA co-treated rats,motor and memory abilities were reduced,surface expression of sortin andα-synuclein in dopaminergic neurons was reduced,and total sortilin and totalα-synuclein were increased in CPA-treated rats.MN9D cells were incubated with 500 nM CPA alone or in combination with 10μM SP600125(JNK inhibitor)for 48 hours.Quantitative real-time polymerase chain reaction analysis of sortilin andα-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone,but the combination of CPA and SP600125 could inhibit this expression.Predictions made using Jasper,PROMO,and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents.A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction.After sortilin expression was inhibited by sh-SORT1,expression of p-JUN and c-JUN was detected by western blot analysis.Long-term adenosine A1 receptor activation levels upregulatedα-synuclein expression at the post-transcriptional level by affecting sortilin expression.The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind toα-synuclein.Co-immunoprecipitation revealed an interaction between sortilin andα-synuclein in MN9D cells.Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression andα-synuclein accumulation,and dramatically improved host cognition and kineticism.This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan(approval No.AUP#20070090)in March 2007 and the Animals Ethics Committee of University of South China(approval No.LL0387-USC)in June 2017.
基金supported by the National Natural Science Foundation of China(Nos.81202935 and 81773893)the National Major Scientific and Technological Special Project of China for “Significant New Drugs Development”(No.2017ZX09301060-001)+1 种基金the Natural Science Foundation of Hubei Province,China(No.2015CFB302)Fundamental Research Funds for the Central Universities “South-Central University for Nationalities”(No.CZY20025)。
文摘We investigated the liver protective activity of dandelion polyphenols(DP)against acetaminophen(APAP;Paracetamol)-induced hepatotoxicity.Mice were acclimated for 1 week and randomly divided into the following groups(n=9 per group):Control,APAP,APAP+DP(100 mg·kg^–1),APAP+DP(200 mg·kg^–1),and APAP+DP(400 mg·kg^–1)groups.Mice were pretreated with DP(100,200,and 400 mg·kg^–1)by oral gavage for 7 d before being treated with 350 mg·kg^–1 APAP for 24 h to induced hepatotoxicity.Severe liver injury was observed,and hepatotoxicity was analyzed after 24 h by evaluation of biochemical markers,protein expressions levels,and liver histopathology.Pretreatment with DP was able to restore serum liver characteristics(aspartate transaminase,AST;alanine aminotransferase,ALT;alkaline phosphatase,AKP),improve redox imbalance(superoxide dismutase,SOD;glutathione,GSH;malondialdehyde,MDA),and decrease inflammatory factors(tumor necrosis factor-α,TNF-α;interleukin-1β,IL-1β).Pretreatment with DP also significantly inhibited the expression levels of nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2).Furthermore,DP pretreatment could inhibit the apoptosis of liver cells caused by APAP through up-regulation of Bcl-2 and down-regulation of Bax and caspase-9 protein.DP also down-regulated p-JNK protein expression levels to inhibit APAP-induced mitochondrial oxidative stress and up-regulated the expression of Nrf-2 and its target gene HO-1.The histopathological staining demonstrated that DP pretreatment could inhibit APAP-induced hepatocyte infiltration,congestion,and necrosis.Our results demonstrate that DP pretreatment could protect against APAP-induced hepatic injury by activating the Nrf-2/HO-1 pathway and inhibition of the intrinsic apoptosis pathway.
基金supported by the grants from the Shanghai Municipal Science and Technology Commission(No.14430721400)National Natural Science Foundation(Nos.81700421 and 81670442)Clinical innovative research funding of Shanghai Jiaotong University School of Medicine(No.PY2018-IIC-05)。
文摘Background:Arteriosclerosis obliterans(ASO)is a major cause of adult limb loss worldwide.Autophagy of vascular endothelial cell(VEC)contributes to the ASO progression.However,the molecular mechanism that controls VEC autophagy remains unclear.In this study,we aimed to explore the role of the GRB2 associated binding protein 1(GAB1)in regulating VEC autophagy.Methods:In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression.Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima.Gain-and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1.Results:The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor(0.80 vs.0.20,t=6.43,P<0.05).The expression level of GAB1 mRNA(1.00 vs.0.24,t=7.41,P<0.05)and protein(0.72 vs.0.21,t=5.97,P<0.05)was significantly decreased in ASO group as compared with the control group.Loss of GAB1 led to a remarkable decrease in LC3II(1.19 vs.0.68,t=5.99,P<0.05),whereas overexpression of GAB1 significantly led to a decrease in LC3II level(0.41 vs.0.93,t=7.12,P<0.05).Phosphorylation levels of JNK and p38 were significantly associated with gain-and loss-of-function of GAB1 protein.Conclusion:Loss of GAB1 promotes VEC autophagy which is associated with ASO.GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.
基金supported by research grants from theMajor State Basic ResearchDevelopment Program(973 Program)(grant number 2012CB518900)the National Natural Science Foundation of China(grant numbers 31230005,31270206,31200134,and 81171525)+1 种基金the National Mega Project on Major Infectious Disease Prevention(grant numbers 2012ZX10002006-003 and 2012ZX10004-207)the Chinese Foundation for Hepatitis Prevention and Control(grant number CFHPC20132153).
文摘HepatitisBvirus(HBV)infection causes acuteand chronic liver diseases,but is not directly cytopathic.Liver injury results fromrepeated attempts of the cellular immune response system to control the viral infection.Here,we investigate the roles of cellular factors and signaling pathways involved in the regulation of HBV replication to reveal the mechanism underlying HBV infection and pathogenesis.Weshowthat collagen triple helix repeat containing 1(CTHRC1)expression is elevated in HBV-infected patients andin HBV-transfected cells through epigenetic modification and transcriptional regulation.CTHRC1 facilitates HBV replication in cultured cells and BALB/c mice by activating the PKCa/ERK/JNK/c-Jun cascade to repress the IFN/JAK/STAT pathway.HBV-activated CTHRC1 downregulates the activityof typeI interferon(IFN),theproductionof IFN-stimulatedgenes(ISGs),andthephosphorylationofsignal transducerandactivator of transcription 1/2(STAT1/2),whereas it upregulates the phosphorylation and ubiquitination of type I IFN receptors(IFNARa/b).Thus,our results showthat HBV uses a novelmechanismto hijack cellular factors and signal cascades in order to evade host antiviral immunity and maintain persistent infection.We also demonstrate that CTHRC1 has a novel role in viral infection.
