Aim Inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of K...Aim Inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of KB- α （IKB-α） -nuclear factor-KB （NF-KB） cascade, whereas interferon-γ （IFN-γ） acts through Janus kinase （ JAK）- signal transducer and activator of transcription 1 （ STAT1 ） signals. Heat shock factor 1 （ HSF1 ）, a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α（TNF-α） and interleukin-6 （IL-6）. But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide （NO） content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation （CHIP） was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.展开更多
Objective C1q/TNF-related protein(CTRP)1 was initiallyidentified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.Previously,we showed that CTRP1promotes the development of ather...Objective C1q/TNF-related protein(CTRP)1 was initiallyidentified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.Previously,we showed that CTRP1promotes the development of atherosclerosis by increasing endothelial adhesiveness.Here,we sought to investigate whether CTRP1 also influences vascular dilatory functions.展开更多
OBJECTIVE To study the function of neuronal nitric oxide synthase(nNOS) in the dentate gyrus(DG) in the pathology of epilepsy.METHODS The expression of nNOS in the DG was measured by qPCR and Western blotting in mice ...OBJECTIVE To study the function of neuronal nitric oxide synthase(nNOS) in the dentate gyrus(DG) in the pathology of epilepsy.METHODS The expression of nNOS in the DG was measured by qPCR and Western blotting in mice 3 and 12 h,1,7,14,and 60 d after treatment with pilocarpine(280 mg·kg-1,ip,one time).We constructed a type of lentiovirus encoding the full length cDNA of nNOS(LV-nNOS-GFP) and injected it and LV-GFP(1 μL) into the DG of the hippocampus 7 d after pilocarpine-induced seizure.The occurrence of epileptic spikes and spontaneous seizure(SRS)were monitored through electroencephalo-graph(EEG) and the protein expression was confirmed by Western blotting.We also constructed a lentioviral vehicle to interfere the expression of nNOS mRNA,which was named as LV-n NOSRNAi-GFP.A volume of 1 μL of LV-nNOS-RNAiGFP or LV-GFP was injected into the DG of the hippocampus 7 d before pilocarpine-induced seizure followed by EEG record and protein detection 2 months later.By EEG,we compared the susceptibility of nNOS knockout and wild-type mice to seizure induction and the development of epilepsy.In addition,we measured the influence of nNOS knockout on the excitability of dentate cells including mEPSC and mIPSC by using patch clamp technique.RESULTS Western blotting and qPCR measurement showed that the mRNA and protein expression of nNOS in the DG was not significantly changed in pilocarpinetreated mice compared with control mice.But the both m RNA and protein expression of nNOS decreased 7,14 and 60 d after treatment with pilocarpine(280 mg·kg-1,ip,one time).With infection of LV-nNOS-GFP in the DG,the decreased level of nNOS was recovered 7 d after seizure induction and the frequency of epileptic spikes and SRS were reversed by nNOS overexpression.We found that nNOS knockout caused a higher susceptive level to seizure induction by pilocarpine.Re-expression of nNOS in the DG of nNOS knockout mice relived the severity of epilepsy.By patch clamp recording,we found that there was no significant difference in the amplitude of mEPSC and mIPSC between nNOS knockout and wild-type mice,but the frequency of mEPSC was increased in nN OS knockout mice.Consistently,knockdown of nNOS by injection of LV-nNOS-RNAi-GFP into the DG caused higher frequency of epileptic spikes and SRS 2 months after pilocarpine-induced seizure.CONCLUSION Neurons expressing nNOS in the DG play an important role in the development of epilepsy.展开更多
文摘Aim Inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of KB- α （IKB-α） -nuclear factor-KB （NF-KB） cascade, whereas interferon-γ （IFN-γ） acts through Janus kinase （ JAK）- signal transducer and activator of transcription 1 （ STAT1 ） signals. Heat shock factor 1 （ HSF1 ）, a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α（TNF-α） and interleukin-6 （IL-6）. But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide （NO） content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation （CHIP） was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.
文摘Objective C1q/TNF-related protein(CTRP)1 was initiallyidentified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.Previously,we showed that CTRP1promotes the development of atherosclerosis by increasing endothelial adhesiveness.Here,we sought to investigate whether CTRP1 also influences vascular dilatory functions.
基金National Natural Science Foundation of China(81571269)Science Technology Innovation Fund of Nanjing Medical University (2017NJMUCX008).
文摘OBJECTIVE To study the function of neuronal nitric oxide synthase(nNOS) in the dentate gyrus(DG) in the pathology of epilepsy.METHODS The expression of nNOS in the DG was measured by qPCR and Western blotting in mice 3 and 12 h,1,7,14,and 60 d after treatment with pilocarpine(280 mg·kg-1,ip,one time).We constructed a type of lentiovirus encoding the full length cDNA of nNOS(LV-nNOS-GFP) and injected it and LV-GFP(1 μL) into the DG of the hippocampus 7 d after pilocarpine-induced seizure.The occurrence of epileptic spikes and spontaneous seizure(SRS)were monitored through electroencephalo-graph(EEG) and the protein expression was confirmed by Western blotting.We also constructed a lentioviral vehicle to interfere the expression of nNOS mRNA,which was named as LV-n NOSRNAi-GFP.A volume of 1 μL of LV-nNOS-RNAiGFP or LV-GFP was injected into the DG of the hippocampus 7 d before pilocarpine-induced seizure followed by EEG record and protein detection 2 months later.By EEG,we compared the susceptibility of nNOS knockout and wild-type mice to seizure induction and the development of epilepsy.In addition,we measured the influence of nNOS knockout on the excitability of dentate cells including mEPSC and mIPSC by using patch clamp technique.RESULTS Western blotting and qPCR measurement showed that the mRNA and protein expression of nNOS in the DG was not significantly changed in pilocarpinetreated mice compared with control mice.But the both m RNA and protein expression of nNOS decreased 7,14 and 60 d after treatment with pilocarpine(280 mg·kg-1,ip,one time).With infection of LV-nNOS-GFP in the DG,the decreased level of nNOS was recovered 7 d after seizure induction and the frequency of epileptic spikes and SRS were reversed by nNOS overexpression.We found that nNOS knockout caused a higher susceptive level to seizure induction by pilocarpine.Re-expression of nNOS in the DG of nNOS knockout mice relived the severity of epilepsy.By patch clamp recording,we found that there was no significant difference in the amplitude of mEPSC and mIPSC between nNOS knockout and wild-type mice,but the frequency of mEPSC was increased in nN OS knockout mice.Consistently,knockdown of nNOS by injection of LV-nNOS-RNAi-GFP into the DG caused higher frequency of epileptic spikes and SRS 2 months after pilocarpine-induced seizure.CONCLUSION Neurons expressing nNOS in the DG play an important role in the development of epilepsy.
