OBJECTIVE To explore the effect of total flavonoids of Rhododendra simsii(TFR)on improving cerebral ischemia/reperfusion injury(CIRI)and its relationship with STIM/Orai-regulated operational Ca^(2+)influx(SOCE)pathway...OBJECTIVE To explore the effect of total flavonoids of Rhododendra simsii(TFR)on improving cerebral ischemia/reperfusion injury(CIRI)and its relationship with STIM/Orai-regulated operational Ca^(2+)influx(SOCE)pathway.METHODS Oxygen-glucose deprivation/reoxygenation(OGD/R)PC12 cells were used to simulate CIRI in vitro,and the intracellular Ca^(2+)concentration and apoptosis rate of PC12 cells were detected by laser confocal microscope and flow cytometry,respectively.The regulation of STIM/Orai on SOCE was analyzed by STIM/Orai gene silencing and STIM/O rai gene overexpression.The CIRI model was established by MCAO in SD rats.The activities of inflammatory cytokines IL^(-1),IL-6 and TNF-αin serum were detected by ELISA.The pathological changes of ischemic brain tissue and the infarction of rat brain tissue were detected by HE staining and TTC staining.The protein and mRNA expression levels of STIM1,STIM2,Orai1,caspase-3 and PKB in brain tissue were detected by Western blotting and RT-qPCR,respectively.RESULTS The results of in vitro experiment showed that the fluorescence intensity of Ca^(2+)and apoptosis rate in PC12 cells treated with TFR were significantly lower than those in OGD/R group,and this trend was enhanced by SOCE antagonist 2-APB.STIM1/STIM2/Orai1 gene silencing significantly reduced apoptosis and Ca^(2+)overload in OGD/R model,while TFR combined with overexpression of STIM1/STIM2/Orai1 aggravated apoptosis and Ca2+overload.In the in vivo experiment,TFR significantly reduced the brain histopathological damage,infarction of brain tissue,the contents of IL^(-1),IL-6 and TNF-αin the serum in MCAO rats and down-regulated the expression of STIM1,STIM2,Orai1 and caspase-3 protein and mRNA in the brain tissue,and up-regulated the expression of PKB.The above effects were enhanced by the addition of 2-APB.CONCLUSION The above results indicate that TFR may reduce the contents of inflammatory factors and apoptosis,decrease Ca2+overload and ameliorate brain injury by inhibiting SOCE pathway mediated by STIM and Orai,suggesting that it has a protective effect against subacute CIRI.展开更多
In all six members of TRPV channel subfamily,there is an ankyrin repeat domain(ARD)in their intracellular N-termini.Ankyrin(ANK)repeat,a common motif with typi-cally 33 residues in each repeat,is primarily involved in...In all six members of TRPV channel subfamily,there is an ankyrin repeat domain(ARD)in their intracellular N-termini.Ankyrin(ANK)repeat,a common motif with typi-cally 33 residues in each repeat,is primarily involved in protein-protein interactions.Despite the sequence similarity among the ARDs of TRPV channels,the struc-ture of TRPV3-ARD,however,remains unknown.Here,we report the crystal structure of TRPV3-ARD solved at 1.95Åresolution,which reveals six-ankyrin repeats.While overall structure of TRPV3-ARD is similar to ARDs from other members of TRPV subfamily;it,however,features a noticeable fi nger 3 loop that bends over and is stabilized by a network of hydrogen bonds and hydrophobic pack-ing,instead of being fl exible as seen in known TRPV-ARD structures.Electrophysiological recordings demonstrated that mutating key residues R225,R226,Q255,and F249 of fi nger 3 loop altered the channel activities and pharmacol-ogy.Taken all together,our findings show that TRPV3-ARD with characteristic fi nger 3 loop likely plays an im-portant role in channel function and pharmacology.展开更多
基金National Natural Science Foundation of China(81173596)and Major Project of Natural Science Foundation of the Department of Education of Anhui Province(KJ2019ZD32)。
文摘OBJECTIVE To explore the effect of total flavonoids of Rhododendra simsii(TFR)on improving cerebral ischemia/reperfusion injury(CIRI)and its relationship with STIM/Orai-regulated operational Ca^(2+)influx(SOCE)pathway.METHODS Oxygen-glucose deprivation/reoxygenation(OGD/R)PC12 cells were used to simulate CIRI in vitro,and the intracellular Ca^(2+)concentration and apoptosis rate of PC12 cells were detected by laser confocal microscope and flow cytometry,respectively.The regulation of STIM/Orai on SOCE was analyzed by STIM/Orai gene silencing and STIM/O rai gene overexpression.The CIRI model was established by MCAO in SD rats.The activities of inflammatory cytokines IL^(-1),IL-6 and TNF-αin serum were detected by ELISA.The pathological changes of ischemic brain tissue and the infarction of rat brain tissue were detected by HE staining and TTC staining.The protein and mRNA expression levels of STIM1,STIM2,Orai1,caspase-3 and PKB in brain tissue were detected by Western blotting and RT-qPCR,respectively.RESULTS The results of in vitro experiment showed that the fluorescence intensity of Ca^(2+)and apoptosis rate in PC12 cells treated with TFR were significantly lower than those in OGD/R group,and this trend was enhanced by SOCE antagonist 2-APB.STIM1/STIM2/Orai1 gene silencing significantly reduced apoptosis and Ca^(2+)overload in OGD/R model,while TFR combined with overexpression of STIM1/STIM2/Orai1 aggravated apoptosis and Ca2+overload.In the in vivo experiment,TFR significantly reduced the brain histopathological damage,infarction of brain tissue,the contents of IL^(-1),IL-6 and TNF-αin the serum in MCAO rats and down-regulated the expression of STIM1,STIM2,Orai1 and caspase-3 protein and mRNA in the brain tissue,and up-regulated the expression of PKB.The above effects were enhanced by the addition of 2-APB.CONCLUSION The above results indicate that TFR may reduce the contents of inflammatory factors and apoptosis,decrease Ca2+overload and ameliorate brain injury by inhibiting SOCE pathway mediated by STIM and Orai,suggesting that it has a protective effect against subacute CIRI.
基金This work was supported by research grants from the National Natural Science Foundation of China to KWW(Grant Nos.30970919 and 81221002)the National Basic Research Program(973 Program)to KWW(No.2013CB531300).
文摘In all six members of TRPV channel subfamily,there is an ankyrin repeat domain(ARD)in their intracellular N-termini.Ankyrin(ANK)repeat,a common motif with typi-cally 33 residues in each repeat,is primarily involved in protein-protein interactions.Despite the sequence similarity among the ARDs of TRPV channels,the struc-ture of TRPV3-ARD,however,remains unknown.Here,we report the crystal structure of TRPV3-ARD solved at 1.95Åresolution,which reveals six-ankyrin repeats.While overall structure of TRPV3-ARD is similar to ARDs from other members of TRPV subfamily;it,however,features a noticeable fi nger 3 loop that bends over and is stabilized by a network of hydrogen bonds and hydrophobic pack-ing,instead of being fl exible as seen in known TRPV-ARD structures.Electrophysiological recordings demonstrated that mutating key residues R225,R226,Q255,and F249 of fi nger 3 loop altered the channel activities and pharmacol-ogy.Taken all together,our findings show that TRPV3-ARD with characteristic fi nger 3 loop likely plays an im-portant role in channel function and pharmacology.
