Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine.Its primary therapies include clearing heat and detoxification,activating blood circulation,and re...Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine.Its primary therapies include clearing heat and detoxification,activating blood circulation,and removing blood stasis.Lonicera japonica flos(LJF)has long been known as an excellent antipyretic and antidote.Luteoloside(Lut)is one of the main components of LJF and exhibits antioxidant,anti-inflammatory,and cytoprotective properties.However,the protection of Lut against iron overload injury and its underlying mechanisms remain unclear.Therefore,HUVECs were exposed to 50μmol·L^(−1)iron dextran for 48 h to establish an iron overload damage model and the effects of Lut were assessed.Our results showed that 20μmol·L^(−1)Lut not only increased cell viability and weakened LDH activity,but also significantly up-regulated DDAHⅡexpression and activity,increased p-eNOS/eNOS ratio and NO content,and reduced ADMA content in HUVECs exposed to iron overload.Furthermore,Lut significantly attenuated intracellular/mitochondrial ROS generation,improved SOD,CAT,and GSH-Px activities,reduced MDA content,maintained MMP,inhibited mPTP opening,prevented cyt c from mitochondria released into cytoplasm,reduced cleaved-caspase3 expression,and ultimately decreased cell apoptosis induced by iron overload.The effects of Lut were similar to those of L-arginine(an ADMA competitive substrate),cyclosporin A(a mPTP blocker agent),and edaravone(a free radical scavenger)as positive controls.However,addition of pAD/DDAHⅡ-shRNA adenovirus reversed the above beneficial effects of Lut.In conclusion,Lut can protect HUVECs against iron overload injury via the ROS/ADMA/DDAHⅡ/eNOS/NO pathway.The mitochondria are the target organelles of Lut’s protective effects.展开更多
OBJECTIVE To investigate whether micro RNA(mi RNA)mi R-21 regulates dimethylarginine dimethylaminohydrolase 1(DDAH1)expression through binding 3′-UTR regiondirectly in human umbilical venous endothelial cells(HUVECs)...OBJECTIVE To investigate whether micro RNA(mi RNA)mi R-21 regulates dimethylarginine dimethylaminohydrolase 1(DDAH1)expression through binding 3′-UTR regiondirectly in human umbilical venous endothelial cells(HUVECs)and to explore whether DDAH1-V2/V3 transcripts can function as micro RNA sponge,thereby modulating DDAH1-V1 expression.METHODS The DDAH13′-UTR containing mi R-21 recognizing sequence was cloned into Pmir GLO dual-luciferase mi RNA target expression plasmid to construct PmirGLO-mi R-21.The plasmid and mi R-21(at concentrations of 25,50,100 nmol·L-1,respectively)or negative control(100 nmol·L-1)were co-transfected into HUVECs,luciferase activity was detected at 24 h.HUVECs were incubated with 2μg·m L-1actinomycin D for the indicated time after mi R-21(25 nmol.L-1)transfection,half-lives of DDAH1 m RNA were determined.HUVECs were transfected with Pmir GLO-mi R-21 alone or co-transfected with mi R-21 for 24 h,DDAH1 transcripts m RNA and DDAH1protein expression were determined.RESULTS Mi R-21decreased luciferase activity of Pmir GLO-mi R-21 in a dose-dependent manner(P<0.05 for 25 nmol·L-1mi R-21,P<0.01 for 50 nmol·L-1and 100 nmol·L-1mi R-21),and mi R-21 inhibitor increased reporter activity of PmirGLO-mi R-21 and m RNA expression of all DDAH1 three transcript variants significantly(P<0.05,respectively).The degree of increase in endogenous DDAH1 m RNA expression by mi R-21 inhibitor was more obvious for DDAH1-V3.Overexpression of mi R-21 decreased m RNA expression and m RNA half-life time of all DDAH1 transcripts significantly(P<0.05),and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1and-V3 with or without mi R-21 transfection(P<0.05,respectively).Mi R-21(100 nmol·L-1)decreased DDAH1protein expression significantly(P<0.05),which was reversed by Pmir GLO-mi R-21 transfection(P<0.05).Transfection of Pmir GLO-mi R-21 alone increased intracellular mi R-21 expression by approximately 5.6-fold,but only showed a trend of increase in DDAH1 protein expression.CONCLUSION Our results confirmed DDAH13′-UTR as a target for mi R-21,and endogenous mi R-21showed increased inhibitory effect on DDAH1-V3 transcript.DDAH1 3′-UTR,especially for DDAH1-V3,may function as mi R-21 sponge to regulate DDAH1 protein expression.Modulation of mi R-21-DDAH1 interaction may provide a new approach for tackling cardiovascular diseases.展开更多
目的探讨DDAH2C-449G基因多态性与动脉粥样硬化血栓形成性脑梗死(atherothrombotic infarct of brain)的关系。方法共纳入345例脑梗死患者和201例对照组人群,以DDAH2C-449G基因为遗传标志,采用聚合酶链式反应(PCR)和限制性片段长度多态...目的探讨DDAH2C-449G基因多态性与动脉粥样硬化血栓形成性脑梗死(atherothrombotic infarct of brain)的关系。方法共纳入345例脑梗死患者和201例对照组人群,以DDAH2C-449G基因为遗传标志,采用聚合酶链式反应(PCR)和限制性片段长度多态性技术(RFLP)检测DDAH2C-449G基因多态性。结果动脉粥样硬化血栓形成性脑梗死GG基因型、G等位基因频率较对照组高(P<0.05)。结论DDAH2C-449G基因多态性可能与动脉粥样硬化血栓形成性脑梗死发病有关,G等位基因可能是动脉粥样硬化血栓形成性脑梗死的易感基因。展开更多
基金the National Natural Science Foundation of China(Nos.21467017,81660583,81673431,and 81803534)Jiangxi Applied Research and Cultivation Program(No.20181BBG78059).
文摘Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine.Its primary therapies include clearing heat and detoxification,activating blood circulation,and removing blood stasis.Lonicera japonica flos(LJF)has long been known as an excellent antipyretic and antidote.Luteoloside(Lut)is one of the main components of LJF and exhibits antioxidant,anti-inflammatory,and cytoprotective properties.However,the protection of Lut against iron overload injury and its underlying mechanisms remain unclear.Therefore,HUVECs were exposed to 50μmol·L^(−1)iron dextran for 48 h to establish an iron overload damage model and the effects of Lut were assessed.Our results showed that 20μmol·L^(−1)Lut not only increased cell viability and weakened LDH activity,but also significantly up-regulated DDAHⅡexpression and activity,increased p-eNOS/eNOS ratio and NO content,and reduced ADMA content in HUVECs exposed to iron overload.Furthermore,Lut significantly attenuated intracellular/mitochondrial ROS generation,improved SOD,CAT,and GSH-Px activities,reduced MDA content,maintained MMP,inhibited mPTP opening,prevented cyt c from mitochondria released into cytoplasm,reduced cleaved-caspase3 expression,and ultimately decreased cell apoptosis induced by iron overload.The effects of Lut were similar to those of L-arginine(an ADMA competitive substrate),cyclosporin A(a mPTP blocker agent),and edaravone(a free radical scavenger)as positive controls.However,addition of pAD/DDAHⅡ-shRNA adenovirus reversed the above beneficial effects of Lut.In conclusion,Lut can protect HUVECs against iron overload injury via the ROS/ADMA/DDAHⅡ/eNOS/NO pathway.The mitochondria are the target organelles of Lut’s protective effects.
基金The project supported by National Natural Science Foundation of China(81170091,81373489,81422052)Special Topic of the Major Subject of National Science and Technology(2013ZX09509-107)+1 种基金Provincial Natural Science Foundation of Hunan(13JJ1010)Funds for Hunan Education Department Program(12K006)
文摘OBJECTIVE To investigate whether micro RNA(mi RNA)mi R-21 regulates dimethylarginine dimethylaminohydrolase 1(DDAH1)expression through binding 3′-UTR regiondirectly in human umbilical venous endothelial cells(HUVECs)and to explore whether DDAH1-V2/V3 transcripts can function as micro RNA sponge,thereby modulating DDAH1-V1 expression.METHODS The DDAH13′-UTR containing mi R-21 recognizing sequence was cloned into Pmir GLO dual-luciferase mi RNA target expression plasmid to construct PmirGLO-mi R-21.The plasmid and mi R-21(at concentrations of 25,50,100 nmol·L-1,respectively)or negative control(100 nmol·L-1)were co-transfected into HUVECs,luciferase activity was detected at 24 h.HUVECs were incubated with 2μg·m L-1actinomycin D for the indicated time after mi R-21(25 nmol.L-1)transfection,half-lives of DDAH1 m RNA were determined.HUVECs were transfected with Pmir GLO-mi R-21 alone or co-transfected with mi R-21 for 24 h,DDAH1 transcripts m RNA and DDAH1protein expression were determined.RESULTS Mi R-21decreased luciferase activity of Pmir GLO-mi R-21 in a dose-dependent manner(P<0.05 for 25 nmol·L-1mi R-21,P<0.01 for 50 nmol·L-1and 100 nmol·L-1mi R-21),and mi R-21 inhibitor increased reporter activity of PmirGLO-mi R-21 and m RNA expression of all DDAH1 three transcript variants significantly(P<0.05,respectively).The degree of increase in endogenous DDAH1 m RNA expression by mi R-21 inhibitor was more obvious for DDAH1-V3.Overexpression of mi R-21 decreased m RNA expression and m RNA half-life time of all DDAH1 transcripts significantly(P<0.05),and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1and-V3 with or without mi R-21 transfection(P<0.05,respectively).Mi R-21(100 nmol·L-1)decreased DDAH1protein expression significantly(P<0.05),which was reversed by Pmir GLO-mi R-21 transfection(P<0.05).Transfection of Pmir GLO-mi R-21 alone increased intracellular mi R-21 expression by approximately 5.6-fold,but only showed a trend of increase in DDAH1 protein expression.CONCLUSION Our results confirmed DDAH13′-UTR as a target for mi R-21,and endogenous mi R-21showed increased inhibitory effect on DDAH1-V3 transcript.DDAH1 3′-UTR,especially for DDAH1-V3,may function as mi R-21 sponge to regulate DDAH1 protein expression.Modulation of mi R-21-DDAH1 interaction may provide a new approach for tackling cardiovascular diseases.
文摘目的探讨DDAH2C-449G基因多态性与动脉粥样硬化血栓形成性脑梗死(atherothrombotic infarct of brain)的关系。方法共纳入345例脑梗死患者和201例对照组人群,以DDAH2C-449G基因为遗传标志,采用聚合酶链式反应(PCR)和限制性片段长度多态性技术(RFLP)检测DDAH2C-449G基因多态性。结果动脉粥样硬化血栓形成性脑梗死GG基因型、G等位基因频率较对照组高(P<0.05)。结论DDAH2C-449G基因多态性可能与动脉粥样硬化血栓形成性脑梗死发病有关,G等位基因可能是动脉粥样硬化血栓形成性脑梗死的易感基因。
