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
Choline acetyltransferase(ChAT)-positive neurons in neural stem cell(NSC)niches can evoke adult neurogenesis(AN)and restore impaired brain function after injury,such as acute ischemic stroke(AIS).However,the relevant ...Choline acetyltransferase(ChAT)-positive neurons in neural stem cell(NSC)niches can evoke adult neurogenesis(AN)and restore impaired brain function after injury,such as acute ischemic stroke(AIS).However,the relevant mechanism by which ChAT+neurons develop in NSC niches is poorly understood.Our RNA-seq analysis revealed that dimethylarginine dimethylaminohydrolase 1(DDAH1),a hydrolase for asymmetric NG,NG-dimethylarginine(ADMA),regulated genes responsible for the synthesis and transportation of acetylcholine(ACh)(Chat,Slc5a7 and Slc18a3)after stroke insult.The dual-luciferase reporter assay further suggested that DDAH1 controlled the activity of ChAT,possibly through hypoxia-inducible factor 1α(HIF-1α).KC7F2,an inhibitor of HIF-1α,abolished DDAH1-induced ChAT expression and suppressed neurogenesis.As expected,DDAH1 was clinically elevated in the blood of AIS patients and was positively correlated with AIS severity.By comparing the results among Ddah1 general knockout(KO)mice,transgenic(TG)mice and wild-type(WT)mice,we discovered that DDAH1 upregulated the proliferation and neural differentiation of NSCs in the subgranular zone(SGZ)under ischemic insult.As a result,DDAH1 may promote cognitive and motor function recovery against stroke impairment,while these neuroprotective effects are dramatically suppressed by NSC conditional knockout of Ddah1 in mice.展开更多
Dimethylarginine dimethylaminohydrolase 1(DDAH1)is an important regulator of plasma asymmetric dimethylarginine(ADMA)levels,which are associated with insulin resistance in patients with nonalcoholic fatty liver diseas...Dimethylarginine dimethylaminohydrolase 1(DDAH1)is an important regulator of plasma asymmetric dimethylarginine(ADMA)levels,which are associated with insulin resistance in patients with nonalcoholic fatty liver disease(NAFLD).To elucidate the role of hepatic DDAH1 in the pathogenesis of NAFLD,we used hepatocyte-specific Ddah1-knockout mice(Ddah1HKO)to examine the progress of high-fat diet(HFD)-induced NAFLD.Compared to diet-matched flox/flox littermates(Ddah1f/f),Ddah1HKO mice exhibited higher serum ADMA levels.After HFD feeding for 16 weeks,Ddah1HKO mice developed more severe liver steatosis and worse insulin resistance than Ddah1f/f mice.On the contrary,overexpression of DDAH1 attenuated the NAFLD-like phenotype in HFD-fed mice and ob/ob mice.RNA-seq analysis showed that DDAH1 affects NF-kB signaling,lipid metabolic processes,and immune system processes in fatty livers.Furthermore,DDAH1 reduces S100 calcium-binding protein A11(S100A11)possibly via NF-kB,JNK and oxidative stress-dependent manner in fatty livers.Knockdown of hepatic S100a11 by an AAV8-shS100a11 vector alleviated hepatic steatosis and insulin resistance in HFD-fed Ddah1HKO mice.In summary,our results suggested that the liver DDAH1/S100A11 axis has a marked effect on liver lipid metabolism in obese mice.Strategies to increase liver DDAH1 activity or decrease S100A11 expression could be a valuable approach for NAFLD therapy.展开更多
目的探讨壳寡糖对爆震伤致小鼠急性肺损伤(ALI)保护作用,并阐明其可能的作用机制。方法将30只昆明小鼠随机分为对照组、ALI组和ALI+壳寡糖组,检测肺干/湿重比变化;HE染色观察肺组织病理改变;ELISA检测血清炎症因子肿瘤坏死因子-α(TNF-...目的探讨壳寡糖对爆震伤致小鼠急性肺损伤(ALI)保护作用,并阐明其可能的作用机制。方法将30只昆明小鼠随机分为对照组、ALI组和ALI+壳寡糖组,检测肺干/湿重比变化;HE染色观察肺组织病理改变;ELISA检测血清炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-4、IL-6和IL-10的变化;Western blot、Real time PCR和免疫荧光检测炎症相关因子和通路相关蛋白DDAH1、ADMA和p38的表达。结果与ALI组相比,壳寡糖可以显著降低爆震伤导致的肺干/湿重比、炎症细胞浸润和炎症相关因子TNF-α、IL-1β、IL-4、IL-6和IL-10表达,升高抑炎因子IL-10表达,差异均有统计学意义(P<0.05);壳寡糖显著提高爆震伤导致的肺组织DDAH1蛋白表达,降低ADMA和p38蛋白表达,差异均有统计学意义(P<0.05)。结论壳寡糖对爆震伤导致的小鼠ALI有保护作用,其可能通过抑制DDAH1表达,促进ADMA,进而激活MAPK通路来实现。展开更多
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.
基金This work was supported by the National Natural Science Foundation of China(32171237,82070250,82171301,82370275,32071126)Beijing Natural Science Foundation(7222010)the Fundamental Research Funds for the Central Universities.Thanks for the support of the undergraduate research training programs of Capital Medical University(XSKY2023,XSKY2022,XSKY2021),China.We sincerely acknowledged that Professor Jianwei Jiao from the Institute of Zoology,Chinese Academy of Sciences,kindly provided the Nestin-Cre(C57BL/6.Cg-Tg(Nes-Cre)1Kln/J)mice.We sincerely appreciate for the technical service and support from Tissue Gnostics Asia Pacific Limited in the image caption and data analysis of immunohistochemical staining analysis.
文摘Choline acetyltransferase(ChAT)-positive neurons in neural stem cell(NSC)niches can evoke adult neurogenesis(AN)and restore impaired brain function after injury,such as acute ischemic stroke(AIS).However,the relevant mechanism by which ChAT+neurons develop in NSC niches is poorly understood.Our RNA-seq analysis revealed that dimethylarginine dimethylaminohydrolase 1(DDAH1),a hydrolase for asymmetric NG,NG-dimethylarginine(ADMA),regulated genes responsible for the synthesis and transportation of acetylcholine(ACh)(Chat,Slc5a7 and Slc18a3)after stroke insult.The dual-luciferase reporter assay further suggested that DDAH1 controlled the activity of ChAT,possibly through hypoxia-inducible factor 1α(HIF-1α).KC7F2,an inhibitor of HIF-1α,abolished DDAH1-induced ChAT expression and suppressed neurogenesis.As expected,DDAH1 was clinically elevated in the blood of AIS patients and was positively correlated with AIS severity.By comparing the results among Ddah1 general knockout(KO)mice,transgenic(TG)mice and wild-type(WT)mice,we discovered that DDAH1 upregulated the proliferation and neural differentiation of NSCs in the subgranular zone(SGZ)under ischemic insult.As a result,DDAH1 may promote cognitive and motor function recovery against stroke impairment,while these neuroprotective effects are dramatically suppressed by NSC conditional knockout of Ddah1 in mice.
基金supported by grants from National Natural Science Foundation of China(82070250,32200631)Beijing Natural Science Foundation(5222029,China)+1 种基金China Postdoctoral Science Foundation(2022T150640)the Fundamental Research Funds for the Central Universities。
文摘Dimethylarginine dimethylaminohydrolase 1(DDAH1)is an important regulator of plasma asymmetric dimethylarginine(ADMA)levels,which are associated with insulin resistance in patients with nonalcoholic fatty liver disease(NAFLD).To elucidate the role of hepatic DDAH1 in the pathogenesis of NAFLD,we used hepatocyte-specific Ddah1-knockout mice(Ddah1HKO)to examine the progress of high-fat diet(HFD)-induced NAFLD.Compared to diet-matched flox/flox littermates(Ddah1f/f),Ddah1HKO mice exhibited higher serum ADMA levels.After HFD feeding for 16 weeks,Ddah1HKO mice developed more severe liver steatosis and worse insulin resistance than Ddah1f/f mice.On the contrary,overexpression of DDAH1 attenuated the NAFLD-like phenotype in HFD-fed mice and ob/ob mice.RNA-seq analysis showed that DDAH1 affects NF-kB signaling,lipid metabolic processes,and immune system processes in fatty livers.Furthermore,DDAH1 reduces S100 calcium-binding protein A11(S100A11)possibly via NF-kB,JNK and oxidative stress-dependent manner in fatty livers.Knockdown of hepatic S100a11 by an AAV8-shS100a11 vector alleviated hepatic steatosis and insulin resistance in HFD-fed Ddah1HKO mice.In summary,our results suggested that the liver DDAH1/S100A11 axis has a marked effect on liver lipid metabolism in obese mice.Strategies to increase liver DDAH1 activity or decrease S100A11 expression could be a valuable approach for NAFLD therapy.
文摘目的探讨壳寡糖对爆震伤致小鼠急性肺损伤(ALI)保护作用,并阐明其可能的作用机制。方法将30只昆明小鼠随机分为对照组、ALI组和ALI+壳寡糖组,检测肺干/湿重比变化;HE染色观察肺组织病理改变;ELISA检测血清炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-4、IL-6和IL-10的变化;Western blot、Real time PCR和免疫荧光检测炎症相关因子和通路相关蛋白DDAH1、ADMA和p38的表达。结果与ALI组相比,壳寡糖可以显著降低爆震伤导致的肺干/湿重比、炎症细胞浸润和炎症相关因子TNF-α、IL-1β、IL-4、IL-6和IL-10表达,升高抑炎因子IL-10表达,差异均有统计学意义(P<0.05);壳寡糖显著提高爆震伤导致的肺组织DDAH1蛋白表达,降低ADMA和p38蛋白表达,差异均有统计学意义(P<0.05)。结论壳寡糖对爆震伤导致的小鼠ALI有保护作用,其可能通过抑制DDAH1表达,促进ADMA,进而激活MAPK通路来实现。
