低剂量辐射诱导小鼠睾丸细胞中IRE1α和XBP1 mRNA及其蛋白的表达
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摘要
目的:检测小鼠睾丸细胞中肌醇需求酶1α(IRE1α)和X盒结合蛋白1(XBP1)mRNA和蛋白的表达,探讨IRE1-XBP1通路激活在低剂量辐射(LDR)诱导小鼠睾丸细胞内质网应激中的作用。方法:50只健康雄性ICR小鼠,随机分为10组,经75 mGy X射线全身照射后不同时间(0、3、6、12和24h)及不同剂量(0、50、75、100和200mGy)X射线照射后12h处死,分别采用实时定量PCR法和Western blotting法检测小鼠睾丸细胞中IRE1α、Total-XBP1(T-XBP1)和Spliced-XBP1(S-XBP1)mRNA表达水平及蛋白表达强度。结果:75mGy X射线照射后0~24h,小鼠睾丸细胞中IRE1α、T-XBP1和S-XBP1mRNA(除了3h时T-XBP1和S-XBP1mRNA)表达水平均随时间延长而升高,并在6h时达到峰值,而后逐渐降低;与0h组比较,6、12和24h组IRE1αmRNA、6和12h组T-XBP1mRNA及S-XBP1mRNA表达水平均明显升高(P<0.05或P<0.01)。与0h组比较,不同时间组IRE1α和S-XBP1蛋白表达强度升高,6和12h组IRE1α蛋白表达强度升高最明显,24h组S-XBP1蛋白表达强度升高最明显,而T-XBP1蛋白表达强度稍有降低。0~200mGy照射后12h,小鼠睾丸细胞中IRE1α、T-XBP1和S-XBP1mRNA表达水平升高,75mGy X射线照射后升高达峰值后逐渐降低,甚至降至0mGy以下;与0mGy组比较,75和200mGy组小鼠睾丸细胞中IRE1α、75mGy组小鼠睾丸细胞中T-XBP1和S-XBP1mRNA表达水平明显升高(P<0.05或P<0.01)。与0mGy组比较,75mGy组IRE1α和S-XBP1蛋白表达强度升高,75和200mGy组小鼠睾丸细胞中S-XBP1蛋白表达强度升高最明显,而T-XBP1蛋白表达强度无明显变化。结论:LDR可诱导小鼠睾丸细胞中IRE1α和S-XBP1mRNA表达水平和蛋白表达强度增加,从而激活内质网应激中的IRE1-XBP1信号通路。
Objective:To detect the expressions of inositol-requiring enzyme-1α(IRE1α)and X box-binding protein-1(XBP1)mRNA and proteins in mouse testis cells,and to explore the role of IRE1-XBP1 pathway activation in the endoplasmic reticulum stress induced by low dose radiation(LDR)in the mouse testis cells.Methods:A total of 50 heatly male ICR mice were randomly divided into 10 groups.After the mice were irradiated with 75 mGy X-ray in whole body at different time(0,3,6,12,and 24 h)or with different doses(0,50,75,100,and 200 mGy)of X-ray for12 h,the expression levels of IRE1α,T-XBP1 and S-XBP1 mRNA and proteins were measured by real-time quantitative PCR and Western blotting method,respectively.Results:The expression levels of IRE1α,T-XBP1 and S-XBP1 mRNA(except T-XBP1 and S-XBP1 mRNA at 3 h after irradiation)in the testis cells of the mice after irradiated with 75 mGy X-ray for 0-24 hwere increased with the time prolongation,and reached to the maximum value at 6 hafter irradiation,then were gradually decreased.Compared with 0 hgroup,the expression levels of IRE1αmRNA(6,12,and 24 hafter irradiation),T-XBP1 mRNA and S-XBP1 mRNA(6 and 12 hafter irradiation)were significantly increased(P<0.05 or P<0.01);the expression intensities of IRE1αand S-XBP1 proteins were increased;the expression intensities of IRE1α(6 and 12 h after irradiation)and S-XBP1(24 hafter irradiation)proteins were increased most significantly,but the expression intensity of T-XBP1 protein was slightly decreased.The expression levels of IRE1α,T-XBP1 and S-XBP1 mRNA in the testis cells of the mice after irradiated with 0-200 mGy for 12 hwere increased,and reached to the maximum values after 75 mGy irradiation,then they were gradually decreased,even below 0 mGy.Compared with 0 mGy group,the expression levels of IRE1α mRNA in the testis cells of the mice in 75 and 200 mGy groups and the expression levels of T-XBP1 mRNA and S-XBP1 mRNA in 75 mGy group were significantly increased(P<0.05 or P<0.01);the expression intensities of IRE1αprotein in 75 mGy group and S-XBP1 protein in 75 and 200 mGy groups were increased mostly,while the expression intensity of T-XBP1 protein had no obvious change.Conclusion:LDR can induce the increase of IRE1αand S-XBP1 mRNAs and proteins,and activate the IRE1-XBP1 pathway in endoplasmic reticulum stress.
Objective:To detect the expressions of inositol-requiring enzyme-1α(IRE1α)and X box-binding protein-1(XBP1)mRNA and proteins in mouse testis cells,and to explore the role of IRE1-XBP1 pathway activation in the endoplasmic reticulum stress induced by low dose radiation(LDR)in the mouse testis cells.Methods:A total of 50 heatly male ICR mice were randomly divided into 10 groups.After the mice were irradiated with 75 mGy X-ray in whole body at different time(0,3,6,12,and 24 h)or with different doses(0,50,75,100,and 200 mGy)of X-ray for12 h,the expression levels of IRE1α,T-XBP1 and S-XBP1 mRNA and proteins were measured by real-time quantitative PCR and Western blotting method,respectively.Results:The expression levels of IRE1α,T-XBP1 and S-XBP1 mRNA(except T-XBP1 and S-XBP1 mRNA at 3 h after irradiation)in the testis cells of the mice after irradiated with 75 mGy X-ray for 0-24 hwere increased with the time prolongation,and reached to the maximum value at 6 hafter irradiation,then were gradually decreased.Compared with 0 hgroup,the expression levels of IRE1αmRNA(6,12,and 24 hafter irradiation),T-XBP1 mRNA and S-XBP1 mRNA(6 and 12 hafter irradiation)were significantly increased(P<0.05 or P<0.01);the expression intensities of IRE1αand S-XBP1 proteins were increased;the expression intensities of IRE1α(6 and 12 h after irradiation)and S-XBP1(24 hafter irradiation)proteins were increased most significantly,but the expression intensity of T-XBP1 protein was slightly decreased.The expression levels of IRE1α,T-XBP1 and S-XBP1 mRNA in the testis cells of the mice after irradiated with 0-200 mGy for 12 hwere increased,and reached to the maximum values after 75 mGy irradiation,then they were gradually decreased,even below 0 mGy.Compared with 0 mGy group,the expression levels of IRE1α mRNA in the testis cells of the mice in 75 and 200 mGy groups and the expression levels of T-XBP1 mRNA and S-XBP1 mRNA in 75 mGy group were significantly increased(P<0.05 or P<0.01);the expression intensities of IRE1αprotein in 75 mGy group and S-XBP1 protein in 75 and 200 mGy groups were increased mostly,while the expression intensity of T-XBP1 protein had no obvious change.Conclusion:LDR can induce the increase of IRE1αand S-XBP1 mRNAs and proteins,and activate the IRE1-XBP1 pathway in endoplasmic reticulum stress.
引文
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[14]Tavernier SJ,Osorio F,van ders Sarren L,et al.Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival[J].Nat Cell Biol,2017,19(6):698-710.
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[17]Zhang B,Wang Y,Pang X,et al.ER stress induced by ionising radiation in IEC-6cells[J].Int J Radiat Biol,2010,86(6):429-435.
[18]Homma T,Fujii J.Heat stress promotes the downregulation of IRE1αin cells:An atypical modulation of the UPR pathway[J].Exp Cell Res,2016,349(1):128-138.
[19]Son B,Kwon T,Lee S,et al.CYP2E1 regulates the development of radiation-induced pulmonary fibrosis via ER stress-and ROS-dependent mechanisms[J].Am J Physiol Lung Cell Mol Physiol,2017,313(5):L916-L929.
[20]Peschek J,Acosta-Alvear D,Mendez AS,et al.A conformational RNA zipper promotes intron ejection during non-conventional XBP1 mRNA splicing[J].EMBO Rep,2015,16(12):1688-1698.
[2]Morgan WF,Bair WJ.Issues in low dose radiation biology:the controversy continues.A perspective[J].Radiat Res,2013,179(5):501-510.
[3]Till JE,Beck HL,Grogan HA,et al.A review of dosimetry used in epidemiological studies considered to evaluate the linear no-threshold(LNT)dose-response model for radiation protection[J].Int J Radiat Biol,2017,7(5):1-17.
[4]Sun Y,Hawkins PG,Bi N,et al.Serum microRNA signature predicts response to high-dose radiation therapy in locally advanced non-small cell lung cancer[J].Int J Radiat Oncol Biol Phys,2018,100(1):107-114.
[5]Zhao Y,Kong C,Chen X,et al.Repetitive exposure to low-dose X-irradiation attenuates testicular apoptosis in type 2diabetic rats,likely via Akt-mediated Nrf2 activation[J].Mol Cell Endocrinol,2016,422(6):203-210.
[6]Jiang X,Hong Y,Zhao D,et al.Low dose radiation prevents doxorubicin-induced cardiotoxicity[J].Oncotarget,2018,9(1):332-345.
[7]Liu G,Gong P,Zhao H,et al.Effect of low-level radiation on the death of male germ cells[J].Radiat Res,2006,165(4):379-389.
[8]Elmhiri G,Gloaguen C,Grison S,et al.DNA methylation and potential multigenerational epigenetic effects linked to uranium chronic low-dose exposure in gonads of males and females rats[J].Toxicol Lett,2018,282(2):64-70.
[9]方芳,龚平生,宋祥福,等.低剂量电离辐射诱导小鼠睾丸细胞内质网应激及PERK-CHOP信号通路的激活[J].中华男科学杂志,2012,18(9):777-782.
[10]Jiang M,Yu S,Yu Z,et al.XBP1(X-box-binding protein-1)-dependent o-glcNAcylation is neuroprotective in ischemic stroke in young mice and its impairment in aged mice is rescued by thiamet-G[J].Stroke,2017,48(6):1646-1654.
[11]杨艳明,方芳,刘念,等.低剂量电离辐射诱导小鼠睾丸细胞中caspase-9表达的变化[J].中华放射医学与防护杂志,2014,34(3):185-187.
[12]Grewenig A,Schuler N,Rübe CE.Persistent DNA damage in spermatogonial stem cells after fractionated low-dose irradiation of testicular tissue[J].Int J Radiat Oncol Biol Phys,2015,92(5):1123-1131.
[13]Tr9ndle I,Westernstr9er B,Wistuba J,et al.Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts[J].Mol Hum Reprod,2017,23(3):141-154.
[14]Tavernier SJ,Osorio F,van ders Sarren L,et al.Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival[J].Nat Cell Biol,2017,19(6):698-710.
[15]Cubillos-Ruiz JR,Bettigole SE,Glimcher LH.Tumorigenic and immunosuppressive effects of endoplasmic reticulum stress in cancer[J].Cell,2017,168(4):692-706.
[16]Iurlaro R,Mu1oz-Pinedo C.Cell death induced by endoplasmic reticulum stress[J].FEBS J,2016,283(14):2640-2652.
[17]Zhang B,Wang Y,Pang X,et al.ER stress induced by ionising radiation in IEC-6cells[J].Int J Radiat Biol,2010,86(6):429-435.
[18]Homma T,Fujii J.Heat stress promotes the downregulation of IRE1αin cells:An atypical modulation of the UPR pathway[J].Exp Cell Res,2016,349(1):128-138.
[19]Son B,Kwon T,Lee S,et al.CYP2E1 regulates the development of radiation-induced pulmonary fibrosis via ER stress-and ROS-dependent mechanisms[J].Am J Physiol Lung Cell Mol Physiol,2017,313(5):L916-L929.
[20]Peschek J,Acosta-Alvear D,Mendez AS,et al.A conformational RNA zipper promotes intron ejection during non-conventional XBP1 mRNA splicing[J].EMBO Rep,2015,16(12):1688-1698.