Trx1 抑制内质网应激参与运动改善心梗后骨骼肌质量减少

目的:探讨运动调节硫氧还蛋白1(thioredoxin,Trx1)表达改善心肌梗死(心梗,myocardial infarction,MI)诱导的骨骼肌氧化应激和内质网应激(endoplasmic reticulum stress,ERS),抑制骨骼肌细胞凋亡,改善骨骼肌质量减少的可能机制,为研究运动改善心衰诱导骨骼肌萎缩的病理分子机制及筛选相关治疗靶点提供实验依据。方法:3月龄C57B6L小鼠,随机分为假手术组(Sham)、心梗组(MI)和心梗运动组(ME),每组8只。MI和ME组小鼠结扎左冠状动脉前降支建立心梗模型,Sham组小鼠只开胸不结扎,作为对照。ME组小鼠进行6周有氧运动训练。训练结束后,检测胫骨前肌相对质量、肌细胞横截面积、活性氧(reactive oxygen species,ROS)水平和抗氧化能力,检测肌组织肌肉环指蛋白1(muscle ring finger-1,MuRF1)、肌萎缩盒F因子(muscle atrophy F-box,MAFbx)、Trx1、硫氧还蛋白互作蛋白(thioredoxin interacting protein)和ERS相关蛋白表达及细胞凋亡现象。H2O2处理C2C12细胞,进行Trx1重组蛋白(TXN)或/和Trx1抑制剂(PX-12)干预,检测ERS相关蛋白表达和细胞凋亡水平。结果:与Sham组相比,MI组小鼠胫骨前肌肌肉相对质量和细胞横截面积显著下降(P<0.01),ROS和丙二醛(Malondialdehyde,MDA)水平显著升高(P<0.01),超氧化物歧化酶(superoxide dismutase,SOD)活性显著降低(P<0.01),MuRF1、MAFbx、TXNIP、ASK1、GRP78、磷酸化IRE1α、ATF6、CHOP、cleaved Caspase12(c-Casp-12)和JNK蛋白表达均升高(P<0.05,P<0.01),Trx1蛋白表达和Bcl-2/Bax比值显著降低(P<0.01),TUNEL阳性细胞显著增加(P<0.01);与MI组相比,运动显著增加心梗小鼠肌肉相对质量(P<0.01)和细胞横截面积(P<0.05),降低ROS和MDA水平(P<0.05),提升SOD活性(P<0.01),下调MuRF1、MAFbx、TXNIP、ASK1、磷酸化IRE1α、ATF6、ATF4、CHOP和c-Casp-12蛋白表达(P<0.05,P<0.01),上调Trx1和GRP78蛋白表达(P<0.01),降低骨骼肌细胞凋亡现象(P<0.05)。正常细胞PX-12或TXN干预可显著上调ATF6、GRP78和c-Casp-12蛋白表达(P<0.01);H2O2可显著增加C2C12成肌细胞TXNIP、ATF6、GRP78、CHOP和c-Casp-12蛋白表达和细胞凋亡水平(P<0.01);TXN干预可降低H2O2处理细胞中ATF6、GRP78和CHOP蛋白表达和细胞凋亡水平(P<0.01),PX-12干预显著增加H2O2处理细胞凋亡水平(P<0.01),降低TXN的有效保护效应。结论:有氧运动可遏制心梗诱导的骨骼肌质量减少现象,其机制可能与降低蛋白降解,上调Trx1表达改善ERS水平抑制细胞凋亡有关。Trx1改善ERS水平可能是其抑制细胞凋亡的途径之一。

硫氧还蛋白在凋亡途径中的作用机制

硫氧还蛋白(Trx)是体内广泛存在的氧化还原蛋白,其家族中两种重要的硫氧还蛋白:硫氧还蛋白1(thioredoxin1,Trx1)和硫氧还蛋白2(thioredoxin2,Trx2)都含有保守的-Cys-Gly-Pro-Cys-还原序列。由于Trx具有调节细胞生长增殖和抗凋亡的作用,因此Trx在凋亡途径中的作用机制就成为了对抗肿瘤的研究热点。

低温通过冷诱导RNA结合蛋白/硫氧还蛋白1通路减轻心肺复苏后神经元氧化应激损伤

目的探讨心跳骤停心肺复苏后脑损伤(BIC)模型中,治疗性低温(TH)对神经元氧化应激损伤的作用及相关机制。方法雄性SD大鼠60只,7~10周,体重280~320 g。采用随机数字表法分为五组:假手术组(S组)、BIC组、低温治疗组(TH组)、沉默冷诱导RNA结合蛋白(CIRP)后低温治疗组(THC组)和空载后低温治疗组(THN组),每组12只。采用经食管连续快速起搏诱发室颤,心跳骤停4 min后,行心肺复苏术(CPR),建立BIC模型。于BIC模型制备后1 d,采用免疫荧光法检测海马活性氧簇(ROS)荧光强度,Western blot法检测海马CIRP、硫氧还蛋白1(Trx1)、磷酸化凋亡信号调节激酶1(ASK1)蛋白含量及其下游p38和c-Jun N-末端激酶(JNK)磷酸化含量。于BIC模型制备后3 d,采用原位末端标记(TUNEL)法检测海马神经元凋亡细胞百分比。结果与S组比较,BIC模型制备后1 d BIC组ROS荧光强度明显升高,CIRP含量明显降低,p-ASK1、p-p38含量明显升高;TH组和THN组ROS荧光强度均明显升高,CIRP含量明显升高,p-JNK含量明显降低;THC组ROS荧光强度明显升高,CIRP、Trx1含量明显降低,p-ASK1、p-p38含量明显升高(P<0.05)。与BIC组比较,BIC模型制备后1 d TH组和THN组ROS荧光强度明显降低,CIRP、Trx1含量明显升高,p-ASK1和p-JNK含量明显降低;TH组p-p38含量明显降低;THC组ROS荧光强度明显降低,p-ASK1和p-p38含量明显升高(P<0.05)。与THC组比较,BIC模型制备后1 d TH组和THN组ROS荧光强度明显降低,CIRP、Trx1含量明显升高,p-ASK1、p-p38和p-JNK含量明显降低(P<0.05)。与S组比较,BIC模型制备后3 d BIC组、TH组、THC组和THN组神经元凋亡细胞百分比明显升高(P<0.05)。与BIC组比较,BIC模型制备后3 d TH组、THC组和THN组凋亡细胞百分比明显降低(P<0.05)。与THC组比较,BIC模型制备后3 d TH组和THN组凋亡细胞百分比明显降低(P<0.05)。结论TH可以减轻BIC模型制备后神经元氧化应激反应以及减少神经元凋亡,沉默CIRP后该作用减弱,显示TH复苏神经元的机制可能与激活CIRP/Trx1/ASK1通路有关。

Effects of Hyperoxia on Cytoplasmic Thioredoxin System in Alveolar Type Epithelial Cells of Premature Rats

This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 （Trx1） and thioredoxin reductase-1 （TrxR1） in alveolar type Ⅱ epithelial cells （AECⅡ） of premature rats. Pregnant Sprague-Dawley rats were sacrificed on day 19 of gestation. AECⅡ were isolated and purified from the lungs of premature rats. When cultured to 80% confluence, in vitro cells were randomly divided into air group and hyperoxia group. Cells in the hyperoxia group were continuously exposed to 95% O2/5% CO2 and those in the air group to 95% air/5% CO2. After 12, 24 and 48 h, cells in the two groups were harvested to detect their reactive oxygen species （ROS）, apoptosis, TrxR1 activity and the expressions of Trx1 and TrxR1 by corresponding protocols, respectively. The results showed that AECⅡ exposed to hyperoxia generated excessive ROS and the apoptosis percentage in the hyperoxia group was increased significantly at each time points as compared with that in the air group （P0.001）. Moreover, TrxR1 activity was found to be markedly depressed in the hyperoxia group in comparison to that in the air group （P0.001）. RT-PCR showed the expressions of both Trx1 and TrxR1 mRNA were significantly increased in AECⅡ exposed to hyperoxia for 12 and 24 h （P0.01）, respectively. At 48 h, the level of Trx1 mRNA as well as that of TrxR1 mRNA in the hyperoxia group was reduced and showed no significant difference from that in the air group （P0.05）. Western blotting showed the changes of Trx1 protein expressions in the hyperoxia group paralleled those of Trx1 mRNA expressions revealed by RT-PCR. It was concluded that hyperoxia can up-regulate the protective Trx1/TrxR1 expressed by AECⅡ in a certain period, however, also cause dysfunction of the cytoplasmic thioredoxin system by decreasing TrxR1 activity, which may contribute to the progression of oxidative stress and cell apoptosis and finally result in lung injury.

Transcriptomic analysis:the protection of over-expression thioredoxin reductase 1 in Parkinson’s disease

Background Parkinson’s disease(PD)is the second most common neurodegenerative disease.The pathologic characteristic feature is the loss of dopaminergic neurons in the substantia nigra(SN).However,the biochemical mechanisms are unclear.A large number of studies have shown that oxidative damage is the primary cause of PD.Hence,antioxidants could become a suitable option to treat PD.The thioredoxin(Trx)system represents a useful,potentially disease-relevant oxidation-reduction system.Thioredoxin reductase 1(TR1)is a significant component of the Trx system.Methods The overexpression lentivirus(LV)or LV-TR1 in the TR1-A53T model of PD by the stereotactic brain,and successful overexpression of LV or LV-TR1 in the MPP^(+)-induced cellular model by LV or LV-TR1 transfection.Results We confirmed that interleukin-7 mRNA levels increased in MPP^(+)compared to that in the control and MPP^(+)-TR1 groups using quantitative polymerase chain reaction.Theγ-H_(2)AX level was increased in the Tg-A53T group compared to that in the TR1-A53T group by western blotting.The expression of Na^(+)-K^(+)-ATP was decreased in the MPP^(+)group compared to that in the control and MPP^(+)-TR1 groups by high content screening.Tg-A53T(the C57BL/6 mice transferred with mutant human a-syn);TR1-A53T(A53T mice which were injected TR1-LV 2µl in SNc on two sides with minipump).The mice were fed for 10 months.control(the N2a cells cultivated with DMEM);MPP^(+)(the N2a cells dealt with MPP^(+)(1 mM)48 h),MPP^(+)-LV(the N2a cells over-expressed LV for 24 h then dealt with MPP^(+)(1 mM)48 h).MPP^(+)-TR1(the N2a cell over-expressed TR1-LV for 24 h then dealt with MPP^(+)(1 mM)48 h).From the Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,we confirmed that the overexpression of TR1 in SN pars compacta cells decreased oxidative stress,apoptosis,DNA damage,and inflammatory response and increased NADPH,Na^(+)-K^(+)-ATP,and immune response in this PD model.Conclusions Our study shows that overexpressed TR1 can be developed as a neuroprotective agent for PD.Therefore,our findings demonstrate a new targeted protein for the treatment of PD.

Up-regulation of Thioredoxin 1 by aerobic exercise training attenuates endoplasmic reticulum stress and cardiomyocyte apoptosis following myocardial infarction

Exercise training(ET)has been reported to reduce oxidative stress and endoplasmic reticulum(ER)stress in the heart following myocardial infarction(MI).Thioredoxin 1(Trx1)plays a protective role in the infarcted heart.However,whether Trx1 regulates ER stress of the infarcted heart and participates in ET-induced cardiac protective effects are still not well known.In this work,H9c2 cells were treated with hydrogen peroxide(H_(2)O_(2))and recombinant human Trx1 protein(TXN),meanwhile,adult male C57B6L mice were used to establish the MI model,and subjected to a six-week aerobic exercise training(AET)with or without the injection of Trx1 inhibitor,PX-12.Results showed that H_(2)O_(2)significantly increased reactive oxygen species(ROS)level and the expression of TXNIP,CHOP and cleaved caspase12,induced cell apoptosis;TXN intervention reduced ROS level and the expression of CHOP and cleaved caspase12,and inhibited cell apoptosis in H_(2)O_(2)-treated H9c2 cells.Furthermore,AET up-regulated endogenous Trx1 protein expression and down-regulated TXNIP expression,restored ROS level and the expression of ER stress-related proteins,inhibited cell apoptosis as well as improved cardiac fibrosis and heart function in mice after MI.PX-12 partly inhibited the AET-induced beneficial effects in the infarcted heart.This study demonstrates that Trx1 attenuates ER stress-induced cell apoptosis,and AET reduces MI-induced ROS overproduction,ER stress and cell apoptosis partly through up-regulating of Trx1 expression in mice with MI.

Broken electron transfer pathway in enzyme:Gold clusters inhibiting TrxR1/Trx via cell studies and theory simulations

Thioredoxin reductase 1(TrxR1)is over activity in tumor cell to maintain their redox balance.Although gold clusters have great potential in antitumor drug as they could well inhibit TrxR1,the molecular mechanism has not been disclosed yet.In this work,we revealed gold clusters can well inhibit the activity of TrxR1 in lung tumor cells and further disclosed the inhibition mechanism by using computational simulation methods.We firstly inferred the binding sites of gold in the hydrophobic cavities on TrxR1.The simulation results show that the gold ion(released from Au cluster)interact with–SH of Cys189 in TrxR1,this greatly increase the distance between the C-terminal redox center of TrxR1 and the Trx redox center,thereby destroy the electron transfer pathway between them.Our electron transfer destroying mechanism is different from the previous hypothesis that gold binds to the Sec498 of TrxR1 which has never been proved by experimental and theory studies.This work provides a new understanding of the gold clusters to inhibit TrxR1 activity.