左、右归丸通过调节AMPK/mTOR通路抑制大鼠股骨成脂化的实验研究

目的基于AMPK/m TOR信号通路初步探讨左、右归丸对绝经后骨质疏松(postmenopausal osteoporsis,PMOP)大鼠成脂分化的调节机制。方法将60只雌性SD大鼠随机分为空白组(KB)、假手术组(SHAM)、模型组(OVX)、左归丸组(ZGW)、右归丸组(YGW)、补佳乐组(BJL)。除KB、SHAM组外,其余大鼠均摘除双侧卵巢,SHAM组大鼠摘除双侧卵巢周围等量脂肪,灌胃12周。应用数字化全身骨密度仪检测大鼠右侧股骨骨密度(bone mineral density,BMD),应用实时荧光定量PCR法检测大鼠右侧股骨PPARγ、AMPK、m TORC1 mRNA的表达,采用Western blot法检测大鼠右侧股骨PPARγ、C/EBPα、C/EBPβ蛋白的表达以及AMPKα、m TOR蛋白磷酸化水平。结果左、右归丸能明显升高PMOP大鼠右侧股骨BMD(P0.01),降低大鼠股骨成脂相关mRNA与蛋白的表达(P0.01或P0.05),降低AMPK mRNA的表达与蛋白磷酸化水平(P0.01),升高m TORC1 mRNA的表达与蛋白磷酸化水平(P0.01或P0.05);与ZGW组相比,YGW组PMOP大鼠右侧股骨BMD没有明显差异(P0.05),股骨成脂分化相关mRNA与蛋白的表达明显升高(P0.01),且AMPK mRNA的表达明显升高(P0.01),m TORC1 mRNA的表达与蛋白磷酸化水平降低(P0.01或P0.05)。结论左、右归丸通过AMPK/m TOR通路改善了PMOP大鼠股骨成脂分化过度,其作用机制可能与调节AMPK、m TOR mRNA的表达与蛋白磷酸化水平有关。     

中老年2型糖尿病患者血清FGF21、FGF23水平与骨量异常关系的研究

目的 探讨中老年2型糖尿病（type 2 diabetes mellitus,T2DM）患者血清成纤维细胞生长因子21（FGF21）、23（FGF23）水平与骨量异常的关系。方法 随机纳入中老年T2DM患者250例,根据骨密度结果将研究对象分为骨量正常组和骨量异常组（骨量减少、骨质疏松）,收集患者的性别、年龄、糖尿病病程等资料,测定空腹血糖、糖化血红蛋白、血钙、血磷、25羟维生素D、肝肾功、血脂、血清FGF21及FGF23水平,分析T2DM患者血清FGF21、FGF23水平与骨量异常的关系。结果 与骨量正常组相比,骨量异常组患者血清FGF21（P＜0.05）、FGF23水平均降低,但后者比较差异不具有统计学意义（P＞0.05）。排除年龄、收缩期血压（SBP）、体质量指数（body mass index, BMI）、评估的肾小球滤过率（eGFR）及甘油三酯（TG）影响后,协方差分析显示骨量异常组血清FGF21水平（pg/mL）仍低于骨量正常组（P＜0.05）。以T2DM是否合并骨量异常为因变量,年龄、BMI、eGFR、TG、FGF21、FGF23、SBP为自变量,Logistic回归分析显示,年龄、SBP是骨量异常的独立危险因素,BMI、FGF21是骨量异常的保护因素。结论 中老年T2DM合并骨量异常患者血清FGF21、FGF23水平较骨量正常者降低,低血清FGF21水平可能是中老年T2DM患者骨量减少的危险因素。     

2型糖尿病患者血清FGF21、骨连结蛋白和骨转换指标的相关性研究

目的测量患有2型糖尿病(type 2 diabetes mellitus,T2DM)的早期糖尿病肾病患者的骨转换标志物(bone turnover markers,BTM)水平,并研究BTM与血清成纤维细胞生长因子-21(FGF21)和骨粘连蛋白(OC)的关联。方法纳入患有T2DM的80例男性和150例女性,测量其临床特征、BTM、OC和FGF21水平。结果96例(41.7%)患者出现尿蛋白异常。异常尿蛋白组血清OC(P<0.05)和FGF21(P<0.05)水平明显高于正常尿蛋白组患者。异常尿蛋白组患者血清P1NP水平略低(P<0.05),但调整FBG、PBG和HbA1c后差异消失。血清FGF21水平与eGFR独立且负相关,但与uACR无关。而OC与uACR独立且正相关。血清FGF21水平与P1NP独立地呈负相关。结论持续的高血糖可能会抑制骨形成,OC和FGF21均与T2DM患者的早期肾病相关。     

细胞外基质刚度通过FAK/ROCK-1/YAP通路调控尿道成纤维细胞活化

目的:探讨基质刚度对尿道成纤维细胞(UFBs)活化的影响及其机制。方法:2020年6月至2021年2月从福建医科大学附属第一医院泌尿外科手术患者获得的尿道瘢痕组织中提取原代UFBs;用聚二甲基矽氧烷分别以60∶1(Soft)、40∶1(Moderate)、30∶1(Stiff)的比例加入固化剂,制备不同刚度的基质凝胶,...     

麻黄碱调控AMPK/NF-κB信号通路改善大鼠膝骨关节炎的实验研究

目的 探讨麻黄碱(Ephedrine)调控腺苷酸活化蛋白激酶(AMPK)/核转录因子-κB(NF-κB)信号通路对大鼠膝骨关节炎(KOA)的影响。方法 建立KOA大鼠模型,将大鼠分为Sham组、KOA组、Ephedrine组(40.0 mg/kg)、Ephedrine(40.0 mg/kg)+Compound C组(0.2 mg/kg),药物分组干预后,分别检测大鼠膝关节宽度与疼痛阈值,番红O-固绿染色观察软骨组织病理变化并进行Mankin评分,ELISA法检测关节液TNF-α、IL-1β、COX-2水平,免疫组化检测软骨组织IL-1β、MMP-13蛋白表达,Western blot检测AMPK/NF-κB通路相关蛋白水平。结果 与Sham组比较,KOA组软骨组织损伤严重,膝关节宽度、Mankin评分、关节液TNF-α、IL-1β、COX-2水平及软骨组织IL-1β、MMP-13、p-NF-κB p65/NF-κB p65水平显著升高,疼痛阈值及软骨组织p-AMPK/AMPK水平显著降低(P<0.05);与KOA组比较,Ephedrine组软骨组织病理损伤减轻,膝关节宽度、Man...     

紫草素调控PI3K/Akt/mTOR通路对人胃癌MGC803细胞自噬和凋亡的影响

目的：研究紫草素对人胃癌MGC803细胞自噬和凋亡的影响及作用机制。方法 ：取对数生长期MGC803细胞,设空白对照组、紫草素组、紫草素+IGF-1(PI3K激活剂)组、紫草素+LY294002(PI3K抑制剂)组。药物干预24 h后,CCK-8法检测细胞增殖抑制率,流式细胞术检测细胞凋亡,GFP-LC3质粒转染法观察细胞自噬小体,Western blot法检测磷脂酰肌醇3激酶（PI3K）、p-PI3K、蛋白激酶B(Akt)、p-Akt、哺乳动物雷帕霉素靶蛋白（mTOR）、p-mTOR、Caspase-3、cleaved Caspase-3、LC3、Beclin-1的表达。结果：与空白对照组比较,紫草素组细胞增殖抑制率、凋亡率升高（P<0.05）,自噬小体数量明显增多,PI3K、Akt、mTOR磷酸化水平降低（P<0.05）,cleaved Caspase-3/Caspase-3、LC3-Ⅱ/LC3-Ⅰ及Beclin-1表达量升高（P<0.05）。IGF-1可明显逆转紫草素对MGC803细胞增殖抑制、凋亡、自噬,PI3K、Akt、mTOR磷酸化和cleaved Cas...     

二甲双胍调控PI3K/Akt/mTOR通路减轻糖皮质激素诱导的成骨细胞凋亡

目的探讨二甲双胍(Met)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对糖皮质激素地塞米松(Dex)诱导的成骨细胞凋亡的影响。方法将体外培养的小鼠胚胎成骨细胞前体细胞MC3T3-E1分为对照组(正常培养)、Dex组(以Dex处理)、Met组(以Dex和Met共同处理)、Met+IGF-1组(以Dex、Met和PI3K/Akt/m TOR通路活化剂IGF-1共同处理)、Met+NVP-BEZ235组(以Dex、Met和PI3K/Akt/m TOR通路抑制剂NVP-BEZ235共同处理),采用免疫印迹法(WB)检测MC3T3-E1细胞中PI3K、Akt、磷酸化(p)-Akt、m TOR和p-mTOR蛋白表达水平,通过噻唑蓝(MTT)法检测MC3T3-E1细胞存活率、流式细胞术检测MC3T3-E1细胞凋亡率、实时荧光定量PCR检测MC3T3-E1细胞中Bcl-2和Bax mRNA表达水平、Caspase-3活性测定试剂盒检测MC3T3-E1细胞Caspase-3活性、JC-1探针检测MC3T3-E1细胞线粒体膜电位变化。结果与对照组比较,Dex组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显降低,而细胞凋亡率、Bax mRNA表达水平和Caspase-3活性均明显升高(P0.05);与Dex组比较,Met组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显升高,而细胞凋亡率、Bax mRNA表达水平、Caspase-3活性明显降低(P0.05);给予IGF-1作用后Met对MC3T3-E1细胞的作用效果明显增强,而给予NVP-BEZ235作用后Met对MC3T3-E1细胞的作用效果明显减弱(P0.05)。结论 Met可通过激活PI3K/Akt/m TOR通路抑制线粒体凋亡途径,减轻糖皮质激素Dex诱导的成骨细胞凋亡。     

基于ERK/mTOR信号通路探讨六味地黄丸对氧化应激状态下成骨细胞自噬的影响

目的 从ERK/mTOR信号通路观察六味地黄丸对氧化应激状态下MC3T3-E1细胞自噬的影响,探讨其治疗绝经后骨质疏松症的作用机制。方法 用过氧化氢（H2O2）模拟氧化应激状态,六味地黄丸（LWDH）含药血清干预MC3T3-E1细胞,将细胞分为Model组（H2O2干预）、Blank组（空白血清）、LWDH组（LWDH含药血清）、Rap组（mTOR通路抑制剂）、U0126组（ERK通路抑制剂）、Rap+LWDH组（mTOR通路抑制剂、LWDH含药血清）、U0126+LWDH组（ERK通路抑制剂、LWDH含药血清）;以不做干预的Control组作为对照。通过细胞活性氧(reactive oxygen species,ROS)检测细胞ROS水平;检测自噬蛋白LC3B及ERK/mTOR信号通路相关蛋白mTOR、p-mTOR、ERK1/2和p-ERK1/2的表达。结果 ROS结果显示,与Model组相比,LWDH组、Rap组、U0126等各组细胞的ROS水平皆降低（P＜0.05）。Western blot结果显示,与Model组相比,LWDH组、Rap组、LWDH+Rap组蛋白LC3Ⅱ/Ⅰ增高（P＜0.05）;与Model组相比,LWDH组、Rap组与Rap+LWDH组的p-mTOR蛋白表达下调（P＜0.05）,LWDH组、U0126组的p-ERK1/2蛋白表达下调（P＜0.05）。结论 六味地黄丸治疗绝经后骨质疏松症的机制可能与抑制ERK/mTOR信号通路诱导氧化应激状态下成骨细胞自噬相关。     

姜黄素调控TLR4/mTOR信号通路对肝癌细胞增殖、侵袭和迁移的作用

目的：分析姜黄素影响肝癌（LC）细胞系BEL-7402体外侵袭、转移与增殖及其潜在分子机制。方法：对BEL-7402细胞实施浓度不等的姜黄素（0、30、60μmol/L）干预,经由四甲基偶氮唑蓝（MTT）技术对细胞的增殖情况展开测定,Transwell法对细胞侵袭与迁移活性展开测定;用姜黄素（60μmol/L）处理BEL-7402细胞,Western blot法检测细胞内Toll样受体4(TLR4)/哺乳动物雷帕霉素靶蛋白（mTOR）表达情况。结果：MTT实验显示,在BEL-7402细胞增殖能力上,相较空白对照组,姜黄素1组、2组皆显著偏低（P<0.05）。Transwell侵袭和迁移实验结果显示,相较于空白对照组,姜黄素1组和姜黄素2组BEL-7402细胞侵袭能力和迁移能力均显著降低（P<0.05）。Western blot结果表明,姜黄素2组BEL-7402内mTOR、TLR4蛋白表达量显著下调（P<0.05）。结论：对于LC细胞BEL-7402的增殖、转移与侵袭,姜黄素具抑制功能,其机制和下调信号途径TLR4/mTOR可能相关。     

右归丸通过PI3K/Akt/mTOR信号通路对膝骨关节炎模型鼠软骨组织保护作用的研究

目的研究右归丸对膝骨关节炎(knee osteoarthritis,KOA)模型大鼠磷脂酰肌醇3-激酶(phosphatidyl inositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)信号通路的影响。方法采用改良Hulth法制备大鼠KOA模型,模型制备成功6周后用右归丸进行干预,灌胃2个月后取材。HE法观察各组大鼠软骨组织病理形态改变并进行Mankin评分,实时荧光定量PCR法检测各组大鼠软骨组织白细胞介素-1β(interleukin-1β,IL-1β)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)和基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)的表达变化,Western blot法检测各组大鼠软骨组织磷脂酰肌醇3-激酶(phosphatidyl inositol 3-kinase,PI3K)、磷酸化的蛋白激酶B(phosphorylated protein kinase B,pAkt)、磷酸化的哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,pmTOR)和Beclin1的蛋白表达。结果与假手术组比较,模型组大鼠软骨组织Makin评分明显升高,软骨组织IL-1β、MMP-3和MMP-13的基因表达均明显升高,软骨组织PI3K、pAkt和pmTOR的蛋白表达均明显升高(P0. 01);而模型组大鼠软骨组织Beclin1的蛋白表达明显降低(P0. 01);模型组关节软骨边缘严重破坏,软骨细胞排列紊乱。与模型组相比,右归丸组大鼠软骨组织Makin评分明显降低,软骨组织IL-1β、MMP-3和MMP-13的基因表达均明显降低,软骨组织PI3K、pAkt和pmTOR的蛋白表达均明显降低,Beclin1的蛋白表达明显升高(P0. 05或P0. 01),其软骨结构趋于正常,软骨细胞分布偶见不均,关节软骨表面欠光滑。结论右归丸可能是通过抑制IL-1β、MMP-3、MMP-13、PI3K、p Akt、pmTOR的表达和增强Beclin1的表达来达到治疗KOA的目的。     

连翘苷调节相关信号通路对地塞米松诱导的成骨细胞自噬和凋亡的影响

目的 探讨连翘苷(phillyrin, PHN)调节磷脂酰肌醇3-激酶(PI3K)/丝氨酸苏氨酸激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对地塞米松(dexamethasone, DEX)诱导的成骨细胞自噬和凋亡的影响。方法 将MC3T3-E1细胞分为对照组(NOR组)、DEX组(10μmol/L DEX处理MC3T3-E1细胞)、L-PHN组(5μmol/L PHN处理MC3T3-E1细胞)、M-PHN组(10μmol/L PHN处理MC3T3-E1细胞)、H-PHN组(20μmol/L PHN处理MC3T3-E1细胞)、ZSTK474组(用20μmol/L PHN和2μmol/L的PI3K/AKT/mTOR信号通路抑制剂ZSTK474处理MC3T3-E1细胞);MTT法检测细胞毒性和细胞活力;流式细胞术检测MC3T3-E1细胞凋亡;透射电子显微镜(TEM)观察自噬小体;Western blot法检测MC3T3-E1细胞中自噬、凋亡和PI3K/AKT/mTOR通路相关蛋白表达;ALP活性及ALP染色检测MC3T3-E1细胞分化能力。结果 0～80μmol/L PHN对...     

Sulforaphane ameliorates oxidative stress induced vascular calcification by regulating Nrf-2 and Sirt-1

Objective To investigate the role and mechanism of sulforaphane (SFN) in vascular calcification induced by oxidative stress. Methods The uremic vascular calcification model was established by treating rat vascular smooth muscle cells (RASMCs) with β-glycerophosphate. RASMCs were divided into 6 groups: normal control (NC) group, 1 μmol/L SFN group, 5 μmol/L SFN group, calcification group, 1 μmol/L SFN+calcification group, 5 μmol/L SFN+calcification group, and were all cultured for 72 h. Cell viability was measured by MTT. RASMCs calcification was visualized by Von Kossa staining. Calcium content was quantified by the microplate test, and mRNA level of FGF-23 was tested by real-time PCR. The expressions of OPN, Runx-2, Nrf-2 and Sirt-1 were evaluated by Western blotting. Confocal microscope was employed to observe mitochondria damage in RASMCs and the production of ROS in RASMCs was measured by reactive oxygen species assay. Results (1) SFN did not affect cell viability of the NC group, but both low dosage and high dosage increased the cell viability of calcification group (all P＜0.05). (2) Compared with calcification group, SNF treatment decreased the calcium concentration, intracellular calcium deposition and the mRNA level of FGF-23 (all P＜0.05). (3) Compared with calcification group, SNF treatment decreased the fluorensence intensity, mitochondria injury and the protein expressions of OPN and Runx-2, but increased the protein expressions of Nrf-2, Sirt-1 and cleaved caspase-3 (all P＜0.05). Conclusion SNF can effectively protect RASMCs against vascular calcification induced by oxidative stress, since it prevents the ROS production and mitochondria dysfunction through Nrf-2 and Sirt-1.     

ERK信号通路在右美托咪定减轻小鼠神经母细胞瘤细胞氧化应激损伤中的作用

目的观察右美托咪定对过氧化氢(H_2O_2)所致小鼠神经母细胞瘤细胞(mouse neuroblastoma N2acells,N2a)氧化应激损伤的影响,探讨ERK信号通路的作用。方法在N2a细胞培养基中加入一定浓度的H_2O_2建立N2a细胞氧化应激损伤模型。将细胞分为五组:对照组(C组)、右美托咪定组(D组)、H_2O_2组(H组)、H_2O_2+右美托咪定组(HD组)、H_2O_2+右美托咪定+ERK抑制剂组(HDP组)。H组、HD组和HDP组给予200μmol/L的H_2O_2,HD组在H_2O_2处理前30min加入100ng/ml右美托咪定,HDP组在H_2O_2处理前30 min加入100ng/ml右美托咪定和20μmol/LERK抑制剂PD98059,D组在相应时点加入100ng/ml右美托咪定,C组给予等容量生理盐水。在H_2O_2刺激1h,检测细胞上清SOD活性,并分析ERK磷酸化水平;H_2O_2刺激4h,观察细胞生存、细胞形态学变化。结果与C组比较,H组N2a细胞生存率明显降低,细胞形态明显损伤,SOD活性明显下降(P0.05);与H组比较,HD组的细胞生存率明显升高,细胞形态明显改善,SOD活性明显升高,ERK磷酸化水平明显增强(P0.05);与HD组比较,HDP组细胞生存率明显降低,细胞形态明显恶化,SOD活性明显降低(P0.05)。C组和D组细胞生存率、细胞形态、SOD活性及ERK磷酸化水平差异无统计学意义。结论右美托咪定预处理能够减轻H_2O_2所致的N2a细胞氧化应激损伤,其机制可能是通过激活细胞内ERK信号通路和提高SOD活性。     

Progesterone augments epirubicin-induced apoptosis in HA22T/VGH cells by increasing oxidative stress and upregulating Fas/FasL

Background

Although epirubicin, an anthracycline drug, is widely used to treat hepatocellular carcinoma, its therapeutic efficacy is disappointing. Thus, the efficacy of epirubicin may be improved when combined with other drugs. This study investigated the therapeutic potential of combination of progesterone and epirubicin in the treatment of the human hepatoma cell line HA22T/VGH and the possible mechanisms through which this combination might induce apoptosis.

Materials and methods

HA22T/VGH cells were treated without or with 25 μM progesterone and/or 0.5 μM epirubicin and analyzed for oxidative stress, redox status, Fas/FasL expression, caspase activity, and apoptosis.

Results

HA22T/VGH cells treated with epirubicin increased the production of reactive oxygen species and nitric oxide, the expression of Fas, FasL, and Fas-associated death domain, and the activities of caspase-8 and caspase-3. Epirubicin treatment also decreased glutathione resulting in the induction of apoptosis. Treatment with progesterone alone increased nitric oxide production, but it did not affect the other parameters. However, when HA22T/VGH cells were treated with progesterone and epirubicin, the effects of epirubicin were enhanced.

Conclusions

Our observations suggest that progesterone enhances the efficacy of epirubicin. The increased efficacy is potentially attributed to progesterone's enhancement of epirubicin-induced oxidative stress, thereby reducing redox status. In addition, progesterone sequentially upregulates Fas/FasL to induce the caspase-8 and caspase-3 pathways, thereby resulting in increased apoptosis. The combination had a greater effect on the induction of HA22T/VGH cell apoptosis and could potentially serve as a more effective treatment for hepatocellular carcinoma than epirubicin alone.     

Fibroblast growth factor 2-induced cytoplasmic asparaginyl-tRNA synthetase promotes survival of osteoblasts by regulating anti-apoptotic PI3K/Akt signaling

Fibroblast growth factor 2 (FGF2), the potent bone anabolic agent, regulates the bone development, as well as the growth, remodeling and healing of the fracture. The intracellular signaling of FGF2 leads to activation of genes involved in cell proliferation, migration, differentiation and survival. However, little is known about FGF2-regulated proteins in the osteoblasts. Therefore, in this study, protein profiling in FGF2-treated MC3T3-E1 preosteoblast cells was evaluated using proteomic technologies. Six proteins including asparaginyl-tRNA synthetase (NARS), eukaryotic translation termination factor 1 (ETF1), GDP-forming succinyl-CoA synthetase (SUCLG2), heat shock protein 84 (HSP 84), sorting nexin 9 (SNX9) and α glucosidase 2α neutral subunit (GANAB) were increased more than 3-fold after the FGF2 treatment. Also, two proteins including β-tropomyosin and tropomyosin 2 were decreased to 2-folds. Among these proteins, asparaginyl-tRNA synthetase (NARS), a member of aminoacyl-tRNA synthetases (AARS), was strikingly up-regulated more than 900-fold. The overexpression of NARS significantly increased the proliferation of both the MC3T3-E1 and the primary mouse calvarial cells. In contrast, significant reduction of the basal expression of NARS by siNARS remarkably suppressed the proliferation and induced the death of cell. After the siNARS treatment, the resistance to apoptosis induced by serum deprivation was also significantly reduced. The level of p-Akt was also reduced and the activity of caspase 3 significantly enhanced. In addition, NARS-induced protection against apoptosis was abolished by the treatment of PI3K inhibitors, wortmannin and LY294002. In conclusion, we suggest that NARS is one of the important mediators of FGF2 induced survival signaling in osteoblasts through the activation of PI3K/Akt survival pathway.     

Mettl3介导m6A甲基化修饰对过氧化氢诱导的脊髓神经元氧化应激和凋亡的影响

【摘要】 目的：探讨Mettl3介导m6A甲基化修饰对过氧化氢（H2O2）诱导的脊髓神经元氧化应激和凋亡的影响。方法：分离新生（24h内）SD大鼠脊髓,经消化、离心后重悬沉淀,差速贴壁30min后收集未贴壁细胞培养于Neurobasal培养基中,经免疫荧光鉴定呈β3-tubulin阳性,确认为脊髓神经元。将脊髓神经元随机分为6组：空白组,细胞不予任何处理,正常培养;转染对照组,细胞转染阴性对照siRNA（si-NC）;转染组,细胞转染Mettl3 siRNA（si-Mettl3）;诱导组,细胞采用300μmol/L的H2O2处理;转染对照诱导组,细胞先转染si-NC,然后采用300μmol/L H2O2处理;转染诱导组,细胞先转染si-Mettl3,然后采用300μmol/L H2O2处理。实时荧光定量聚合酶链反应（RT-qPCR）检测各组细胞Mettl3的mRNA表达水平,Western blot检测Mettl3、Bax、Bcl-2和cleaved Caspase-3的蛋白表达水平,免疫荧光检测Mettl3表达,酶标仪检测各组整体m6A水平,并检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平,流式细胞仪检测细胞凋亡。结果：与空白组相比,诱导组的m6A甲基化修饰水平显著提高（P<0.05）,Mettl3的mRNA和蛋白表达水平显著性上调（P<0.05）,IL-1β（73.39±8.82ng/L vs 125.58±15.31ng/L）、IL-6（63.34±7.12ng/L vs 101.28±12.49ng/L）、TNF-α（103.29±12.19ng/L vs 204.37±23.65ng/L）和MDA（4.01±0.67pmol/L vs 9.23±1.05pmol/L）水平显著性升高（P<0.05）,SOD（28.37±3.72U/mg vs 17.23±2.05U/mg）和GSH-Px（158.19±19.26U/mg vs 83.35±9.05U/mg）水平显著性降低（P<0.05）,且Bax和cleaved Caspase-3的蛋白表达水平显著性上调（P<0.05）,Bcl-2的蛋白表达水平显著性下调（P<0.05）,细胞凋亡率显著升高[（8.30±0.68）% vs （34.29±3.16）%,P<0.05];与诱导组相比,转染诱导组的m6A甲基化修饰水平显著性降低（P<0.05）,Mettl3的mRNA和蛋白表达水平显著性下调（P<0.05）,IL-1β（125.58±15.31ng/L vs 96.28±11.27ng/L）、IL-6（101.28±12.49ng/L vs 84.56±10.24ng/L）、TNF-α（204.37±23.65ng/L vs 147.15±18.46ng/L）和MDA（9.23±1.05pmol/L vs 7.28±0.96pmol/L）水平显著性降低（P<0.05）,SOD（17.23±2.05U/mg vs 24.01±2.76U/mg）和GSH-Px（83.35±9.05U/mg vs 121.48±15.47U/mg）水平显著升高（P<0.05）,且Bax和cleaved Caspase-3的蛋白表达水平显著性下调（P<0.05）,Bcl-2的蛋白表达水平显著性上调（P<0.05）,细胞凋亡率显著性降低[（34.29±3.16）% vs （23.57±2.01）%,P<0.05]。与空白组相比,转染组的炎性因子、氧化应激和凋亡水平均无显著性差异（P>0.05）。结论：H2O2可上调脊髓神经元中m6A甲基化修饰水平,并可诱导氧化应激和细胞凋亡;抑制Mettl3表达能够降低H2O2诱导的脊髓神经元m6A甲基化修饰水平,进而缓解氧化应激和细胞凋亡。     

转录因子NF-E2相关因子2-氧化反应元件通路在抗心肌再灌注损伤中的作用

背景 转录因子NF-E2相关因子2(Nf-E2 related factor-2,Nrf2)抗氧化反应元件(antioxidant response element,ARE)通路广泛分布于机体心血管系统中,激活后可上调内源性抗氧化系统减轻心肌的氧化损伤. 目的 阐述Nrf2-ARE通路作为抗心肌缺血/再灌注损伤(ischemia/reperfusion injury,I/RI)的潜在靶点,并探讨其可能的保护机制.内容 Nrf2-ARE通路处于氧化应激、炎症反应的中心地位,介导编码抗氧化蛋白和二项解毒酶基因的基础表达和诱导表达;多种外源性化合物可以激活Nrf2-ARE通路,在转录水平上调节抗氧化蛋白及二项解毒酶基因的表达,增强内源性抗氧化系统的能力从而在减少氧自由基产生、抗炎症反应、减轻钙超载、抗心肌细胞凋亡等方面减轻心肌I/RI.趋向 激活Nrf2-A RE通路可为临床抗I/RI提供新的策略.