二甲双胍对高糖诱导的肾小管上皮细胞TGF-β/ERK/MMP-9通路及上皮间质转化的影响

目的探讨二甲双胍对高糖诱导的肾小管上皮HK-2细胞上皮间质转化(EMT)的影响及其机制。方法以0、7.5、15.0、30.0、60.0、120.0mmol/L二甲双胍作用48h后,采用噻唑蓝(MTT)法检测HK-2细胞活力以筛选合适的二甲双胍作用浓度;将体外培养的HK-2细胞分为对照组(5.5 mmol/L D-葡萄糖)、高渗组(24.5 mmol/L甘露醇和5.5 mmol/L D-葡萄糖)、高糖组(30 mmol/L D-葡萄糖)、高糖+7.5 mmol/L二甲双胍组(30 mmol/L D-葡萄糖+7.5 mmol/L二甲双胍)和高糖+15 mmol/L二甲双胍组(30 mmol/L D-葡萄糖+15 mmol/L二甲双胍),倒置显微镜观察各组HK-2细胞形态,免疫印迹法(Western blotting)检测各组HK-2细胞中α-平滑肌肌动蛋白(α-SMA)、E-钙黏附蛋白(E-cadherin)、转化生长因子-β(TGF-β)、细胞外信号调节激酶(ERK)、磷酸化(p)-ERK、基质金属蛋白酶9(MMP-9)蛋白表达水平,实时荧光定量PCR(qRT-PCR)检测α-SMA、E-cadherin、TGF-β、ERK、MMP-9 mRNA表达水平。结果与对照组相比,30.0、60.0mmol/L二甲双胍作用后HK-2细胞活力明显升高,120.0mmol/L二甲双胍作用后HK-2细胞活力明显降低(P0.05),而7.5、15.0mmol/L二甲双胍作用后HK-2细胞活力差异无统计学意义(P0.05);与对照组比较,高渗组细胞形态无明显改变,且细胞中α-SMA、E-cadherin、TGF-β、MMP-9蛋白和mRNA表达水平以及p-ERK/ERK蛋白、ERK mRNA表达水平差异均无统计学意义(P0.05),但高糖组细胞失去原有形态变为长梭形,且细胞中α-SMA、TGF-β蛋白和mRNA表达水平以及p-ERK/ERK蛋白、ERK mRNA表达水平明显升高,而MMP-9、E-cadherin蛋白和mRNA表达水平明显降低(P0.05);与高糖组比较,高糖+7.5mmol/L二甲双胍组、高糖+15.0mmol/L二甲双胍组细胞形态由长梭形逐渐变成圆形或椭圆形,且α-SMA、TGF-β蛋白和mRNA表达水平以及p-ERK/ERK蛋白、ERK mRNA表达水平明显降低,而MMP-9、E-cadherin蛋白和mRNA表达水平明显升高(P0.05),且高糖+15.0 mmol/L二甲双胍组细胞上述指标变化幅度大于高糖+7.5 mmol/L二甲双胍组。结论二甲双胍可抑制高糖诱导的肾小管上皮HK-2细胞EMT,其作用机制可能与抑制TGF-β/ERK/MMP-9通路活化有关。     

Sirt3/Sod2信号通路介导的补骨脂酚抗糖尿病大鼠主动脉凋亡的机制研究

目的 探讨补骨脂酚在糖尿病主动脉组织中的保护作用及其机制,明确Sirt3/Sod2信号通路在此过程中的作用。方法 将SD大鼠随机分成对照组、糖尿病组和糖尿病+补骨脂酚组,将内皮细胞分为高脂高糖组、高脂高糖+3-TYP组、高脂高糖+补骨脂酚组和高脂高糖+补骨脂酚+3-TYP组。采用OGTT试验检测大鼠的空腹血糖。采用HE染色观察主动脉壁病理变化。采用荧光染色观察主动脉Sirt3表达变化。采用Western blotting检测主动脉组织及内皮细胞Sirt3、Ac-Sod2、Cleaved caspase-3、Bax、Bcl-2蛋白的表达情况。并用Sirt3的抑制剂3-TYP来研究补骨脂酚抗糖尿病大鼠主动脉凋亡的作用机制。结果 糖尿病大鼠主动脉壁组织排列紊乱,细胞减少,Sirt3及Bcl-2蛋白表达水平明显降低,Ac-sod2、Cleaved caspase-3及Bax蛋白表达水平明显升高。补骨脂酚可显著改善高血糖引起的主动脉壁组织排列紊乱情况,升高Sirt3及Bcl-2蛋白的表达水平,降低Ac-sod2、Cleaved caspase-3及Bax蛋白的表达水平。在高脂高糖损伤内皮细胞试验...     

黄芪甲苷通过激活Sirt3抑制血管紧张素Ⅱ诱导的心肌肥大及氧化应激

目的研究Sirt3在黄芪甲苷抑制血管紧张素Ⅱ诱导的心肌肥大和氧化应激中的作用。方法α-actinin染色检测心肌细胞大小;MitoSOX染色检测ROS;Real time PCR检测心肌肥大标记物ANP、BNP和β-MHC的mRNA水平;Western blot法检测心肌细胞肥大信号通路蛋白水平。结果100nmol·L-1血管紧张素Ⅱ处理心肌细胞48 h,心肌细胞面积与ANP、BNP、β-MHC mRNA水平显著增加,ROS水平及NOX-2、NOX-4表达显著增加,Sirt3表达显著降低;黄芪甲苷预处理心肌细胞1 h显著抑制血管紧张素Ⅱ诱导的上述效应。Sirt3-siRNA干预Sirt3蛋白水平,显著抑制黄芪甲苷的心肌肥厚抑制作用以及抗氧化作用,上调ROS水平,促进心肌肥大。结论黄芪甲苷通过激活Sirt3抑制血管紧张素Ⅱ诱导的心肌肥大以及氧化应激。     

二甲双胍抑制炎症相关AMPK/Sirt1/NF-κB信号通路改善肾小管上皮细胞衰老的分子机制

摘 要 目的：研究二甲双胍对D 半乳糖诱导肾小管上皮细胞（NRK 52E）衰老的保护作用及其对腺苷酸激活蛋白激酶/细胞沉默调节蛋白1/核转录因子 κB（AMPK/Sirt1/NF-κB）信号通路的影响。 方法: Western blot检测不同浓度D 半乳糖（50,100,200 mmol·L-1）和二甲双胍（10,30,60 mmol·L-1）干预后细胞衰老标志物Klotho、P27和P16蛋白表达水平及AMPK/Sirt1/NF-κB信号通路蛋白表达量;细胞计数试剂盒（CCK8）法检测NRK 52E细胞增殖活性。 结果: 与对照组比较,D 半乳糖干预能显著升高Klotho和Ac NF-κB蛋白表达水平,显著降低P27、P16、p AMPK和Sirt1蛋白表达水平（P＜0.05）,且D 半乳糖呈浓度依赖性。不同浓度二甲双胍干预后,与D 半乳糖组（100 mmol·L-1）比较,Klotho和Ac NF-κB蛋白表达水平显著降低,P27、P16、p AMPK和Sirt1蛋白表达水平显著升高（P＜0.05）,且二甲双胍呈浓度依赖性,而细胞增殖活性差异无统计学意义（P＞0.05）。 结论: 在D 半乳糖诱导的NRK 52E细胞衰老模型中,二甲双胍在体外具有抗衰老的作用,并且通过抑制炎症相关AMPK/Sirt1/NF-κB信号通路而缓解衰老过程。     

恩格列净与二甲双胍联合治疗2型糖尿病的药物经济学评价

目的：对恩格列净与二甲双胍联合治疗2型糖尿病（T2DM）进行经济学评价。方法：在恩格列净与二甲双胍联合治疗T2DM的3期随机对照临床试验（RCT）的基础上,建立恩格列净和二甲双胍治疗T2DM的Markov模型,模拟10 mg恩格列净联合二甲双胍、二甲双胍单用治疗T2DM无并发症、T2DM并发症及死亡的动态变化。以质量调整生命年（QALYs）为健康产出指标,以3倍2019年国内生产总值（gross domestic product,GDP）作为意愿支付阈值（WTP）,使用Markov模型进行回乘分析和队列模拟获得2种治疗方案的长期效果与成本,并对成本、效用及贴现进行敏感性分析,检验结果稳定性。结果：在模拟为期10年的疾病进展后,与二甲双胍相比,10 mg恩格列净与二甲双胍联合治疗方案的增量成本-效用比（ICRE）为96 256.582元/QALYs,小于WTP,增加的成本可以接受。敏感度分析显示,10 mg恩格列净与二甲双胍联合方案具有成本-效用的概率是60.300%。结论：对于T2DM患者,10 mg恩格列净与二甲双胍的联合方案属于优势方案,增加成本可以接受,但经济性概率不高。     

二甲双胍对高糖培养H9c2细胞Connexin43表达的影响

目的研究二甲双胍(metformin,Met)对高糖培养下的H9c2细胞缝隙连接蛋白43(connexin43,Cx43)表达的影响及其机制。方法高糖培养的大鼠H9c2心肌细胞分别加入3和5μmol·L-1的两种不同浓度的二甲双胍继续培养24 h。MTT实验检测H9c2细胞活力;LDH释放实验检测细胞毒力;细胞免疫荧光实验检测Cx43的表达和分布;荧光法检测细胞内活性氧(ROS)水平;Western blot检测Cx43、pAMPK、AMPK和GAPDH的表达。结果 Met能增加H9c2细胞活力,降低细胞内ROS水平,对LDH释放差异无显著性;Met使AMPK磷酸化水平明显升高,增加心肌Cx43表达,改善Cx43的分布。结论 Met可能通过激活AMPK途径,增加心肌细胞Cx43的表达和减少细胞内ROS的产生,进而发挥心血管的保护效应。     

肉桂多糖的单糖组成分析及其降血糖作用研究

目的:分析肉桂多糖的单糖组成,并探讨其对糖尿病小鼠糖脂代谢的影响。方法:采用PMP-HPLC法分析肉桂多糖的单糖组成。采用腹腔注射链脲佐菌素的方法建立糖尿病小鼠模型,将成模小鼠随机分成5组:模型组,二甲双胍组,肉桂多糖低、中、高剂量组。给药4周后检测各组小鼠的空腹血糖值(FBG)、血清生化指标、血清胰岛素(INS)、肝糖原和肌糖原含量;HE染色法观察胰腺病理学特征;qRT-PCR法检测GLUT2、PEPCK、GCK mRNA表达量;Western blot检测PEPCK、GCK蛋白表达量。结果:肉桂多糖主要由甘露糖、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖组成,各单糖摩尔比依次为1.00∶2.79∶24.30∶2.12∶2.19。与模型组相比,各给药组的FBG和LDL-C水平显著下降,肝糖原和肌糖原水平显著上升;肉桂多糖高剂量组的TC、TG和LDL-C水平显著下降,HDL-C和INS水平显著上升。高剂量肉桂多糖可显著上调糖尿病小鼠肝脏中GCK、GLUT2的表达量,显著下调PEPCK表达量。结论:建立的PMP-HPLC分析方法操作简单可行,准确性、重复性良好。肉桂多糖对链脲佐菌素诱导的糖尿病小鼠具有降血糖作用,其降糖机制可能与调节糖脂代谢有关。     

黄芪甲苷在高糖诱导的内皮细胞损伤中的作用研究

目的探讨黄芪甲苷(astragaloside Ⅳ,AS-IV)对高糖诱导的内皮细胞损伤的保护作用及机制。方法体外培养人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs),使用高浓度葡萄糖(30 mmol/L)诱导细胞损伤,采用实时聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)检测炎症因子肿瘤坏死因子(tumor necrosis factor,TNF)-α、白介素(interleukin,IL)-6和IL-1β的表达。使用不同浓度黄芪甲苷(10、20、50及100 μmol/L)处理高糖损伤的HUVECs,RT-PCR检测炎症因子TNF-α、IL-6和IL-1β的表达水平。黄芪甲苷干预后,酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测TNF-α、IL-6和IL-1β含量,蛋白质印迹法(western blot,WB)检测自噬相关蛋白LC3及Beclin1的表达。进一步将实验分为高糖组、黄芪甲苷组及3-甲基腺嘌呤(3-methyladenine,3-MA)组,RT-PCR检测TNF-α、IL-6和IL-1β的信使RNA(messenger RNA,mRNA)表达水平、WB法检测自噬相关蛋白LC3和Beclin1表达。结果高糖可显著诱导炎症因子TNF-α、IL-6和IL-1β的表达,50 μmol/L黄芪甲苷为最佳抑制炎症反应的浓度。黄芪甲苷组自噬相关蛋白LC3及Beclin1的蛋白水平显著上调,差异具有统计学意义(P<0.05)。与黄芪甲苷组相比,3-MA组自噬相关蛋白LC3和Beclin1的蛋白水平显著降低,TNF-α、IL-6、IL-1β的mRNA表达水平显著上调,差异具有统计学意义(P<0.05)。结论黄芪甲苷可保护内皮细胞免于炎症损伤,其作用机制可能与细胞自噬有关。     

基于NLRP3/GSDMD信号通路研究人参皂苷Rb1对高糖诱导的胰岛β细胞焦亡的影响

目的: 研究人参皂苷Rb1(GRb1)抑制高糖诱导的胰岛β细胞焦亡及对NLRP3/GSDMD信号通路的调节机制。方法: 高糖(50 mmol·L^-1)诱导大鼠胰岛细胞瘤细胞(rat insulinoma cells, INS-1)焦亡模型,并构建NLRP3过表达质粒转染INS-1细胞,采用倒置显微镜观察GRb1对细胞形态变化的影响;采用ELISA法检测GRb1对细胞上清液中乳酸脱氢酶(LDH)、白细胞介素-1β(IL-1β)、胰岛素水平的影响;采用Western blot法和qRT-PCR法检测GRb1对INS-1细胞中核苷酸结合域样受体蛋白3(nucleotide binding domain like receptor protein 3, NLRP3)和切割蛋白D(gasdermin D, GSDMD)表达的影响。结果: 在高糖环境下,GRb1可减轻INS-1细胞形态学改变,显著增加INS-1细胞胰岛素分泌,降低细胞上清液中IL-1β、LDH水平(P＜0.05),且呈剂量依赖性;GRb1呈剂量依赖性抑制高糖诱导的INS-1细胞中GSDMD及上游调节因子NLRP3的表达(P＜0.05);过表达NLRP3后可显著逆转高糖环境下GRb1对INS-1细胞的部分保护性作用(P＜0.05)。结论: GRb1能够改善高糖诱导的INS-1细胞焦亡,减轻炎症反应,其机制可能与抑制NLRP3/GSDMD信号通路有关,从而防治糖尿病及其并发症的发生。     

阿卡波糖、二甲双胍、吡格列酮对非酒精性脂肪性肝病大鼠肝组织肿瘤坏死因子-α及细胞色素P4502E1的影响

目的观察阿卡波糖、二甲双胍、吡格列酮对非酒精性脂肪性肝病(NAFLD)大鼠肝脏中肿瘤坏死因子-α(TNF-α)、细胞色素P4502E1(CYP2E1)的影响。方法将SD大鼠随机分为正常对照组(普通饲料喂养)、非酒精性脂肪肝组(高脂饮食喂养)、阿卡波糖干预组[高脂饮食喂养加阿卡波糖100 mg/(kg.d)灌胃]、二甲双胍干预组[高脂饮食喂养加二甲双胍500 mg/kg.d)灌胃]、吡格列酮干预组[高脂饮食喂养加吡格列酮15 mg/(kg.d)灌胃]。饲养12周末处死大鼠,检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)活性以及总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)含量,测量空腹血糖(FBG)及胰岛素(FINS)水平,计算胰岛素抵抗指数(HOMA-IR);HE染色观察肝脏病理形态学的变化;Real time PCR和免疫组化法检测肝组织中TNF-α及CYP2E1 mRNA及蛋白表达的变化。结果 3种药物干预组均可降低ALT,AST,ALP活性(P<0.05),减少TC,TG,FFA含量(P<0.05),改善大鼠肝脏病理形态学改变,降低肝脏中TNF-α及CYP2E1 mRNA及蛋白的表达(P<0.01)。二甲双胍、吡格列酮两组间无显著差异(P>0.05),阿卡波糖组的作用明显低于前两者(P<0.01)。结论阿卡波糖、二甲双胍、吡格列酮均可减少TNF-α及CYP2E1的表达,改善非酒精性脂肪肝的病理变化,其中二甲双胍、吡格列酮作用相似,阿卡波糖作用较弱。     

Effects of atorvastatin on fine particle-induced inflammatory response, oxidative stress and endothelial function in human umbilical vein endothelial cells

The study is to explore the toxicity of organic extracts and water-soluble fraction of fine particles on human umbilical vein endothelial cells (HUVECs). The exposure doses were 100, 200 and 400 μg/ml, respectively, for two kinds of fractions. Moreover, atorvastatin was used for intervention study. HUVECs were stimulated by 400 μg/ml organic and water soluble extracts, respectively, immediately followed by treatment with atorvastatin in concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L, respectively. Cell viability, malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), reactive oxygen species (ROS) and the expression of interleukin-6 beta (IL-6), tumor necrosis factor-α (TNF-α), endothelin-1 and P-selectin were determined in cells. The results showed that MDA and ROS increased in HUVECs after exposed to organic extracts and water-soluble fraction, whereas cell viability, NO and SOD decreased. The mRNA expression of IL-6, TNF-α, endothelin-1 (ET-1) and P-selectin increased after exposed to different fractions. Meanwhile, at the same exposure dose, water-soluble fraction caused more significant increase of MDA, IL-6, TNF-α and P-selectin and decrease of cell viability and NO when compared to organic extracts. Compared to no atorvastatin group, the levels of MDA, ROS and the expression of IL-6, TNF-α, ET-1 and P-selectin decreased in HUVECs in adding atorvastatin group, but cell viability, NO and SOD increased, which indicated that atorvastatin attenuated fine particle-induced inflammatory response, oxidative stress and endothelial damage. The results hinted that the inflammatory response, oxidative stress and endothelial dysfunction might be the mechanisms of cardiovascular injury induced by different fractions of ambient fine particles.     

Upregulation of Sirt1 by tyrosol suppresses apoptosis and inflammation and modulates extracellular matrix remodeling in interleukin-1β-stimulated human nucleus pulposus cells through activation of PI3K/Akt pathway

Intervertebral disc degeneration (IDD) is the major pathogenesis of lower back pain. Tyrosol is a polyphenolic compound that exhibits anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Herein, we explored the effects and mechanisms of tyrosol on IDD progression in interleukin (IL)-1β-stimulated human nucleus pulposus cells (HNPCs). Cell viability and apoptosis were detected by CCK-8 and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was examined to evaluate inflammation. The mRNA expression of matrix metalloproteinases (MMPs) (MMP-3/9/13), collagen type II, SRY-related high mobility group box 9 (SOX-9), and aggrecan was measured by qRT-PCR. Protein levels of silent information regulator 2 homolog 1 (Sirt1), phosphorylated protein kinase B (p-Akt), Akt, collagen type II, SOX-9, and aggrecan were determined by western blot. Results showed that tyrosol attenuated IL-1β-induced viability reduction, apoptosis, and caspase-3/7 activity in HNPCs. The increase in the production of TNF-α, IL-6, NO, and PGE2 in IL-1β-treated HNPCs was abolished by tyrosol treatment. Tyrosol treatment reversed IL-1β-induced upregulation of MMP-3, MMP-9, and MMP-13, and downregulation of collagen II, SOX-9, and aggrecan in HNPCs. Additionally, tyrosol treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in IL-1β-stimulated HNPCs. Sirt1 was upregulated by tyrosol, and Sirt1 silencing inhibited Akt phosphorylation in HNPCs. Sirt1 knockdown attenuated the effects of tyrosol on IL-1β-induced apoptosis, inflammation, and ECM remodeling in HNPCs. In summary, upregulation of Sirt1 by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1β-stimulated HNPCs through activation of PI3K/Akt pathway.     

雷公藤多苷对高糖诱导人肾小管上皮细胞凋亡及CXCL10/CXCR3轴的影响

目的 探讨雷公藤多苷（TG）对高糖诱导人肾小管上皮细胞HK-2凋亡及CXC趋化因子配体10(CXCL10)/CXC趋化因子受体3(CXCR3)轴的影响。方法 体外培养人肾小管上皮细胞HK-2,并分为对照组（含5.5 mmol/L葡萄糖培养基）、高糖组（含25 mmol/L葡萄糖培养基）和12.5、25、50 mg/L TG组（分别用12.5、25、50 mg/L的TG和25 mmol/L葡萄糖培养基）。四甲基偶氮唑盐比色法检测HK-2细胞活力;流式细胞术检测HK-2细胞凋亡情况;2’,7’-二氯二氢荧光素二乙酸酯法检测HK-2细胞活性氧（ROS）水平;黄嘌呤氧化酶法检测HK-2细胞超氧化物歧化酶（SOD）水平;酶联免疫吸附试验检测HK-2细胞炎性因子肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)水平;蛋白免疫印迹法检测HK-2细胞凋亡蛋白[胱天蛋白酶（Caspase）-3、Caspase-9]以及CXCL10、CXCR3蛋白表达。结果 与对照组比较,高糖组HK-2细胞活力、SOD水平显著降低,凋亡率、ROS、TNF-α、TGF-β1水平及Caspase-3、Casp...     

Antithrombotic and profibrinolytic activities of phloroglucinol

Phloroglucinol is the monomeric units of phlorotannins abundant in brown algae. Several biological effects of phloroglucinol have been reported, however, antithrombotic and profibrinolytic activities of phloroglucinol have not been studied yet. In this study, the anticoagulant properties of phloroglucinol were determined by assays of activated partial thromboplastin time (aPTT), prothrombin time (PT) and cell based thrombin and activated factor X (FXa) generation activities. And the effects of phloroglucinol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human endothelial cells (HUVECs). I found that phloroglucinol prolonged aPTT and PT significantly and inhibited thrombin and FXa generation in HUVECs. Furthermore, phloroglucinol inhibited TNF-α induced PAI-1 production. I then used pathway inhibitors to investigate which step of the TNF-α induced signaling pathway was targeted by phloroglucinol. I observed that the c-Jun N-terminal kinase (JNK) inhibitor increased the inhibitory effects of phloroglucinol, whereas the nuclear factor factor-κB (NF-κB) and the extracellular signal-regulated kinase (ERK) inhibitor did not. Therefore these results suggest that phloroglucinol possess antithrombotic and profibrinolytic activities and that NF-κB and ERK pathways are possible targets of phloroglucinol in the regulation of TNF-α stimulated PAI-1 production in HUVECs.     

叶黄素通过SIRT1/NLRP3信号通路提高人视网膜色素上皮细胞存活率的机制研究 #br#

目的 探讨叶黄素对高浓度葡萄糖诱导后人视网膜色素上皮细胞（ARPE-19细胞）内过氧化应激反应、炎 症反应与细胞增殖活性的改善作用及调控机制。方法 使用不同浓度的叶黄素和高浓度葡萄糖同时处理ARPE-19 细胞后，应用 CCK-8 检测细胞增殖活性变化，应用酶联免疫吸附试验检测炎性细胞因子水平变化，应用 CMH 2DCFDA探针法检测活性氧（ROS）水平变化，应用荧光定量PCR检测SIRT1与NLRP3 mRNA表达水平变化。结果 在高葡萄糖环境下，ARPE-19细胞内炎性细胞因子白细胞介素（IL）-1β、IL-6、IL-18、肿瘤坏死因子（TNF）-α和ROS 的表达水平显著高于对照组（P＜0.05），细胞存活率低于对照组（P＜0.01）。与葡萄糖组比较，叶黄素组SIRT1 mRNA 表达水平和细胞存活率显著上调，而 NLRP3 mRNA 表达水平显著下调（P＜0.05）。结论 叶黄素可通过 SIRT1/ NLRP3信号通路改善人视网膜色素上皮细胞内的过氧化应激反应与炎症反应，进而提高细胞存活率。     

蛹虫草提取物对高糖诱导内皮细胞衰老的影响

目的：探讨蛹虫草乙酸乙酯提取物（CME）对高糖诱导人脐静脉内皮细胞（HUVECs）衰老的影响。方法：用高糖（40mmol／L）诱导建立HUVECs衰老体外模型,以冬虫夏草提取物（CSE）（50μg／mL）为阳性对照,观察CME（12．5～100μg／mL）对内皮细胞衰老的干预作用。四甲基偶氮唑蓝（MTT）比色法检测细胞增殖;SA—β-半乳糖酐酶（SA—β—gal）染色法检测细胞衰老特异性的SA—β—gal活性;流式细胞术检测细胞周期分布和细胞内活性氧（ROS）含量。结果：经高糖干预24、48、72h后,HUVECs增殖活性OD值明显低于对照组;而且在高糖孵育96h后,SA—β-gal染色阳性细胞率明显增加;孵育48h后,处于G0／G1期细胞的百分率和ROS相对水平明显增加（P〈0．01）。与高糖组相比,CME（25～50μg／mL）孵育48和72h后细胞OD值显著提高（P〈0．05）;CME（12．5～100μg／mL）孵育96h后SA-β-gal染色阳性细胞率显著降低（P〈0．01）;孵育48h后Go／G1期的细胞百分率（P〈0．01）、细胞内ROS水平显著降低（P〈0．05）;CME25、50／μg／mL效果优于CME12．5、100〉g／mL（P〈0．05）,且与CSE50ptg／mL作用相近（P〉0．05）。结论：蛹虫草乙酸乙酯提取物可促进内皮细胞生长增殖,可能与其降低细胞内ROS水平,减轻细胞氧化应激损伤有关,从而减轻内皮细胞衰老。     

2-amino-phenoxazine-3-one attenuates glucose-induced augmentation of embryonic form of myosin heavy chain, endothelin-1 and plasminogen activator inhibitor-1 in human umbilical vein endothelial cells

The aim of this study was to investigate the changes in mRNA level of embryonic form of myosin heavy chain (SMemb), endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1), which are considered to be involved in the angiogenesis and atherosclerosis in diabetic blood vessels, in human umbilical vein endothelial cells (HUVECs) caused by high ambient glucose, and the effects of 2-aminophenoxazine-3-one (Phx-3), which was produced by the reaction of bovine hemoglobin with o-aminophenol, on them. The mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were extensively upregulated in HUVECs treated with high concentration of glucose (15 mM), compared with those in the cells with normal concentration of glucose (5 mM). The migration activity of HUVECs evaluated by the cell migration assay was accelerated by 15 mM glucose. When 10 microM Phx-3, at the concentration of which the proliferation of HUVECs was not affected, was administered to HUVECs with 15 mM glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were significantly downregulated to the normal levels in the cells. However, when 10 microM Phx-3 was administered to HUVECs with 5 mM of glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were not affected. The migration activity of HUVECs, which was accelerated by high glucose, was reversed by 10 microM Phx-3. The present results suggest that Phx-3 may be a drug to prevent the high glucose-associated endothelial damage, vascular angiogenesis in diabetic patients, by inhibiting the expression of angiogenic factors, such as SMemb, ET-1 and PAI-1, in the endothelial cells.     

复方金银花外洗液体外抗炎作用的实验研究

目的对复方金银花外洗液的体外抗炎作用进行研究。方法采用MTT法测定复方金银花外洗液对RAW264.7细胞活性的影响;采用ELISA法观察RAW264.7细胞上清液中TNF-α、IL-6的分泌情况;采用Western-blot法检测TNF-α、IL-6蛋白的表达情况。结果 MTT结果显示,复方金银花外洗液在0~210μL/mL对RAW264.7细胞活力无显著影响(P>0.05);同时,金银花外洗液能够抑制LPS诱导的RAW264.7细胞的增殖活性,且呈剂量依赖性。ELISA检测结果显示,模型组细胞培养上清中TNF-α、IL-6含量显著升高,与正常组相比差异有统计学意义(P<0.01),复方金银花外洗液中、高剂量组培养液上清中TNF-α、IL-6含量低于模型组(P<0.05)。Western blot结果显示,模型组中TNF-α、IL-6的蛋白含量明显高于正常组(P<0.01),不同浓度外洗液处理后,炎症因子TNF-α、IL-6的蛋白表达水平显著降低,与模型组比较差异有统计学意义(P<0.05)。结论复方金银花外洗液在细胞水平的主要抗炎作用与抑制炎症因子TNF-α、IL-6的蛋白表达水平有关。