Mechanism of ligustrazini against thrombosis

Objective To investigate the mechanism of Chinese medicine ligustrazini against thrombosis, and the effects of ligustrazini on plasminogen activator inhibitor (PAI-1) expression in normal endothelial cells and lipopolysaccharide (LPS) stimulated endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. PAI-1 protein in HUVEC conditioned medium was measured by Sandwich enzyme-linked immunosorbent assay (ELISA), and PAI-1 mRNA expression was determined by Northern blot analysis. Using electrophoretic mobility shift assay (EMSA), we observed HUVEC nuclear factor-kappa B (NF-κB) nuclear translocation. Results LPS treatment of cultured HUVECs resulted in a significant increase in PAI-1 protein and mRNA expression by these cells. However, when HUVECs were incubated with LPS plus ligustrazini, the upregulation of PAI-1 by LPS was abated. Moreover, ligustrazini could decrease the basal level of PAI-1 protein and mRNA in HUVECs as compared with control. Nuclear extracts prepared from HUVECs stimulated by LPS demonstrated that binding to the NF-kB oligo nucleotide increased as compared with the unstimulated cells, but ligustrazini did not change those binding in the absence or presence of LPS. Conclusions Ligustrazini inhibited both basal and LPS-induced PAI-1 protein and mRNA expression in endothelial cells, and the modulation of PAI-1 in HUVECs by ligustrazini might have other mechanisms rather than NF-kB     

Inhibiting NF-κB increases cholesterol efflux from THP-1 derived-foam cells treated with AngII via up-regulating the expression of ATP-binding cassette transporter A1

Objective: To study the role of nuclear factor-kappa B(NF-κB) in cholesterol efflux from THP-1 derived-foam cells treated with AngiotensinⅡ(AngⅡ). Methods:Cultured THP-1 derived-foam cells were treated with AngⅡ or preincubated with tosyl-phenylalanine chloromethyl-ketone(TPCK) NF-κB inhibitor. The levels of activated NF-κB in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results:Addition of TPCK to the cells before AngⅡ stimulation attenuated the response of NF-κB p65 nuclear translocation induced by AngⅡ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%(P < 0.05) and 41.1%(P < 0.05) respectively, when compared with AngⅡgroup. In accordance, the ABCA1 mRNA and protein were increased by 30% and 19%(P < 0.05) respectively, when compared with AngⅡ group. Conclusion:AngⅡ can down-regulate ABCA1 in THP-1 derived-foam cells via NF-κB, which leads to less cholesterol efflux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.     

Inhibiting NF-κB increases cholesterol efflux from THP-1 derived-foam cells treated with AngⅡ via up-regulating the expression of ATP-binding cassette transporter A1

Objective: To study the role of nuclear factor-kappa B(NF-κB) in cholesterol efflux from THP-1 derived-foam cells treated with AngiotensinⅡ(AngⅡ). Methods:Cultured THP-1 derived-foam cells were treated with AngⅡ or preincubated with tosyl-phenylalanine chloromethyl-ketone(TPCK) NF-κB inhibitor. The levels of activated NF-κB in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results:Addition of TPCK to the cells before AngⅡ stimulation attenuated the response of NF-κB p65 nuclear translocation induced by AngⅡ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%(P < 0.05) and 41.1%(P < 0.05) respectively,when compared with AngⅡgroup. In accordance,the ABCA1 mRNA and protein were increased by 30% and 19%(P < 0.05) respectively,when compared with AngⅡ group. Conclusion:AngⅡ can down-regulate ABCA1 in THP-1 derived-foam cells via NF-κB,which leads to less cholesterol efflux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.     

Blockage of PPARδ increases the expression of inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods :Lentivirus-mediated RNA interference (RNAi) was used to block the expression of PPARS in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α(TNFα, 20 ng/ml) for 4 h. The expression of PPAR8, nuclear factor κB (NFκB) and C reactive protein (CRP) were determined by Western blot analysis. Results:The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα, but had no effect on normal cells. Conclusion: PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.     

EFFECT OF OXIDIZED-LDL ON NF-κB NUCLEAR TRANSLOCATION IN AORTIC SMOOTH MUSCLE CELLS ORIGINATED FROM RATS OF DIFFERENT AGES

Objective To investigate the molecular mechanism of atherosclerosis that related to age. Methods Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-κB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells(SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL high density lipoprotein(HDL) originated fi‘om rats of 2 and l0 months old respectively. Fat stain was used to identify the lipid intake in SMCs. Results The optimal stimulation time ofox-LDL to SMCs was 12 hours. NF-KB intensity increased in most nuclei of SMCs that originated fi‘om rats of either 2 or l0 months old co-cultured with ox-LDL. The intensity of NF-KB and the amount of intracellular lipid taken in SMCs were more obvious in cells fi‘om 10-month-old rats than fi‘om the younger ones.Change of PDGF-B expression in SMCs was not remarkable in each group of rats. Conclusions The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear translocation of NF-KB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-κB.     

RIP3在necroptosis信号通路中的胞内定位

目的 研究necroptosis信号通路中受体相互作用蛋白3（RIP3）的胞内定位.方法 SD大鼠原代皮质神经元细胞培养12天,zVAD 20 μmol/L预处理30 min,TNFα 30 ng/ml处理不同时间,Western blot检测胞质、胞核中RIP3蛋白水平,结合免疫荧光观察RIP3的胞内定位.结果 随着TNFα作用时间的延长,胞质、胞核中RIP3蛋白水平均呈上升趋势,胞质中RIP3蛋白水平在8 h达高峰,随后下降,胞核中RIP3蛋白水平在12 h达高峰,即在12 h出现核转位的高峰（P＜0.05）.结论 正常细胞中RIP3存在于胞质,necroptosis时发生核转位.     

受体相互作用蛋白家族在炎症中的作用研究进展

受体相互作用蛋白（RIP）家族是一组苏氨酸/丝氨酸蛋白激酶,具有相对保守的激酶结构域和不同的非激酶结构域,参与固有免疫应答、炎症等生理和病理过程。近年来研究表明,RIP家族通过参与坏死复合物的形成介导细胞坏死、触发炎症反应,其中RIP1和RIP3与细胞坏死的关系尤为密切。细胞坏死是一种精密调控的细胞死亡方式。通过TNF信号通路和Toll样受体信号通路传递的死亡信号可招募并磷酸化混合谱系激酶结构域蛋白,最终导致细胞崩解死亡,而细胞崩解后释放的胞内物质可触发炎症反应。本文着重阐述RIP家族介导细胞坏死和炎症发生所涉及的主要信号通路及分子机制,简述了RIP家族在相关炎症性疾病中的重要作用,并对RIP作为炎症性疾病治疗靶点的可能性进行了展望。     

蟾蜍灵通过程序性坏死途径诱导人胃癌细胞SGC-7901死亡

目的 探讨蟾蜍灵（Bufalin）诱导人胃癌SGC-7901细胞死亡过程中程序性坏死途径的发生机制。方法 体外培养SGC-7901细胞,于对照组加入RPMI 1640培养液,实验组加入不同浓度的蟾蜍灵（50、100、150、200 nmol/L）,对照组与实验组细胞继续培养48 h。MTT法检测细胞活性,DAPI染色法观察细胞核形态改变,透射电镜观察细胞膜及细胞核改变,流式细胞术检测细胞坏死率,Western blotting法检测程序性坏死关键蛋白受体相互作用蛋白激酶1（receptor interacting protein kinase 1,RIP1）的表达。结果 MTT实验结果表明,蟾蜍灵对SGC-7901细胞的生长具有显著的抑制作用（P<0.05）。通过透射电镜可以观察到细胞坏死时的特征性改变,如细胞膜破裂、细胞内空泡的产生以及细胞器的崩解。DAPI染色显示,蟾蜍灵（100 nmol/L）处理细胞48 h后,无明显细胞凋亡特征性改变,如细胞核固缩、核碎裂、凋亡小体形成。与对照组相比细胞坏死率升高（P<0.05）。蟾蜍灵对程序性坏死途径关键蛋白RIP1的表达有促进作用（P<0.05）。结论 蟾蜍灵通过程序性坏死途径诱导SGC-7901细胞死亡。     

RIP1在急性胰腺炎胰腺腺泡细胞凋亡中的作用探讨

目的：探讨细胞凋亡在急性胰腺炎炎症中的作用及受体相互作用蛋白（RIP ）的调控作用。方法将30只C57小鼠分为3组：对照组、急性水肿性胰腺炎（AEP）组、急性坏死性胰腺炎（ANP）组。AEP组连续注射雨蛙素（50μg／kg）13次;ANP组连续注射雨蛙素（50μg／kg）13次,另注射1次脂多糖（15 mg／kg）;对照组注射等量生理盐水7次。末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记法观察胰腺腺泡细胞凋亡,实时荧光定量PCR法检测RIP1 mRNA的表达,蛋白质免疫印迹法检测RIP1蛋白的表达。结果成功建立AEP及ANP小鼠模型。与对照组比较,2个胰腺炎模型组均存在细胞凋亡,且与AEP组比较,ANP组小鼠细胞凋亡减少,差异有统计学意义（P＜0．05）。与对照组比较,AEP组RIP1 mRNA及蛋白表达均升高,而ANP组降低,差异均有统计学意义（ P＜0．05）。结论 RIP1参与急性胰腺炎的发病过程,可能与调控腺泡细胞凋亡有关。     

细胞程序性坏死在急性肺损伤中作用的研究进展

 程序性坏死作为一种新的细胞程序性死亡方式参与急性肺损伤（acute lung injury,ALI）的病理过程,主要在疾病前期由各类高危因素触发,由受体相互作用蛋白激酶1（receptor-interacting protein kinase 1,RIPK1）、RIPK3、混合谱系激酶结构域样蛋白（mixed-lineage kinase domain-like protein,MLKL）介导,导致肺泡上皮细胞、血管内皮细胞、肺泡巨噬细胞等细胞死亡,直接或通过调节机体炎症反应造成严重的肺泡结构损伤及肺水肿,应用抑制剂阻断程序性坏死可以减轻肺损伤。本文就程序性坏死在ALI中的作用进行综述,以期为临床筛选高危患者和治疗提供依据。     

早期胰岛素治疗对糖尿病大鼠肝脏固醇调节级联反应和脂肪沉积的影响

目的 探讨早期胰岛素治疗对2型糖尿病大鼠肝脏固醇调节级联反应和脂肪沉积的影响.方法 高脂饮食联合小剂量链脲佐菌素诱导成2型糖尿病大鼠模型,分为糖尿病组(DM)和早期胰岛素治疗组(INS).INS组大鼠给予低精蛋白锌人胰岛素治疗3周.Western印迹检测肝脏免疫球蛋白结合蛋白(Bip)、胰岛素诱导基因1(Insig1)、氧调节蛋白150(ORP150)、胞质同醇调节因子结合蛋白1(SREBP1)和核SREBP1(nSREBP1)蛋白表达.结果 与正常大鼠比较,DM组Bip和ORP150蛋白表达增加(Bip:0.67±0.02比0.43±0.01;ORP150:1.83±0.03比1.04±0.03,均P<0.05),Insig1蛋白表达下调(0.25±0.02比0.80±0.07,P<0.05),SREBP1和nSREBP1蛋白表达均增加(SREBP1∶1.03±0.14比0.41±0.01;nSREBP1:3.63±0.77比0.96±0.20,均P<0.05).胰岛素治疗后Bip和ORP150蛋白表达减少(Bip:0.41±0.04比0.67±0.02;ORP150:1.11 ±0.04比1.83±0.03,均P<0.05),Insig1蛋白表达上调(0.43 ±0.02比0.25±0.02,P<0.05),SREBP1和nSREBP1蛋白表达均降低(SREBP1:0.46±0.01比1.03±0.14;nSREBP1:1.65±0.18比3.63±0.77,P<0.05).胰岛素治疗增加大鼠内脏脂肪重量(22.4 g±3.6 g比12.0 g±2.6 g,P<0.05).结论 早期胰岛素治疗诱导肝脏脂质向内脏脂肪组织重新分布,内质网应激减轻和SREBP1表达及其活性下调可能是胰岛素降低肝脏异位脂质沉积的分子机制之一.

Abstract：

Objective To explore the effect of early insulin therapy on sterol regulatory element binding protein 1 ( SREBP1) pathway and lipid accumulation in liver of type 2 diabetic rats ( DM ). Methods A high-fat diet plus a low-dose of streptozotocin (STZ) was administered to the Sprague-Dawley (SD) rats to create a type 2 diabetic animal model. Then the rats were divided into 3 groups: normal control ( NC) , DM (untreated diabetic rats) and INS (a 3-week treatment of NPH insulin initiated from day 3 of STZ injection). Insulin was delivered daily by a 3-week subcutaneous injection (6-8 U/day) . Liver homogenate was prepared. The protein levels of ER stress marker immunoglobulin binding protein ( Bip),oxygen-regulated protein 150 (ORP150) , insulin-induced gene 1 (Insig1) , SREBP1 and nuclear SREBP1 (nSREBPl) were assayed by Western blot. Adipose tissue mass was measured. Results In the DM group,ER (endoplasmic reticulum) stress marker Bip and ORP150 were up-regulated (0.67 ±0.02 vs 0.43 ±0. 01 for Bip; 1. 11 ±0. 04 vs 1. 83 ±0. 03 for ORP150, P <0. 05 for both) and Insigl decreased (0. 25 ±0. 02 vs 0. 80 ± 0. 07, P < 0. 05). And the expressions of SREBP1 and nSREBPl were elevated (1. 03 ±0. 14 vs 0. 41 ± 0.01 for SREBP1; 3. 63 ± 0. 77 vs 0. 96 ± 0. 20 for nSREBPl, P < 0. 05 for both) in comparison with the normal control rats. In the INS group, all aforementioned changes became attenuated or reversed (0.41 ±0.04 vs 0. 67 ±0. 02 for Bip; 1. 83 ±0. 03 vs 1. 11 ±0. 04 for ORP150; 0. 43 ±0.02 vs 0. 25 ± 0. 02 for Insigl; 0. 46 ± 0.01 vs 1. 03 ± 0. 14 for SREBP1; 1.65 ± 0. 18 vs 3. 63 ± 0. 77 for nSREBP1, P <0.05 for all). Furthermore, adipose tissue mass increased (22. 4 g ±3. 6g vs 12.0 g ±2. 6 g, P<0. 05). Conclusion The early insulin therapy induces a fat redistribution from liver to adipose tissue. The mechanism is probably through a reduction of ER stress and a down-regulated pathway of SREBP1 in liver of diabetic rats.     

程序性坏死：缺血再灌注损伤中的新靶点

程序性坏死是新发现的一种细胞死亡方式,在凋亡通路抑制条件下,死亡受体与配体结合可调控非半胱氨酸天冬氨酸蛋白酶依赖的程序性细胞死亡,具备典型的坏死样形态学特征,可触发显著的炎症反应。含死亡域的受体相互作用蛋白激酶1/3在程序性坏死的调节中发挥特异性激酶作用,且可被坏死性抑制剂特异性抑制。程序性坏死在缺血再灌注损伤中的重要作用备受关注,研究已发现其在心、肾、脑组织及视网膜等缺血再灌注损伤中可作为细胞死亡的主要形式。     

NRIP3基因高表达抑制淋巴结阴性乳腺癌转移的研究

目的 探讨核受体相互作用蛋白(NRIP3)在乳腺癌组织中的表达及与淋巴结阴性乳腺癌无远处转移生存的关系。方法 利用BRB Array Tools软件分析165例乳腺癌芯片中NRIP3基因表达水平,Kaplan-Meier法计算不同表达水平的无远处转移生存率,并进行Log-rank检验;应用Cox比例风险回归模型行多因素分析。结果 NRIP3基因低、中、高表达组中位无远处转移生存期（DMFS）分别为（3980±331）d、(4391±246)d、(4518±147)d,Log rank检验生存曲线差异有统计学意义（χ2=7.847,P<0.05）,多因素Cox模型分析显示,NRIP3基因高表达是乳腺癌远处转移独立的保护因素（P<0.01,Exp(B)= 0.387）。雌激素受体（ER）阳性患者中NRIP3高表达率为41.2%（42/102）,ER阴性患者中NRIP3高表达率为20.6%(13/63),差异有统计学意义（χ2=7.427,P<0.05）;ER阳性的乳腺癌患者中,低表达NRIP3基因组的DMFS为(4191±366)d,明显低于中表达组和高表达组（χ2=7.268,P＜0.05）,在ER阴性的乳腺癌患者中差异无统计学意义（χ2=0.524,P＞0.05）。结论 NRIP3基因高表达可抑制淋巴结阴性乳腺癌发生远处转移,其发挥作用与ER阳性表达以及可能的内分泌治疗关系值得进一步研究。     

甲基乙二醛对大鼠海马神经元的毒性作用及脑源性神经营养因子、TrKB表达的影响

目的 探讨葡萄糖降解产物甲基乙二醛(MG)对海马神经元的毒性作用及可能机制.方法取新生24h Sprague-Dawley大鼠海马神经元原代培养至第7天,给予MG干预24h,四甲基偶氮唑蓝(MTT)法检测海马神经元存活率,异硫氰酸荧光素标记的膜联蛋白V联合碘化丙啶(PI)法检测海马神经元的凋亡率,采用实时RT-PCR法及Western印迹法测定脑源性神经营养因子(BDNF)及其受体酪氨酸蛋白激酶(TrkB)mRNA和蛋白表达水平.结果 1.随着MG浓度的增加及干预时间延长,海马神经元存活率逐渐降低,呈浓度依赖性(r=0.946,P<0.01)和时间依赖性(r=0.993,P<0.01).与0h组海马神经元凋亡率(1.633±0.153)%相比,100μM MG干预2h、6h、12h和24h后,其凋亡率逐渐增加[分别为(2.833±0.153)%、(3.367±0.153)%、(4.433±0.404)%和(8.833±0.306)%;均P<0.01];2.与同时间对照组相比,MG干预12h组及24h组海马神经元BDNF mRNA及蛋白表达增加(P<0.05或P<0.01);而MG干预6h组、12 h及24h组TrkB mRNA及蛋白表达减少(P<0.05或P<0.01).结论 MG对海马神经元具有直接毒性作用,可能首先抑制海马神经元TrKB表达,导致BDNF表达代偿性增高,损伤BDNF-TrKB通路,诱导神经元凋亡增加.