虎杖苷抑制肝星状细胞增殖活化的作用和机制研究

目的:研究虎杖苷 (Polydatin) 在体外对HSC-T6细胞的影响,探讨虎杖苷抑制肝纤维化发生的作用及相关机制。方法:体外培养大鼠肝星状细胞HSC-T6,分为对照组和虎杖苷低、中、高浓度（125、250、500 μmol/L）给药组。MTT法检测细胞增殖情况;比色法检测细胞培养上清液中乳酸脱氢酶（LDH）的活性;酶消化法检测细胞培养上清液中羟脯氨酸（Hyp）的含量;蛋白印迹法检测α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（CollagenⅠ）、核因子NF-E2相关因子2（Nrf2）和血红素加氧酶-1（HO-1）蛋白表达水平。多组间比较采用单因素方差分析,若方差齐则采用LSD检验,若方差不齐采用Dunnett T3检验,以P<0.05认为差异有统计学意义。结果:与对照组比较,虎杖苷125、250、500 μmol/L给药组细胞活力显著降低（F=252.01, P<0.001）,Hyp含量明显减少（F=16.50,P<0.05）,α-SMA和CollagenⅠ蛋白表达显著减少（F=19.89、182.91, 均P<0.05）,且随着浓度的增加,抑制作用越强,呈一定的剂量依赖性;与对照组比较,虎杖苷125、250、500 μmol/L给药组Nrf2蛋白表达显著增加（F=23.70,P<0.05）,虎杖苷250、500 μmol/L给药组HO-1蛋白表达显著增加（F=12.40,P<0.01）,且随着浓度增加,上调作用越明显,呈一定的剂量依赖性。结论:虎杖苷通过抑制肝星状细胞增殖活化及胶原合成发挥抑制肝纤维化发生的作用,该作用可能与其上调Nrf2/HO-1通路有关。     

雷帕霉素诱导大鼠肝星状细胞凋亡的研究

目的观察雷帕霉素(RAPA)对大鼠肝星状细胞系rHSCs-99增殖、凋亡的影响。方法将rHSCs-99分成4组:对照组(A组),RAPA 50 nmol/L组(B组),RAPA 100 nmol/L组(C组),RAPA 150 nmol/L组(D组)。RAPA作用72 h后,分别用MTT法检测rHSCs-99增殖,ELISA法检测I型胶原表达,流式细胞仪Annexin-V FICT/PI法检测细胞凋亡情况,Wright-Giemsa染色法观察细胞形态学改变,细胞免疫组化法检测Fas、P53与Bcl-2的表达变化。结果 RAPA作用后,rHSCs-99增殖受到抑制,I型胶原含量降低,凋亡率增高,Fas、P53表达增多,Bcl-2表达减少。结论 RAPA能抑制rHSCs-99的增殖及I型胶原合成。促进rHSCs-99凋亡,其机制可能是通过开启Fas/FasL凋亡途径及上调凋亡相关基因P53、下调Bcl-2的表达来实现。     

肝复康药物血清对肝星状细胞核因子-κB及I、Ⅲ型胶原表达的影响

目的:通过观察中药肝复康中剂量浓度含药血清对肝星状细胞T6(hepatic stellate cell-T6,HSC-T6)细胞株中核因子-κB(NF-κB)、Ⅰ型胶原(TypeⅠCollagen,ColⅠ)及Ⅲ型胶原(TypeⅢCollagen,ColⅢ)基因表达的影响,探讨其抗纤维化的作用及分子机制。方法:常规方法制备肝复康中剂量浓度含药血清,并用肝复康含药血清干预,以逆转录聚合酶链反应(RT-PCR)和Western-Blot方法观察分析NF-κB、ColⅠ和ColⅢ表达的变化并探讨其相关性。结果:经乙醛刺激后的HSC-T6细胞株NF-κB、ColⅠ和ColⅢ表达均上调,肝复康中剂量浓度含药血清处理后可明显下调上述基因的表达。结论:中药肝复康抗纤维化的机制可能与其抑制NF-κB及ColⅠ、ColⅢ的表达有关。     

龙血素B对肝星状细胞增殖及细胞外基质分泌的影响

目的观察龙血素B对大鼠肝星状细胞(HSC)增殖及细胞外基质分泌的影响。方法用不同浓度的龙血素B处理大鼠HSC;MTT法计算药物的抑制率;放射免疫法测定细胞上清中透明质酸(HA)、层粘连蛋白(LN)和Ⅳ型胶原(Ⅳ-C)。结果随药物浓度的升高,龙血素B对HSC-T6增殖的抑制作用逐渐增强,有明显的量效关系。细胞上清中HA、LN和Ⅳ-C的含量与对照组相比降低,其中龙血素B浓度为0.300μg/μL时降低最明显。结论龙血素B可抑制HSC-T6增殖,并不同程度抑制HA、LN和Ⅳ-C的分泌。     

Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6

AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A recepto (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro . METHODS: HSCs were derived from the livers of adul male Sprague-Dawley rats. IL-6 expression was evalu ated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated pro tein kinases (MAPK) and extracellular regulated pro tein kinases (ERK) 1/2 upon induction by IL-17A and suppression by IL-17RA shRNA were examined using Western blotting.RESULTS: IL-6 expression induced by IL-17A was significantly increased compared to control in HSCs (P 0.01 in a dose-dependent manner). Suppression of IL17RA using lentiviral-mediated shRNA inhibited IL-6 expression induced by IL-17A compared to group with only IL-17A treatment (1.44 ± 0.17 vs 4.07 ± 0.43, P 0.01). IL-17A induced rapid phosphorylation of p38 MAPK and ERK1/2 after 5 min exposure, and showed the strongest levels of phosphorylation of p38 MAPK and ERK1/2 at 15 min in IL-17A-treated HSCs. IL-6 mRNA expression induced by IL-17A (100 ng/mL) for 3 h exposure was inhibited by preincubation with specific inhibitors of p38 MAPK (SB-203580) and ERK1/2 (PD-98059) compared to groups without inhibitors preincubation (1.67 ± 0.24, 2.01 ± 0.10 vs 4.08 ± 0.59, P 0.01). Moreover, lentiviral-mediated IL-17RA shRNA 1 inhibited IL-17A-induced IL-6 mRNA expression compared to random shRNA in HSCs (1.44 ± 0.17 vs 3.98 ± 0.68, P 0.01). Lentiviral-mediated IL17RA shRNA 1 inhibited phosphorylation of p38 MAPK and ERK1/2 induced by 15 min IL-17A (100 ng/mL) exposure. CONCLUSION: Down-regulation of the IL-17RA receptor by shRNA decreased IL-6 expression induced by IL-17A via p38 MAPK and ERK1/2 phosphorylation in HSCs. Suppression of IL-17RA expression may be a strategy to reduce the inflammatory response induced by IL-17A in the liver.     

肝细胞中HBV X基因的表达及其共培养的肝星状细胞的增殖和迁移

目的:研究乙型肝炎病毒X(hepatitis B virus X,HBV X)基因在乙型肝炎病毒致肝纤维化中的作用.方法:构建HBV X基因真核表达载体pHBV-X-IRES2-EGFP,将其转染人肝细胞HL-7702后分成2组,一组经G418筛选出稳定表达HBVX基因的肝细胞株(L02/x),另一组予瞬时转染48 h(L02/48x).Real-time PCR、Western blot鉴定2组细胞HBV X基因的表达.与转染空质粒和未转染的肝细胞组对照,将L02/x和L02/48x细胞分别与肝星状细胞(hepatic stellate cells,HSCs)共培养36 h,并检测各组HSCs增殖和迁移情况.结果:Real-time PCR和Western blot实验显示,转染pHBV-X-IRES2-EGFP载体的L02/x和L02/48x细胞均有HBV X基因的表达.与转染空质粒和未转染的肝细胞组对照,与L02/x和L02/48x细胞共培养的HSCs的增殖和迁移均显著增多.结论:HBV X基因在肝细胞中的表达可以促进HSCs发生增殖和迁移,从而在乙型肝炎病毒致肝纤维化过程中起重要作用.     

白藜芦醇抗肝星状细胞活性和抗肝纤维化的实验研究

目的 探讨白藜芦醇调节肝星状细胞(hepatic stellate cells,HSCs)活性及其抗肝纤维化作用.方法 从大鼠肝脏分离纯化培养HSCs;DCFH-DA法检测不同浓度白藜芦醇对HSCs中活性氧的影响;通过CCK-8比色法检测白藜芦醇对HSCs增殖的影响.Western blot检测HSCs的α-肌动蛋白(α-SMA)表达.通过PCR检测白藜芦醇对HSCs活性相关基因表达的影响.给大鼠肝纤维化模型经腹输注白藜芦醇,检测肝组织病理切片,肝纤维化指标.结果 从大鼠活体分离培养HSCs.白藜芦醇可抑制HSCs中活性氧的产生;明显抑制HSCs的α-SMA表达(103 ±7,90 ±7,63 ±4,53 ±3,F=62.179,P ＜0.05)与增殖(0.536±0.052,0.411±0.047,0.327±0.063,0.312±0.032,F=12.776,P＜0.05);抑制HSCs活性相关基因(大鼠生肌调节因子、胶原蛋白Ⅲ及胶原蛋白Ⅰ)的表达(122 ±.5,96±3,68 ±3,60 ±3,F=180.600,P ＜0.05) (100 ±8,82±3,53±3,51 ±2,F =77.451,P＜0.05) (170±3,147±4,92 ±3,90 ±2,F=462.878,P＜0.05).大鼠活体实验显示白藜芦醇可降低肝羟脯氨酸含量及血清胶原蛋白Ⅲ和透明质酸水平(358.3 ±20.2,320.5±15.3,290.3±24.5,F=23.929,P ＜0.05) (32.8±3.1,28.9±1.3,25.3±1.8,F=20.050,P＜0.05)(276.3±17.8,225.3±28.3,195.4±11.2,F=18.585,P＜0.05).结论 白藜芦醇能够抑制大鼠HSCs的活化增殖,对活体肝纤维化具有一定的抑制作用,这可能与白藜芦醇的抗氧化及抑制MyoD的表达作用有关.     

Effects of ticagrelor pretreatment on electrophysiological properties of stellate ganglion neurons following myocardial infarction

Higher sympathetic activity predisposes to malignant ventricular arrhythmias in the context of myocardial infarction (MI). This is, in part, mediated by the electrical activity of the stellate ganglion (SG). The aim of this study is to examine the effects of ticagrelor pretreatment on the electrophysiological properties of SG neurons following MI in rabbits. MI was induced by isoproterenol (ISO) of 150 mg kg^-1 d^-1 (twice at an interval of 24 hours). Ticagrelor pretreatment was administered at low- (10 mg kg^-1 d^-1) or high-dose (20 mg kg^-1 d^-1). Protein and RNA expression were determined by immunohistochemical analysis and real-time PCR, respectively. The activity of sodium channel current (I_Na), delayed rectifier potassium current (I_KDR), M-type potassium current (I_KM) as well as action potentials (APs) from SG neurons were measured by whole-cell patch-clamp. Intracellular calcium concentrations were measured by confocal microscopy. Compared with the control group, the MI group exhibited a greater amplitude of I_Na, I_KDR and I_KM, significantly altered activation and inactivation characteristics of I_Na, no significant alterations in protein or mRNA expression of sodium and M-type potassium channels, along with higher AP amplitude and frequency and intracellular calcium concentrations. Most of these abnormalities were prevented by pretreatment with low- or high-dose ticagrelor. Our data suggest that ticagrelor exerts cardioprotective effects, potentially through modulating the activity of different ion channels in SG neurons.     

超声引导下间断给药星状神经节阻滞术效果和并发症

目的 对比以不同方式行星状神经节阻滞术(SGB)对于疗效及并发症的影响。方法 对127例患者分别于超声引导下间断给药(A组,n=45)、超声引导下连续给药(B组,n=42)及解剖定位盲法穿刺给药(C组,n=40)后行首次SGB,比较组间首次穿刺成功率、SGB成功率、药物跨越警戒点(颈总动脉与颈内静脉交汇处)者占比、霍纳综合征出现和维持时间差异及短期并发症情况;以二分类logistic回归模型分析药物跨越警戒点与声音嘶哑的关系。结果 A、B组首次穿刺成功率及SGB成功率均高于C组(P均<0.05)。B组药物跨越警戒点者占比高于A组(P<0.05)。A、B组出现霍纳综合征时间均早于C组(P均<0.05);A组霍纳综合征维持时间最长,B组次之,C组最短,两两比较差异均有统计学意义(P均<0.05)。术后A组2例、B组8例、 C组12例声音嘶哑,B、C组声音嘶哑发生率明显高于A组(P均<0.05);A组2例、B组2例、C组7例穿刺点疼痛;C组4例食管损伤,其穿刺点疼痛及食管损伤发生率均高于A、B组(P均<0.05)。结论 超声引导下间断给药法SGB较超声引导下连续给药及解剖定位盲法穿刺给药成功率高,维持时间长且并发症少。