粉防己碱抗氯化铯诱发家兔在体心脏早后除极及心律失常的作用

目的 :研究粉防己碱 (Tet)抗氯化铯 (CsCl)诱发触发活动及触发性心律失常作用。方法 :14只家兔分对照组和Tet组 ,Tet组给予Tet 0 5mg kg min共 10min ,对照组给予生理盐水 ,然后给予CsCl1 5mmol kg静注 ,给药前后记录心电图和右室心内膜MAP。结果 :Tet组和对照组均诱发EAD ,但Tet组EAD幅度低于对照组 ,室性心律失常发生率低于对照组。结论 :Tet有抗CsCl诱发EAD及室性心律失常作用。其机制可能是Tet抑制慢钙通道 ,减少内向电流。     

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.     

Tetrandrine stimulates the apoptosis of hepatic stellate cells and ameliorates development of fibrosis in a thioacetamide rat model

AIM To investigate the therapeutic effect of tetrandrine on liver fibrosis induced by thioacetamide in rats in vivo and in vitro.METHODS In vitro study we investigated the effect of tetrandrine on the apoptosis of rat hepatic stellate cells transformed by simian virus 40 (T-HSC/Cl-6), which retains the features of activated cells. In vivo studyhepatic fibrosis was induced in rats by thioacetamide.Tetrandrine was given orally to rats at doses of 5, 10 or 20 mg/kg for 4 wk compared with intraperitoneal injection of interferon-r.RESULTS In vitro study 5, 10 or 25 μg/mL of tetrandrine-induced activation of caspase-3 in t-HSC/Cl-6 cells occurred dose-depenclently. In vivo study tetrandrine treatment as well as interferon-r significantly ameliorated the development of fibrosis as determined by lowered serum levels of aspartate aminotransferase (AST),alanine aminotransferase (ALT), total bilirubin (T-Bil)and the levels of liver hydroxyproline (Hyp), hyaluronic acid (HA), laminin (LN) and also improved histological findings. The effects of tetrandrine at the concentration of 20 mg/kg were better than the other concentration groups.CONCLUSION Tetrandrine promotes the apoptosis of activated HSCs in vitro. Tetrandrine administration can prevent liver fibrosis and liver damage induced by thioacetamide in rats in vivo, indicating that it might exert a direct effect on rat HSCs.     

汉防己甲素联合顺铂对 NCI-H446细胞的影响及相关机制分析

目的：研究汉防己甲素(tetrandrine,TET)联合顺铂(cisplatin,DDP)对人小细胞肺癌细胞株 NCI-H446体外抗肿瘤效果,探讨 TET 增强 NCI-H446细胞对 DDP 敏感性的可能机制。方法体外培养人小细胞肺癌株 NCI-H446;药物作用48 h 后,采用 CCK-8法测定各组细胞活力;Edu 染色观察各组细胞增殖情况;Hoechst 染色检测各组细胞凋亡情况;Western blot 检测各组磷酸化Akt、Bax、Bcl-2、cleaved-caspase-3、LC3等蛋白表达水平变化。结果低浓度的 TET 与 DDP 联合后可有效抑制 NCI-H446细胞株的增殖,诱导其凋亡;联合组半数抑制浓度较单药组显著降低;联合组磷酸化 Akt 表达显著下降,Bax、cleaved-caspase-3表达显著提高,抗凋亡蛋白 Bcl-2表达下降;自噬相关蛋白 LC3表达明显上调。结论低浓度 TET 与顺铂具有协同效应,其机制可能是通过上调促凋亡蛋白 Bax,下调抗凋亡蛋白 Bcl-2来诱导细胞凋亡;并且自噬可能在其中发挥重要作用。     

汉防己甲素抑制矽肺胶原mRNA表达的研究

在制备Ｐｒｏα１（Ｉ），Ｐｒｏα１（ＩＩＩ）胶原ｃＤＮＡ探针的基础上，采用斑点及原位分子杂交技术，观察汉防己甲素对矽组织中Ｉ，ＩＩＩ型胶原基因的ｍＲＮＡ的影响。结果表明染尘２，４个月的矽肺组织中Ｉ，ＩＩＩ型胶原ｍＲＮＡ的含量比正常肺组织明显增加（Ｐ＜０．０５），经汉防己甲素治疗后增高的Ｉ，ＩＩＩ型ｍＲＮＡ含量显著减少（Ｐ＜０．０５）。原位杂交显示，正常肺组织杂交颗粒主要分布于肺泡间隔的成纤维细胞中     

粉防己碱对实验性胸膜炎的防治作用的研究

角叉菜胶（Ｃａｒｒａｇｅｅｎｉｎ，Ｃａｒ）致大鼠胸膜炎后８ｈ，胸膜腔渗出液中中性白细胞游离钙浓度（Ｎｅｕ［Ｃａ＾２＾＋］ｉ）升高，中性白细胞钙调素活性（Ｎｅｕ－ＣａＭＡ）增强，粉防己碱（Ｔｅｔｒａｎｄｒｉｎｅ，Ｔｅｔ）（１０－８０ｍｇ．ｋｇ＾－＾１）于致炎前３０ｍｉｎ和致炎后４ｈ给大鼠灌胃可明显减少炎症胸膜腔渗出液量，蛋白渗出量和白细胞游出数，同时能降低Ｎｅｕ［Ｃａ＾２＾＋］ｉ和Ｎｅｕ－ＣａＭＡ；     

粉防己碱对胎鼠大脑细胞游离钙含量的影响

结果表明,粉防己碱(Tet)非常明显地阻断K+去极化导致的胎鼠大脑[Ca²⁺]i含量的增加。经不同浓度Tet处理的大脑细胞加入KCl后,[Ca²⁺]i含量仍基本维持在静息状态下水平。对[Ca²⁺]i的阻断程度与Tet浓度无关。Tet对L-谷氨酸(L-Glu)所致大脑[Ca²⁺]i升高亦有明显抑制作用。10^-7mol·L^-1浓度的Tet还降低BayK8644引起的[Ca²⁺]i上升高。Tet能抑制高K⁺,二氢吡啶类钙激动剂及兴奋性神经递质导致的胎鼠大脑[Ca²⁺]i含量增加,提示Tet对神经细胞损伤可能有一定保护作用。     

Protective Effect of Tetrandrine and Fructose-1,6-diphos phate on the Model of Focal Cerebral Ischemia in Rats

以大鼠大脑中动脉阻塞２４小时后的梗塞面积和体积为指标，试验了汉防己碱和１，６┐二磷酸果糖对大鼠局部脑缺血的保护作用。在大脑中动脉阻塞后，汉防己碱７．５，１２．０和１５．０ｍｇ·ｋｇ┐１以及１，６┐二磷酸果糖２００和３５０ｍｇ·ｋｇ┐１分别立即腹腔给药，以剂量依赖方式明显减少大鼠脑的梗塞面积和体积。ＭＫ８０１２．０ｍｇ·ｋｇ┐１亦能减少梗塞面积和体积。汉防己碱和１，６┐二磷酸果糖联合用药所产生的保护作用比任何一药物单用时的保护作用都好，提示汉防己碱和１，６┐二磷酸果糖有协同作用。在大脑中动脉阻塞后１小时和２小时给药，汉防己碱和１，６┐二磷酸果糖仍有脑缺血保护作用，但在梗塞后３小时给药则未见任何保护作用。提示中风或脑缺血后，既非５分钟即不可救药，但亦应尽早用药。实验结果表明汉防己碱和１，６┐二磷酸果糖可能是有希望的脑缺血保护剂。     

汉防己甲素防护环孢素A肾毒性的实验研究

本文观察了中药钙拮抗剂汉防己甲素对大鼠环孢素A肾毒性（CsA－NT）的防护作用。雄性Wistar大鼠用CsA50mg·kg－1／d灌胃7天导致急性CsA－NT，表现为血清肌酐（Cr）升高34．8％，内生肌酐清除率（Ccr）下降39．4％，组织学检查发现近曲小管大片空泡变性。同时，肾皮质丙二醛（MDA）含量显著升高，超氧化物歧化酶（SOD）活性显著下降。大鼠每日用CsA灌胃前30分钟，给予汉防己甲素10mg／kg腹腔注射，能使上述指标明显改善，组织学损伤也明显减轻。实验结果提示汉防己甲素能从功能和形态上防护大鼠急性CsA－NT。     

内皮素对瘢痕成纤维细胞增殖和胶原合成作用的实验研究

目的：探讨内皮素（ET）在瘢痕成纤维细胞增殖与胶原合成中的作用及一氧化氮（NO）、粉防已碱（Tet）的拮抗效应。方法：体外培养人增生性瘢痕成纤维细胞，以^3H-TdR掺入法测定细胞增殖；以^3H-脯氨酸掺入量判断细胞胶原合成。结果：ET呈浓度依赖性促进瘢痕成纤维细胞增殖与胶原合成，随着ET浓度（2．5－100ng/ml）的增加，^3H-TdR掺入值较对照组分别增加了1．8、4．0和4．9倍；^3H－脯氨酸掺入值较对照组分别增加了1．1、3．1和3．8倍（P＜0．01）。用NO供体亚硝基乙酰青霉胺（S-nitroso-N-acetyl penicillamine,SNAP)50μg/ml单独处理成纤维细胞，对^3H-TdR掺入值无明显影响（与对照组相比，P＞0．05），但对^3H-脯氨酸掺入值有下调作用；SNAP可拮抗ET－1（25ng/ml）刺激细胞增殖作用（抑制率为22．89％，P＜0．05）；对ET－1的促胶原合成作用无明显影响（P＞0．05）。Tet在不抑制细胞DNA合成的药物浓度（3μg/ml）即可明显减少瘢痕成纤维细胞^3H-脯氨酸掺入值，并能显著降低由ET介导的瘢痕成纤维细胞^3H-TdR掺入值（抑制率为33．21％，P＜0．01）。结论：ET对体外培养的瘢痕成纤维细胞增殖与胶原合成具有显著促进作用，此效应可部分被SNAP、Tet所阻抗。