雷公藤内酯醇对大鼠生殖内分泌及双氢睾丸酮受体的影响

雄性Sprague-Dawley大鼠口服雷公藤内酯醇（75μg/kg/d）共35天，服药28天后，交配实验表明，所有给药大鼠均已产生抗生育作用。给药35天后，给药组动物体重、睾丸、精囊、腹前列腺及垂体前叶的重量，血清及睾丸间质液中的睾酮浓度；垂体促间质细胞激素（LH）、促卵泡激素（FSH）浓度以及睾丸内FSH受体的浓度与对照组相比均无改变；附睾中精子密度及附睾重量明显降低，精子均不活动。在光镜和相差显微镜下观察给药大鼠睾丸生精细胞及附睾上皮细胞没有损伤。然而，在附睾头、尾管腔中的精子分别呈现中度和严重的损伤。如精子头尾断裂，轴丝膨大。给药大鼠附睾头、尾的胸浆及胞核，双氢睾酮受体（DHT）的含量增高，但无显著的统计学意义。腹前列腺胞核受体也无显著差异。而腹前列腺胞浆DHT受体与对照组相比有显著升高（P＜0.01）。实验结果提示雷公藤内酯醇的抗生育作用部位之一在附睾。     

雷公藤甲素的动力学研究

经小鼠、大鼠ig和iv高、中、低三种剂量的动力学研究结果表明:ig后的药一时曲线为开放二室模型;iv为开放三室模型。小鼠的胃肠吸收较大鼠快,T peak分别为0.687、1.037h,体内消除较缓慢,在高剂量下可见AUC增大、C1减少及t1/2β延长,提示临床上在高剂量应用时可能出现的非线性动力学性质。     

雷公藤甲素对KA损伤大鼠星形胶质细胞的影响

目的:观察海人藻酸(Kainic acid,KA)海马内注射后星形胶质细胞的变化及雷公藤甲素(TRP)对其的影响。方法:90只SD大鼠(200～220g)随机分为3组:右侧海马注射生理盐水后生理盐水灌胃作为对照组(NS NS),右侧海马注射海人藻酸后生理盐水灌胃干预组(KA NS),右侧海马注射海人藻酸后雷公藤甲素灌胃干预组(KA TRP)。动物存活1天,3天,5天,7天,14天后免疫组织化学结合图像分析技术观察海马内星形胶质细胞形态和数目的变化。结果:(KA NS)组海马内星形胶质细胞数目明显增多,胞体明显增大,突起变短,变粗,与(NS NS)组相比差别具有显著性(p<0.05)(;KA TRP)组星形胶质细胞数量明显减少,胞体变小,突起变细长,与(KA NS)组相比差别具有显著性(P<0.05)。结论:KA注射后可导致大鼠海马内星形胶质细胞的激活,雷公藤甲素对KA诱导的星形胶质细胞的活化有抑制作用。     

血清中雷公藤甲素的液相色谱/质谱联用法测定研究

目的:建立一种新型、灵敏、准确的人血清中雷公藤甲素的液相色谱/质谱联用测定方法。方法:血清经醋酸乙酯提取后,在Waters C18反相色谱柱(150 mm×3.9 mm i.d.,5μm)上,以醋酸-醋酸铵溶液(5 mmol/L)/乙腈/甲醇(60:30:10,v/v/v)为流动相,以泼尼松龙为内标,采用大气压化学电离(APCI)离子化方式在选择离子监测(SIM)模式下进行检测,定量检测离子为m/z[M-H]-359.1。结果:血清中雷公藤甲素测定的日内与日间精密度分别小于10.7%和11.3%,其在1.0～200.0 ng/ml范围具有良好的线性,检出限为1.0 ng/ml。结论:本方法可用于血清中痕量雷公藤甲素残留的测定。     

雷藤氯内酯醇的半合成研究——雷公藤内酯醇和雷公藤内酯酮的结构改造

通过HCl对雷公藤内酯醇(triptolide)的12-C进行选择性亲核进攻,将雷公藤植物中分离获得的雷公藤内酯醇(triptolide)和雷公藤内酯酮(triptonide)结构,改造为具有强抗炎、免疫抑制和雄性抗生育活性的雷藤氯内酯醇(tripchlorolide)。并通过二维NMR谱和选择性远程DFPT等图谱分析,归属了雷公藤内酯醇、雷公藤内酯酮和表雷公藤内酯醇(epitriptolide)NMR谱的全部碳和氢的信号。     

雷公藤内酯醇对人肾小管上皮细胞抗原呈递功能的影响

目的：观察雷公藤内酯醇对炎症因子刺激下人肾近端小管上皮细胞（HKC细胞）抗原呈递能力和共刺激分子表达的影响。方法：以IFN－γ（200μg/L)和TNF－α（20μg/L)联合刺激HKC细胞或同时加入不同浓度的雷公藤内酯醇,采用流式细胞仪检测细胞表面主要组织相容性二类复合体（MHC－Ⅱ）,共刺激分子B7－1,B7－2及细胞间粘附分子1（ICAM－1）表达情况,细胞内ICAM－1mRNA 表达量采用半定量逆转录PCR法检测。结果：普能培养时HKC细胞低度表达MHC－II分子,大量表达ICAM－1分子,不表达B7－1和B7－2分子,IFN－γ（200μg/L)和TNF－α（20μg/L)联合刺激24h后,小管细胞表面MHC－Ⅱ,ICAM－1,B7分子表达均显著增强,其中MHC－Ⅱ变化最大,RTPCR显示ICAM－1 mRNA表达也明显增加,雷公藤内酯醇可以剂理依赖性地抑制炎症因子引起的细胞MHC－Ⅱ,B7－1以及B7－2分子表达上调,但对刺激引起的ICAM－1蛋白及mRNA表达上调无明显影响。结论：雷公藤内酯醇可以抑制炎症因子刺激下人肾近端小管上皮细胞MHC－Ⅱ及B7分子表达上调,减弱肾小管上皮细胞作为抗原呈递细胞活化T细胞的能力。     

Membrane protein-chimeric liposome-mediated delivery of triptolide for targeted hepatocellular carcinoma therapy

Triptolide (TPL) is a diterpenoid triepoxide with broad antitumor efficacy, while lack of mechanism of action, severe systemic toxicity, and poor water solubility of TPL limited its usage. To unveil the mechanism of action and improve the pharmaceutical properties of TPL, here we explored the molecular mechanism of TPL and then fabricated TPL-loaded membrane protein-chimeric liposomes (TPL@MP-LP) and tested its anticancer efficacy against hepatocellular carcinoma (HCC). CCK8 assay, colony formation assay, EdU assay, and flow cytometry were used to examine the activity of TPL. RNA sequence and gain-and-loss of function assays were used to explore the molecular mechanisms. TPL@MP-LP was characterized by size, zeta potential, polydispersity index, and transmission electron microscopy. Cellular uptake and cell viability assay were performed to evaluate the internalization and anticancer efficacy of TPL@MP-LP in vitro. Biodistribution and in vivo antitumor efficacy of TPL@MP-LP were evaluated on orthotopic HCC mice models. TPL robustly inhibited HCC cells by inducing cell proliferation arrest, apoptosis via the mitochondrial pathway, and necroptosis via RIPK1/RIPK3/MLKL signaling. TPL was successfully loaded into MP-LP, with a drug-loading capacity of 5.62 ± 0.80%. MP-LP facilitated TPL internalization and TPL@MP-LP exerted enhanced anticancer efficacy against Huh7 cells. TPL@MP-LP showed targeting ability to the tumor site. More importantly, TPL@MP-LP treatment suppressed tumor growth but showed minimal damage to liver and renal functions. TPL exerted anticancer effects on HCC via inducing cell proliferation arrest, apoptosis, and necroptosis, and the MP-LP might be a promising delivery strategy to improve the antitumor efficacy while mitigating toxicity of TPL for HCC therapy.     

雷公藤内酯醇滴眼液防治角膜移植免疫排斥反应的实验研究

目的探讨雷公藤内酯醇(T10)滴眼液局部应用对角膜移植免疫排斥反应的影响.方法建立封闭群大鼠角膜移植模型,随机分组A、B、C组为SD-Wistar组间同种异体角膜移植,SD为受体,Wistar为供体,其中A组为空白对照组,B组为o.5mg@L1T10滴眼液组,C组为1mg@L-1T10滴眼液组,D组为SD-SD对照组,即SD大鼠间同种异体移植组和E组SD自体角膜移植对照组.用裂隙灯显微镜记录及比较各组角膜透明度、水肿度、新生血管度、移植排斥指数(RI)以及角膜排斥发生时间.结果术后各组角膜植片透明度、水肿度、新生血管程度以及发生角膜移植排斥时间,A组与C组比较有显著性差异(P＜0.05);D组和E组比较无显著性差异(P＞0.05).结论T10滴眼液可有效防治角膜移植免疫排斥反应.     

Effects of triptolide on proliferation and apoptosis in rat mesangial cells induced by transforming growth factor β1 and related mechanisms

Objective To investigate the effects of triptolide on proliferation, apoptosis and the changes of Ski, Smad3, Smad7 and collagen type Ι (ColΙ) in cultured rat mesangial cells induced by transforming growth factor (TGF)?β1. Methods Cultured HBZY?1 rat mesangial cells were divided into 5 groups: (1)normal control group; (2)TGF?β1 group (10 μg/L); (3)-(5)triptolide (0.4, 2, 10 μg/L)+TGF?β1 (10 μg/L) groups. The cell proliferation was detected by MTT. Apoptosis of mesangial cells was detected by TUNEL assay. The expressions of Ski, Smad3, Smad7 mRNA were examined by real?time quantitative PCR. The expressions of Ski, Smad3, Smad7 and ColΙ protein were detected by Western blotting. The localizations of Ski and Smad3 protein were detected by laser confocal fluorescence microscope. Results Compared with the normal control, TGF?β1 (10 μg/L) significantly stimulated mesangial cells proliferation, while decreased apoptosis. The mRNA and protein expressions of Ski, Smad7, Smad3 and ColΙ protein expression in TGF?β1 group were increased (P＞0.05). In comparison with TGF?β1 group, triptolide could significantly inhibit TGF?β1?induced mesangial cells proliferation in dose?dependent manner, and promote the apoptosis of mesangial cells. In TGF?β1 group, mRNA and protein expresscons of Ski and Smad7 were increased (P<0.05), Smad3 mRNA and protein were decreased (P＞0.05), and ColΙ protein was decreased (P<0.01). In comparison with TGF?β1 group, fluorescence intensity of Ski, Smad3 proteins was significantly increased in cytoplasm, while decreased in nucleus. Conclusions Triptolide can inhibit TGF?β1?induced mesangial cells proliferation through regulating the expressions of Ski, Smad7 mRNA and protein, inhibiting Ski. Smad7 translocation to the nucleus, and down?regulating Smad3 mRNA and protein expression. Triptolide can promote apoptosis of mesangial cells.     

Molecular cloning and functional identification of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Tripterygium wilfordii

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the last step key enzyme of the methylerythritol phosphate (MEP) pathway, synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which is important for regulation of isoprenoid biosynthesis. Here the full-length cDNA of HDR, designated TwHDR (GenBank Accession No. KJ933412.1), was isolated from Tripterygium wilfordii for the first time. TwHDR has an open reading frame (ORF) of 1386 bp encoding 461 amino acids. TwHDR exhibits high homology with HDRs of other plants, with an N-terminal conserved domain and three conserved cysteine residues. TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant. Since loss-of-function E.coli hdr mutant is lethal, the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant. This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme. The expression of TwHDR was induced by methyl-jasmonate (MJ) in T. wilfordii suspension cells. The expression of TwHDR reached the highest level after 1 h of MJ treatment. These results indicate that we have identified a functional TwHDR enzyme, which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T. wilfordii.