Triptolide modulates the sensitivity of K562/A02 cells to adriamycin by regulating miR-21 expression

Context: Multidrug-resistance is a serious obstacle encountered in leukemia treatment. Recent studies have shown microRNA-21 (miR-21) is overexpressed in several types of cancer and contributes to tumor resistance to chemotherapy. In our previous studies, we found triptolide (TPL) could enhance adriamycin-induced cytotoxicity and apoptosis in K562/A02 cells.

Objective: In the present study, we investigated the mechanism of TPL on the sensitivity of K562/A02 cells to adriamycin.

Materials and methods: Cell viability was assessed by methyl thiazolyl tetrazolium (MTT) assay. Expression of mature miR-21 was determined by SYBER green PCR. The miR-21 mimics and inhibitors were chemically synthesized and transfected into K562 cells or K562/A02 cells. PTEN protein levels was determined by western blots. PTEN promoter activity was measured by luciferase assays.

Results: TPL (5 nmol/L) increased the sensitivity of K562/A02 to adriamycin. When adriamycin was combined with 5 nmol/L TPL, the mean apoptotic population of K562/A02 cells was increased from 4.3 to 18.5%, respectively. K562/A02 cells showed a significant reduction in miR-21 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) expressions after TPL treatment. K562/A02 cells that were transfected with the miR-21 inhibitor had a significantly higher PTEN protein level than the control. K562 cells that were pre-treated with PTEN siRNA had increased survival rate compared to the control group.

Discussion and conclusion: Our findings indicated that triptolide modulates the sensitivity of K562/A02 cells to adriamycin by regulating miR-21 expression. Triptolide inhibited miR-21 expression and enhanced PTEN levels in K562/A02 cells.     

低剂量雷公藤甲素预处理减轻小鼠肝脏缺血再灌注损伤的作用及其机制

目的 探讨雷公藤甲素(TPT)预处理减轻小鼠肝脏缺血再灌注损伤的作用及其作用机制.方法 将60只雄性C57BL/6小鼠分成4组,每组15只.(1)TPT手术组:手术方法参照Kobayashi法,术中夹闭肝门静脉左支、肝动脉左支及左肝管,90min后开放血管,建立小鼠肝脏缺血再灌注损伤模型;(2)TPT假手术组:手术方式同TPT手术组,但术中不阻断血流,仅用生理盐水(NS)纱布覆盖切口90 min;(3)NS手术组:手术方式同TPT手术组;(4)NS假手术组:手术方式同TPT假手术组.两TPT组小鼠于术前1周开始腹腔注射TPT0.1 mg·kg-1·d-1,术前1 h加用1次,而两NS组同期仅腹腔注射等体积无菌NS.再灌注后24 h,检测各组小鼠肝功能和观察肝组织病理学变化,采用流式细胞术检测各组TH17细胞占单个核细胞的比例,采用实时聚合酶链反应(PCR)检测各组肝组织中IL-17和ROR-γt mRNA的表达,采用酶联免疫吸附试验(ELISA)检测血清中IL-6、IL-17和TGF-β的含量.结果 TPT手术组肝功能的损伤较NS手术组明显减轻(P＜0.05);NS假手术组、TPT假手术组、NS手术组和TPT手术组TH17细胞占单个核细胞的比例分别为(0.72±00.23)%、(0.41±0.18)%、(4.26±0.82)%和(1.77±0.53)%;两TPT组IL-17和ROR-γt mRNA的表达量以及外周血中IL-6、IL-17和TGF-β的含量,均明显低于相应的NS组(P＜0.05).结论 低剂量TPT预处理能够减轻小鼠肝脏缺血再灌注损伤,这可能与TPT预处理抑制小鼠体内Th17细胞分化、发育及其功能有关.     

鞘内联合给予T10与MK-801对神经病理性痛模型大鼠脊髓背角NR1与CREB蛋白磷酸化的影响

目的 研究鞘内联合给予雷公藤内酯醇(triptolide,T10)与MK-801对神经病理性痛模型大鼠痛行为及脊髓背角N-甲基-D-门冬氨酸(N-methyl-D-aspartic acid,NMDA)受体NR2B亚单位与环磷腺苷效应原件结合蛋白(cAMP-response element binding protein,CREB)磷酸化水平的影响。方法 选取健康雄性SD大鼠36只,随机分为6组:正常对照组(normal),假手术-生理盐水组(sham-saline),L₅脊神经结扎(spinal nerve ligation;SNL)-生理盐水组(SNL-saline),SNL-T10单纯给药组(SNL-T10),SNL-MK-801单纯给药组(SNL-MK-801),SNL-T10+MK-801联合给药组(SNL-T10+MK-801)。模型组和给药组均采用L5SNL构建慢性神经病理性痛模型,生理盐水、T10(10 μg/kg)和MK-801(30 μg/kg)从术后第1 d连续鞘内注射至第7 d,1次/d。利用Von-Frey丝刺激法连续观察造模后大鼠的机械性缩足阈值(paw withdrawl threshold,PWT);应用免疫荧光组织化学染色方法观察腰膨大节段脊髓背角内pCREB蛋白的表达;Western blot方法定量检测NR2B和CREB的磷酸化水平。结果 术后第1 d起,SNL组大鼠术侧后爪PWT明显下降(P＜0.01),且在3 d后基本保持平稳;鞘内单独给予T10或MK-801干预后第7 d术侧后爪的MWT明显提高,与给药前相比有显著性差异(P＜0.05);SNL-T10+MK-801联合给药组大鼠第7 d术侧后爪的PWT提高幅度较SNL-T10和MK-801组明显,与给药前相比差异更加显著(P＜0.01);停止给药后,SNL-T10+MK-801联合给药组镇痛疗效较SNL-T10以及MK-801组持续时间长。免疫荧光组织化学染色显示:pCREB主要表达于神经元内。Western blot检测结果显示:与对照组相比,SNL后第7d脊髓背角内pCREB蛋白的表达明显上调,SNL-T10+MK-801联合给药组大鼠脊髓背角内pNR2B和pCREB蛋白的表达水平要依次低于单独SNL-T10、SNL-MK-801给药组和SNL-saline组。结论 鞘内联合给予T10和MK-801能够有效缓解神经病理性痛模型大鼠的机械性痛行为,并且降低了二者的给药剂量,其机制可能与抑制脊髓背角神经元NR2B/CREB的磷酸化水平密切相关。     

雷公藤内酯醇降调胰腺癌细胞5-脂氧合酶表达作用研究

目的探讨雷公藤内酯醇（TL）对胰腺癌细胞中5 脂氧合酶(5 LOX)蛋白表达及胰腺癌细胞凋亡的影响。方法应用RT PCR法和免疫细胞化学法检测胰腺癌细胞株SW1990中5 LOX表达，同时应用流式细胞术检测胰腺癌细胞SW1990的凋亡指数。结果胰腺癌细胞株SW1990表达5 LOX，正常胰腺组织不表达； TL可抑制SW1990中5 LOX表达，并促进胰腺癌细胞SW1990凋亡，作用随浓度的增加而增强(P＜0.01)；细胞凋亡与5 LOX蛋白的表达相关(P＜0.01)。结论TL可抑制胰腺癌细胞SW1990中5 LOX的表达，并促进胰腺癌细胞凋亡     

Triptolide protects dopaminergic neurons from inflammation-mediated damage induced by lipopolysaccharide intranigral injection

Converging lines of evidence suggest that neuroinflammatory processes may account for the progressive death of dopaminergic neurons in Parkinson's disease (PD). Therefore, anti-inflammatory strategies have attracted much interest for their potential to prevent further deterioration of PD. Our previous study showed that triptolide, a traditional Chinese herbal compound with anti-inflammatory and immunosuppressive properties, protected dopaminergic neurons from lipopolysaccharide (LPS)-induced damage in primary embryonic midbrain cell cultures. To examine further if triptolide can protect dopaminergic neurons from inflammation-mediated damage in vivo, microglial activation and injury of dopaminergic neurons were induced by LPS intranigral injection, and the effects of triptolide treatment on microglial activation and survival ratio and function of dopaminergic neurons were investigated. Our results demonstrated that microglial activation induced by a single intranigral dose of 10 mug of LPS reduced the survival ratio of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNpc) to 29% and the content of dopamine (DA) in striatum to 37% of the non-injected side. Intriguingly, treatment with triptolide of 5 mug/kg for 24 days once per day dramatically improved the survival rate of TH-ir neurons in the SNpc to 79% of the non-injected side. Meanwhile, treatment with triptolide of 1 or 5 mug/kg for 24 days once per day significantly improved DA level in striatum to 70% and 68% of the non-injected side, respectively. Complement receptor 3 (CR3) immunohistochemical staining revealed that triptolide treatment potently inhibited LPS-elicited deleterious activation of microglia in SNpc. The excessive production of cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, was significantly abolished by triptolide administration. These results, together with our previous data in vitro, highly suggest the effectiveness of triptolide in protecting dopaminergic neurons against inflammatory challenge.     

雷公藤内酯醇对胰腺癌细胞株SW1990基因表达谱的影响

目的:应用基因表达谱芯片研究胰腺癌细胞株(SW1990)在雷公藤内酯醇作用后其基因表达的差异性。方法:用Cy5和Cy3两种荧光染料通过逆转录反应将加药组和对照组的SW1990细胞的mRNA分别标记成两种探针,并与载有一组靶基因的表达谱芯片杂交,通过扫描荧光强度,计算机软件分析,寻找经雷公藤内酯醇作用后表达有差异的基因。对芯片结果中两个表达有改变的基因行荧光定量PCR验证。结果:胰腺癌细胞SW1990受雷公藤内酯醇作用4h后,共筛选出11条差异表达基因,且全部下调。结论:雷公藤内酯醇可能部分通过下调C-MYC、ETS2、TGIF、RTP801等基因来发挥其有效的抗肿瘤特性。     

Triptolide induces Bcl-2 cleavage and mitochondria dependent apoptosis in p53-deficient HL-60 cells

Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.     

Synthesis, characterization andin vitro evaluation of triptolide-lysozyme conjugate for renal targeting delivery of triptolide

A triptolide-lysozyme (TP-LZM) conjugate was synthesized to achieve renal specific delivery and to reduce the side effects of triptolide. Triptolide was coupled to lysozyme through succinic via an ester bond with an average coupling degree of 1 mol triptolide per 1 mol lysozyme. The lysozyme can specifically accumulate in the proximal tubular cells of the kidney, making it a potential carrier for targeting drugs to the kidney. The structure of triptolide succinate (TPS) was confirmed by IR, 1H-NMR, MS and UV. The concentrations of triptolide in various samples were determined by reversed-phase high-performance liquid chromatography (HPLC). In this study, the physicochemical and stability profiles of TP-LZM under various conditions were investgated the stability and releasing profiles of triptolide-lysozyme (TP-LZM) under various conditions. In vitro release trails showed triptolide-lysozyme was relatively stable in plasma (less than 30% of free triptolide released) and could release triptolide quickly in lysosome (more than 80% of free triptolide released) at 37 degrees C for 24 h. In addition, the biological activities of the conjugate on normal rat kidney proximal tubular cells (NRK52E) were also tested. The conjugate can effectively reduce NO production in the medium of NRK52E induced by lipopolysaccharide (LPS) but with much lower toxicity. These studies suggest the possibility to promote curative effect and reduce its extra-renal toxicity of triptolide by TP-LZM conjugate.     

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。