Immunosuppressant PG490 (triptolide) induces apoptosis through the activation of caspase-3 and down-regulation of XIAP in U937 cells

PG490 (triptolide) is a natural, biologically active compound extracted from Chinese herb Tripterygium wilfordii. It possesses potent anti-inflammatory and immunosuppressive properties. The mechanism by which triptolide initiates apoptosis remains poorly understood. In the present report, we investigated the effect of triptolide on the apoptotic pathway in U937 human promonocytic cells. We show that triptolide inhibits U937 cells growth by inducing apoptosis. Following treatment of U937 cells with 25 nM triptoride for 24 hr, morphological features of apoptosis and DNA fragmentation were observed. Caspase inhibitors significantly reduced triptolide-induced caspase-3 activation. In addition, apoptosis triggered by triptolide was not associated with the generation of reactive oxygen species, which was not affected by the antioxidant N-acetylcysteine (NAC). The data collectively indicate that the cytotoxic effect of triptolide in U937 cells is attributable to apoptosis mediated by the caspase-3 activation pathway that may be associated with XIAP down-regulation.     

Triptolide inhibits murine-inducible nitric oxide synthase expression by down-regulating lipopolysaccharide-induced activity of nuclear factor-kappa B and c-Jun NH2-terminal kinase

Triptolide (PG490) is a natural, biologically active compound extracted from the Chinese herb Tripterygium wilfordii. It has been shown to possess potent anti-inflammatory and immunosuppressive properties. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, triptolide inhibits nitric oxide (NO) production in a dose-dependent manner and abrogates inducible nitric oxide synthase (iNOS) gene expression. To investigate the mechanism by which triptolide inhibits murine iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAP kinases) and nuclear factor-kappa B (NF-kappa B) in these cells. Addition of triptolide inhibited phosphorylation of c-Jun NH(2)-terminal kinase (JNK) but not that of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase. In addition, triptolide significantly inhibited the DNA binding activity of NF-kappa B. Taken together, these results suggest that triptolide acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of NF-kappa B and JNK activation.     

Triptolide and its expanding multiple pharmacological functions

Triptolide, a diterpene triepoxide, is a major active component of extracts derived from the medicinal plant Tripterygium wilfordii Hook F (TWHF). Triptolide has multiple pharmacological activities including anti-inflammatory, immune modulation, antiproliferative and proapoptotic activity. So, triptolide has been widely used to treat inflammatory diseases, autoimmune diseases, organ transplantation and even tumors. Triptolide cannot only induce tumor cell apoptosis directly, but can also enhance apoptosis induced by cytotoxic agents such as TNF-α, TRAIL and chemotherapeutic agents regardless of p53 phenotype by inhibiting NFκB activation. Recently, the cellular targets of triptolide, such as MKP-1, HSP, 5-Lox, RNA polymerase and histone methyl-transferases had been demonstrated. However, the clinical use of triptolide is often limited by its severe toxicity and water-insolubility. New water-soluble triptolide derivatives have been designed and synthesized, such as PG490-88 or F60008, which have been shown to be safe and potent antitumor agent. Importantly, PG490-88 has been approved entry into Phase I clinical trial for treatment of prostate cancer in USA. This review will focus on these breakthrough findings of triptolide and its implications.     

雷公藤与4种中药饮片配伍对雷公藤内酯醇的影响

目的 研究赤芍、莱菔子、佛手、竹茹与雷公藤配伍后对雷公藤内酯醇的影响.方法 4种饮片分别与雷公藤配伍,加水提取,用HPLC测定雷公藤单提液及其配伍提取液中的雷公藤内酯醇.结果 佛手-雷公藤配伍提取液中雷公藤内酯醇约增加25%(P＜0.05).其余配伍提取液中雷公藤内酯醇无显著变化.结论 佛手和雷公藤配伍能明显增加提取液中雷公藤内酯醇,其余配伍对雷公藤内酯醇无显著影响.     

芳竭丸活血化瘀作用的实验研究

目的对中药新药芳竭丸的活血化瘀作用进行研究。方法采用在冰水中游泳和SC注射肾上腺素方法制造大鼠急性血瘀模型,观察芳竭丸对血液流变性的影响;用肾上腺素制造大鼠肠系膜急性微循环障碍模型,观察芳竭丸对微循环障碍的改善作用;采用体外法,用PT和KPTT试剂盒测定血液凝固时间,观察芳竭丸对凝血酶原时间（PT）及活化部分凝血活酶时间（KPTT）的影响。结果芳竭丸能明显降低大鼠的全血黏度、血浆黏度、血纤维蛋白原含量、血沉及血小板黏附率,减轻血瘀大鼠血液的黏、凝状态,防止血栓形成;可抑制肾上腺素引起的大鼠肠系膜微循环细动脉管径缩小、流速减慢、毛细血管开放量减少、流态改变,并改善这些现象;可显著延长家兔的PT、KPTT。结论芳竭丸有改善血液流变性及微循环障碍的作用,有抑制内源性及外源性凝血系统的作用,提示有活血化瘀作用。     

雷公藤内酯醇不同季节的含量变化

目的：对福建产的雷公藤不同季节不同部位进行雷公藤内酯醇的含量的测定。方法：雷公藤春夏秋冬生长的全根、根皮、去皮根、茎、叶,经无水乙醇提取,中性氧化锃伴和,石油醚回流和氯仿提取,与雷公藤内酯醇分别点样于薄层板上,氯仿-乙醚（2:1）上行展开,喷显色剂,薄层扫描定量检测。结果：雷公藤内酯醇在雷公藤全根、根皮、叶中的含量为夏季最高,去皮根为春季最高,茎为秋季高;夏季的雷公藤不同部位雷公藤内酯醇的含量高低,依次为全根、根皮、去皮根、茎、叶。结论：为合理利用雷公藤资源提供了依据。     

HPLC法测定粉背雷公藤中雷公藤甲素的含量

目的：建立高效液相色谱法测定广西产粉背雷公藤中不同部位雷公藤甲素的含量方法。方法：采用C18色谱柱（250mm×4．6mm,5μm）,流动相为甲醇-水（45：55）,检测波长217nm,流速1mL／min,柱温24℃,进样量为20μL。结果：雷公藤甲素在0．5～10μg／mL范围内呈良好的线性关系;平均加样回收率为99．61％,RSD为1．69％（n=5）;粉背雷公藤根部和茎枝中雷公藤甲素的平均含量分别为14．98和3．64μg/g。结论：本方法简便可行、定量准确、重现性好,可用于粉背雷公藤中雷公藤甲素的质量控制。     

Triptolide affects the differentiation, maturation and function of human dendritic cells

Triptolide is a purified component from a traditional Chinese herb Tripterygium wilfordii Hook F. It has been shown to have anti-inflammatory and immunosuppressive activities by its inhibitory effect on T cells. But the effect of triptolide on dendritic cells (DC) is unknown. Dexamethasone (Dex) is a classic immunosuppressive agent known to suppress the immune response at different levels and has recently found to modulate the development of DC, thereby influencing the initiation of the immune response. In this study, we investigated the affect of triptolide on the differentiation, maturation and function of DC differentiated from human monocytes (MoDC) in vitro in the presence of GM-CSF and IL-4. Dex was included in the study as a reference. Our data show that both triptolide and Dex prevented the differentiation in immature MoDC by inhibiting CD1a, CD40, CD80, CD86 and HLA-DR expression but upregulating CD14 expression, as well as by reducing the capacity of MoDC to stimulate lymphocyte proliferation in the allogeneic mixed lymphocyte reaction. They blocked the maturation of MoDC as totally blocked induction of CD83 expression and absent upregulation of CD40, CD80, CD86 and HLA-DR. In addition, higher concentration of triptolide (20 ng/ml) and 10(-6) M Dex induced apoptosis in MoDC as measured by expression of APO2*7 and DNA fragmentation (TUNEL assay). However, the phagocytic capacity of MoDC was enhanced by triptolide but not Dex. Therefore, the suppression of DC differentiation, the function in immature DCs as well as the inhibition of DC maturation by triptolide may explain some of its immunosuppressive properties. It is suggested that DCs are a primary target of the immunosuppressive activity of triptolide.     

Experimental AA amyloidosis in mice is inhibited by treatment with triptolide, a purified traditional Chinese medicine

The effect of triptolide, which is isolated from Tripterygium Wilfordii, on induction and development of murine AA amyloidosis was studied. In the first experiment, we examined the ability of triptolide to inhibit initiation of amyloidosis. Oral or intraperitoneal administration of 480 microg/kg/day triptolide inhibited splenic amyloid deposition in both rapid and chronic induction models of mouse AA amyloidosis. Moreover, serum amyloid A (SAA) and interleukin (IL)-6 levels were also suppressed remarkably. Triptolide also immediately decreased SAA levels and reduced the incidence of amyloidosis even under conditions of high SAA levels. In the second experiment, we evaluated the influence of triptolide on development and resorption of amyloid deposition. Amyloid deposition was induced in mice by 28 daily injections of casein. After splenic and hepatic biopsies to confirm the presence of amyloid deposits, the mice immediately started to receive daily injections of 480 microg/kg/day triptolide with or without casein. Treatment with triptolide for 35 days and 105 days prevented deposition of amyloid and promoted resorption of splenic amyloid deposits. In conclusion, we show for the first time that triptolide inhibits induction and development of experimental murine amyloidosis. These results suggest that through suppression of IL-6, triptolide can reduce production of SAA. Amyloid deposition is prevented when levels of the amyloid-forming precursor protein SAA are decreased significantly.     

Caspase 3 is involved in the apoptosis induced by triptolide in HK-2 cells

Triptolide, a main active component extracted from the traditional Chinese herbal medicine, Tripterygium wilfordii Hook. f (TWHf), has been shown to possess potent immunosuppressive and anti-inflammatory properties. However, the toxicity of triptolide limits its clinical applications. Here we treated the human proximal tubular epithelial cell line HK-2 cells with triptolide in vitro and investigated its toxic effects. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for viability inhibition and annexin V/propidium iodide (PI) staining for apoptosis/necrosis. The activation of caspase 3 was analyzed by Western Blotting. MTT assay showed triptolide inhibited the viability of HK-2 cells in a time- and dose-dependent manner. Flow cytometry assay showed triptolide caused apoptosis rather than necrosis in HK-2 cells by staining with annexin V/PI. Furthermore, the increase of cleaved p17 fragment, an active form of caspase 3, was detected. These results suggested that triptolide is able to cause cytotoxicity on HK-2 cells, and the mechanism of which is associated with caspase 3.     

The effect of triptolide on CD4+ and CD8+ cells in Peyer's patch of SD rats with collagen induced arthritis

Triptolide is a purified component from a traditional Chinese herb Tripterygium wilfordii Hook F. It has been shown to have anti-inflammatory and immunosuppressive activities by its inhibitory effect on T cells. But the effect of triptolide on Peyer's patch cells is unknown. Enteric mucosal immune system, including Peyer's patch, is regarded as one of the sites for inducing immunity tolerance, and this intolerance effect has been used to induce oral tolerance which can considerably reduce arthritis severity in several models of experimental polyarthritis and RA patients. In this study, we investigated the effect of triptolide on the Peyer's patch cells and peripheral lymphocytes in collagen induced arthritis (CIA) in rats. CIA in rat is a widely studied animal model of inflammatory polyarthritis with similarities to rheumatoid arthritis (RA). Our data show that triptolide could lower the arthritic scores and delay the onset of CIA. There are more Peyer's patches in triptolide treated rats than in control rats, while there is no difference in Peyer's patch numbers between CIA rats and triptolide treated rats. In the Peyer's patch, more CD4+ cells are observed in CIA rats, and the numbers of CD4+ cells in triptolide treated rats and control rats are similar. While more CD8+ cells are observed in triptolide treated rats, and the numbers of CD8+ cells in CIA rats and control rats are similar. In periphery, more CD4+ cells and less CD4+ cells in CIA rats and triptolide treated rats are respectively observed. Therefore, the regulation on Peyer's patch might explain some of the immunosuppressive activities of triptolide, and enteric immune response might be actively involved in CIA pathogenesis. It is suggested that the Peyer's patch is one of the primary targets of the immunosuppressive activity of triptolide.     

Downregulation of lymphocyte activity and human synovial fibroblast growth in rheumatoid arthritis by triptolide

The antirheumatic effects of triptolide, a purified component derived from a Chinese herb, Tripterygium wilfordii Hook f. (TWH), was examined. Peripheral blood mononuclear cells (PBMC), T cells, or human synovial fibroblasts isolated from healthy controls or rheumatoid arthritis (RA) patients were cultured in vitro in the absence or presence of triptolide. Estimated by ELISA, immunoglobulin synthesis in pokeweed mitogen or Staphylococcus aureus Cowan 1 strain stimulated PBMC was significantly impaired by triptolide in a concentration‐dependent manner (1–10 nM). Similarly, proliferation of PBMC in response to phytohemagglutinin (PHA‐M), interleukin‐2, or phorbol 12‐myristate 13‐acetate (PMA)/ionomycin estimated by incorporation of [³H]‐thymidine was inhibited by triptolide. Cell viability was not affected at the immunosuppressive concentrations of triptolide. No abnormality of intracellular Ca²⁺ flux as estimated by flow cytometry was detected in PHA‐M‐stimulated T cells by triptolide. Biosynthesis of cellular protein estimated by incorporation of [³H]‐leucine was significantly reduced in PMA/ionomycin stimulated PBMC by triptolide at concentrations above 7.5 nM. Proliferation of human synovial fibroblasts as estimated by crystal violet staining was significantly inhibited by triptolide at 30 nM. The present data demonstrate that triptolide is a potent immunosuppressant and has an antiproliferative effect on synovial fibroblast. The immunosuppressive activity of triptolide is not due to cytotoxicity, nor is it targeted at the initial membrane signal transduction process and the generation of second messengers. Inhibition of cellular protein synthesis by triptolide during lymphocyte activation may account for its inhibitory activity. The precise mechanism of action of triptolide needs to be defined in order to develop improved versions of the molecule for the potential treatment of RA. Drug Dev. Res. 47:144–153, 1999. © 1999 Wiley‐Liss, Inc.     

正交设计优选雷公藤提取工艺

目的:优选雷公藤提取工艺的最佳条件。方法:采用L9(34)正交试验设计,以雷公藤内酯醇含量、浸出物收率为考察指标,考察提取时间、提取次数、加水量等因素对提取工艺的影响。结果:以加水量为药材量的10倍,煎煮提取时间每次1.5h,共提取2次为雷公藤最佳提取工艺。结论:优选出的最佳工艺条件经验证其结果稳定,指标性成分含量高。     

雷公藤二萜类化学成分及在研新药的研究进展

目的对雷公藤属植物中二萜类化学成分及其在研新药的研究作一综述,提供有关雷公藤属植物研究的借鉴资料。方法查阅近30多年来国内外公开发表的有关雷公藤属植物化学成分的相关文献,对其二萜类结构及结构分类进行论述,同时查阅雷公藤在研新药研发近况。结果雷公藤二萜类主要包括松香烷型和贝壳杉烷型两大类,共118个单体,介绍了研发代号为MC002、LLDT-8及MRx102,3个以雷公藤甲素为先导化合物的在研新药。结论为今后雷公藤属植物的进一步研究提供了参考。     

Triptolide inhibits inducible nitric oxide synthase expression induced by toll-like receptor agonists

Toll-like receptors (TLRs) play an important role in the induction of innate immune responses recognizing many pathogen-associated molecular patterns. The activation of TLRs triggers two downstream signaling pathways; MyD88- (myeloid differential factor 88) and TRIF-(toll-interleukin-1 receptor domain-containing adapter inducing interferon-β) dependent pathways leading to the induction of pro-inflammatory gene products such as inducible nitric oxide synthase (iNOS). The present study investigated the effect of triptolide (TP), a natural component of Tripterygium wilfordii Hook. F, on inflammation by modulating iNOS expression induced by TLR agonists in murine macrophages. TP suppressed iNOS expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3 agonist), and macrophage-activating lipopeptide 2-kDa (TLR2 and TLR6 agonist). All the results suggest that TP can modulate TLR signaling pathways and subsequent chronic inflammatory responses.     

Triptolide inhibits CC chemokines expressed in rat adjuvant-induced arthritis

Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F (TWHF), has been proven to have potent immunosuppressive and anti-inflammatory activities. It has been clinically used to treat patients with rheumatoid arthritis (RA), in which chemokines play an important role in immune and inflammatory responses. To investigate the effect of triptolide on MCP-1, MIP-1alpha and RANTES, we used complete Freund's adjuvant to induce adjuvant-induced arthritis (AA) in rats. AA in rat is a useful experimental model of human RA. Our data show that the thickness of arthritic ankle decreases with administration of triptolide. Both mRNA and protein levels of MCP-1, MIP-1alpha and RANTES in synovial tissue of rats with AA are significantly higher than those in normal rats. mRNA levels of MIP-1alpha and RANTES increase in peripheral blood mononuclear cells of rats with AA in comparison with those in normal rats, whereas no MCP-1 mRNA can be detected. Triptolide can significantly inhibit rat AA induced over-expression of MCP-1, MIP-1alpha and RANTES at both mRNA and protein levels in a dose-dependent manner. These results may contribute to the therapeutic effects of triptolide in rheumatoid arthritis.     

Involvement of CYP3A4/5 and CYP2D6 in the metabolism of aconitine using human liver microsomes and recombinant CYP450 enzymes

Triptolide, the primary active component of Tripterygium wilfordii Hook F, has various pharmacological activities but also a narrow therapeutic window. Cytochrome P450s are proposed to be responsible for the hydroxylation of triptolide in vitro and CYP3A induction by dexamethasone can increase the metabolism of triptolide and decrease the hepatotoxicity in rat. However, triptolide-induced toxicity has not been investigated in an animal model having a suppression of P450 activities. Here we compared the toxicological effects and toxicokinetics of triptolide between liver-specific cytochrome P450 reductase (CPR) knockout (KO) mice (abolished hepatic P450 activities) and wild-type (WT) control mice after a single oral gavage of triptolide at 0.5 mg/kg or 1.0 mg/kg. A low toxic dose of triptolide at 0.5 mg/kg for WT mice resulted in severe toxicities including death in KO mice. Changes in serum biochemistry, hematology and histopathology further indicated much more severe toxicities in multiple organs in KO mice compared to WT mice after triptolide administration. The mono-hydroxylated metabolites of triptolide detected in the blood of WT mice were undetectable in KO mice, accompanied by much higher triptolide levels in the blood and tissues including the liver, kidney, and spleen determined by LC-MS/MS. Taken together, our results confirmed that inactivation of hepatic P450s abolishes the ability in metabolism of triptolide in the liver, subsequently resulting in an increase in bioavailability and toxicity of triptolide in vivo. It is suggested that P450 inhibition/inactivation might pose a significant health risk in the clinic use of triptolide.     

Immunochemical characterization of the functional constituents of Tripterygium wilfordii contributing to its anti-inflammatory property

1. Tripterygium wilfordii (TW) contains bioactive compounds that possess immunosuppressive properties. These compounds are considered to be potential drugs in the treatment of acute graft rejections. However, their structure-activity relationships remain unknown. 2. The aim of the present study was to delineate the molecular moieties of triptolide that could account for its ability to inhibit inflammatory responses. In this context, purified TW active compounds (triptolide and triptonide) and synthetic triptolide derivatives were prepared to investigate the structure-activity relationships of triptolide. To this end, rat splenocytes were treated with increasing concentrations of the compounds and then allogenically stimulated using a mixed lymphocyte reaction to determine their antiproliferative activities. From the results, the IC50 value of each compound was calculated. 3. Modification of the beta-hydroxyl group at the C-14 position of the triptolide molecule significantly affected the immunosuppressive activity of T59, as demonstrated by a sevenfold increase of the IC50. Conversely, reduction of the gamma-butyrolactone group in T60 and T61 completely abrogated the antiproliferative effect. Alterations in the C-14 beta-hydroxyl and gamma-butyrolactone groups also resulted in reduced cytotoxicity. 4. The present findings demonstrate that the C-14 beta-hydroxyl and gamma-butyrolactone moieties of the triptolide molecule are crucial for its anti-inflammatory properties and cytotoxicity and are responsible for the compound's antiproliferative activity.     

Effects of triptolide on the synaptophysin expression of hippocampal neurons in the AD cellular model

Due to the immunoinflammatory pathology of Alzheimer's disease (AD) brain, recent studies have begun to focus attention on the role of anti-inflammatory drugs or immunomodulators in AD. Triptolide isolated from the herb Tripterygium wilfordii Hook F has anti-inflammatory and immunosuppressive activities. In this study, we observed the effects of triptolide on synaptophysin expression in AD cellular model. AD cellular model was established by action of Aβ-stimulated microglial conditioned medium (MCM) on cultured rat hippocampal neurons (HN). Immunocytochemical staining, western blot and RT-PCR were used to observe the effects of triptolide at different dosages on the synaptophysin expression of hippocampal neurons in AD cellular model at different time points during incubation of cultures. After 24 h of cultivation, the expression level of synaptophysin in MCM/HN model group was decreased as compared with normal HN group and MCM/HN control group, and the expression level of synaptophysin in MCM/HN low-dose triptolide group and MCM/HN high-dose triptolide group was increased as compared with MCM/HN model group. It is concluded that triptolide can promote the synaptophysin expression of hippocampal neurons in the AD cellular model.