Triptolide protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats:Implication for immunosuppressive therapy in Parkinson’s disease

Objective Neuroinflammation with microglial activation has been implicated to have a strong association with the progressive dopaminergic neuronal loss in Parkinson＇s disease （PD）. The present study was undertaken to evaluate the activation profile of microglia in 1-methyl-4-phenyl pyridinium （MPP^＋）-induced hemiparkinsonian rats. Triptolide, a potent immunosuppressant and microglia inhibitor, was then examined for its efficacy in protecting dopaminergic neurons from injury and ameliorating behavioral disabilities induced by MPP^＋. Methods The rat model of PD was established by intranigral microinjection of MPP^＋. At baseline and on day 1, 3, 7, 14, 21 following MPP^＋ injection, the degree of microglial activation was examined by detecting the immunodensity of OX-42 （microglia marker） in the substantia nigra （SN）. The number of viable dopaminergic neurons was determined by measuring tyrosine hydroxylase （TH） positive neurons in the SN. Behavioral performances were evaluated by counting the number of rotations induced by apomorphine, calculating scores of forelimb akinesia and vibrissae-elicited forelimb placing asymmetry. Results Intranigral injection of MPP^＋ resulted in robust activa- tion of microglia, progressive depletion of dopaminergic neurons, and ongoing aggravation of behavioral disabilities in rats. Triptolide significantly inhibited microglial activation, partially prevented dopaminergic cells from death and improved behavioral performances. Conclusion These data demonstrated for the first time a neuroprotective effect of triptolide on dopaminergic neurons in MPP^＋ induced hemiparkinsonian rats. The protective effect of triptolide may, at least partially, be related to the inhibition of MPP^＋-induced microglial activation. Our results lend strong support to the use of immunosuppressive agents in the management of PD.

16-Hydroxytriptolide" An Active Compound from Tripterygium wilfordii

From the dried roots and leaves of Tripterygium wilfordii^(1.2),a new diter- penoid triepoxide,16-hydroxytriptolide was isolated,and its structure and stereochemistry elucidated as 16-(S)-hydroxy-triptolide on the basis of spectral data(IR, MS, UV,~1HNMR,^(13)CNMR,2d-NMR,Selective Long-range DEPT)and x-ray crystallographic analysis.This compound showed definite antiinflammatory action,strong immunosuppressive and antifertile activities.In addition,a known compound,triptolide was also isolated and all the spectral signals of^1 HNMR and ^(13)CNMR were assigned.

西北农林科技大学生命科学学院研究团队发现小分子药物triptolide诱导细胞自噬的新机制

2015年12月29日,Oncotarget杂志（IF=6.359）在线发表西北农林科技大学生命科学学院雷鸣教授课题组的最新研究成果“Triptolide induces protective autophagy through activation of the CaMKKβ-AMPK signaling pathway in prostate cancer cells”（DOI：10.18632/oncotarget.6783）。

Targets and molecular mechanisms of triptolide in cancer therapy

Triptolide（TPL/TL） is a natural drug with novel anticancer effects. Preclinical studies indicated that TPL inhibits cell proliferation, induces cell apoptosis, inhibits tumor metastasis and enhances the effect of other therapeutic methods in various cancer cell lines. Multiple molecules and signaling pathways, such as caspases, heat-shock proteins, NF-κB, and deoxyribonucleic acid（DNA） repair-associated factors, are associated with the anti-cancer effect. TPL also improves chemoradiosensitivity in cancer therapy. Phase I trials indicate the potential clinical value of TPL use. However, further trials with larger sample sizes are needed to confirm these results.

Effect of Triptolide on Reproductive Endocrinology and Dihydrotestosterone Receptors in Rats

Triptolide was given orally to adult male Sprague-Dawley rat sat a dosage of 75 μg/kg for 35 days.After 28 days of treatment,the result of mating tests showed that all the drug treated rats were infertile.At the end of drug treatment,the density of caudal spermatozoa and the weight of cpididymis were reduced significantly.All the spermatozoa were immobile.There was no detectable damage of spermatogenesis and epididymal epithelia in triptolide treated rats under microscopical examination.However,modcrate and severe damage of spermatozoa were seen in the corpus and caudal epididymis.The content of cytosolic and nuclear dihydrotestosterone (DHT) receptors in the caput and caudal epididymides was increased but insignificantly as compared with that of the controls.However, the content of DHT receptor in the cytosal of the ventral prostate was elevated very,significantly (P< 0.01).This result suggests that one of the sitcs of action of triptolide might be the epididymis.

Role of Reactive Oxygen Species in Triptolide-induced Apoptosis of Renal Tubular Cells and Renal Injury in Rats

This study investigated the role of reactive oxygen species（ROS） in the pathogenesis of triptolide-induced renal injury in vivo.Rats were randomly divided into 4 groups（n=5 in each）：triptolide group in which the rats were intraperitoneally injected with triptolide solution at a dose of 1 mg/kg of body weight on day 8;control group in which the rats received a single intraperitoneal injection of 0.9% physiological saline on day 8;vitamin C group in which the rats were pretreated with vitamin C by gavage at a dose of 250 mg/kg of body weight per day for 7 days before the same treatment as the control group on day 8;triptolide＋vitamin C group in which the rats were first subjected to an oral administration of vitamin C at a dose of 250 mg/kg of body weight per day for 7 days,and then to the same treatment as the triptolide group on day 8.All the rats were sacrificed on day 10.Blood samples were collected for detection of plasma creatinine（Pcr） and plasma urea nitrogen（PUN） concentrations.Both kidneys were removed.The histological changes were measured by haematoxylin-eosin（HE） staining.The production of ROS was determined by detecting the fluorescent intensity of the oxida-tion-sensitive probe rhodamine 123 in renal tissue.Renal malondialdehyde（MDA） content was meas-ured to evaluate lipid peroxidation level in renal tissue.TUNEL staining was performed to assess apop-tosis of renal tubular cells.Renal expression of apoptosis-related proteins Bcl-2,Bax,Bid,Bad,Fas and FasL,as well as corresponding encoding genes were assessed by Western Blotting and real-time PCR.The results showed that triptolide treatment promoted the generation of a great amount of ROS,up-regulated the expression of Bax,Bid,Bad,Fas and FasL at both protein and mRNA levels,as well as the ratio of Bax to Bcl-2,and caused the apoptosis of renal tubular cells and renal injury.However,pretreatment with an antioxidant,vitamin C,significantly reduced the generation of ROS and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury.It was concluded that ROS plays a critical role in triptolide-induced apoptosis of renal tubular cells and renal injury.The protective administration of vitamin C may help alleviate triptolide-induced renal injury and nephrotoxicity.

Triptolide prolonged allogeneic islet graft survival in chemically induced and spontaneously diabetic mice without impairment of islet function

BACKGROUND: Triptolide (TPT) is a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook. F. It exhibits potent immunosuppressive and anti-inflammatory properties. This study was undertaken to investigate its effects on prolongation of islet allograft survival in rodents. Additionally, we investigated whether TPT would be toxic to islet function in vivo. METHODS: We transplanted BALB/c islets to either chemically induced diabetic C57BL/6 mice or spontaneously diabetic non-obese diabetic (NOD) mice. TPT was injected within 2 weeks or continuously, until rejection, in the two combinations. Then, we evaluated the toxicity of TPT on islet function by daily injection to naive BALB/c or diabetic BALB/c that was cured by syngeneic islet transplantation under the kidney capsule. Mice injected with cyclosporine A (CsA) or vehicle served as controls. Intraperitoneal glucose tolerance tests (IPGTTs) performed at 4 and 8 weeks in the naive BALB/c group, and at 2, 4, 6, and 8 weeks in the syngeneic transplanted group. RESULTS: The medium survival time of islets allograft from TPT treated C57BL/6 and NOD recipients were 28.5 days (range 24-30 days, n=10) and 33.0 days (range 15-47 days, n=6), respectively, and they were significantly different from those of the vehicle treated controls, which were 14.0 days (range 13-16 days, n=6) and 5.0 days (range 4-10 days, n=6), respectively (all P<0.0001). The IPGTT demonstrated that there was no difference between the TPT treated and vehicle treated groups, either in the normal or syngeneic transplanted islet BALB/c mice. However, CsA injection impaired islet function in both normal and syngeneic transplanted mice as early as 4 weeks. CONCLUSION: TPT prolonged islets allograft survival in a chemically induced diabetic or an autoimmune diabetic murine model without impairment of islet function. (Hepatobiliary Pancreat Dis Int 2010; 9: 312-318)

Clinical Observation on Effect of Triptolide Tablet in Treating Patients with Psoriasis Vulgaris

Objective: To investigate the effects of triptolide tablet in the treatment of patients with psoriasis vulgaris. Methods: By an open clinical study of 103 patients with psoriasis vulgaris. Psoriasis area severity index (PASI) was measured and recorded before and after treatment for efficacy evaluation. Results: Of the 103 patients, markedly effective was got in 41 (39.7%), improved in 37 (35.8%) and ineffective in 25 (24.5%), the total effective rate being 75.7%, and the adverse reaction was shown only in few patients with decreased WBC during the treatment period. Conclusion: Triptolide tablet is effective for the treatment of psoriasis vulgaris during the one-year follow-up.

Triptolide抑制角膜免疫反应及TGF-β诱导角膜基质细胞胶原收缩实验研究

雷公藤内酯醇（Triptolide,TP）是中药雷公藤的一种成分,已证实具有多种药理作用如抗炎、免疫调节等[1]。感染性角膜炎是世界范围内,尤其是发展中国家的主要致盲性眼病之一。TNF-α作为Thl细胞分泌产生的炎性细胞因子之一,与IL-2、IFN-1等共同介导细胞免疫[2,3],这些炎性因子及趋化因子可诱导炎性细胞对组织的浸润功能。

Apoptosis of human pancreatic cancer cells induced by Triptolide

AIM: To investigate apoptosis in human pancreatic cancer cells induced by Triptolide (TL), and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax. METHODS: Human pancreatic cancer cell line SW1990 was cultured in DMEM media for this study. MTT assay was used to determine the cell growth inhibitory rate in vitro. Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment. RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax. RESULTS: TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner. TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h, the apoptotic rates of human pancreatic cancer cells increased significantly. RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not. CONCLUSION: TL is able to induce the apoptosis in human pancreatic cancer cells. This apoptosis may be mediated by up-regulating the expression of apoptosis- associated caspase-3 and bax gene.

Triptolide Inhibits Cell Growth and Induces G0-G1 Arrest by Regulating P21wap1/cip1 and P27 kip1 in Human Multiple Myeloma RPMI-8226 Cells

Objective： To investigate the effects of triptolide（TPL） on cell growth, cell cycle and the expressions of p21wapl/cipl and p27kipl. Methods： MTT assay was used to determine the cell viability after triptolide treatment in human multiple myeloma RPMI-8226 cells. The effect on cell cycle distribution was determined by flow cytometry. Semi-quantitative reverse transcription-PCR was used to examine the mRNA expressions of p21wapl/cipl and p27kipl. The protein expressions of p21 wapl/cipl and p27kipl were determined by Western blot. Results： Triptolide of varying concentrations induced cell viability inhibition in dose- and time-related fashion and caused Go- G1 phase arrest of cell cycle progression in RPMI-8226 cells. These effects accompanied with up-modulation of the expressions of p21 wapl/cipl and p27kipl. Conclusion： These results suggest that triptolide inhibit cell proliferation and cell cycle progression via up-regulating p21wapl/cipl and p27kipl and triptolide may exert its anti-cancer activity through this pathway.

Inhibitive effect of triptolide on invasiveness of human fibrosarcoma cells by downregulating matrix metalloproteinase-9 expression

Objective:To explore the molecular mechanisms of antitumor properties of triptolide,a bioactive component isolated from the Chinese herb Tripterygium wolfordii Hook F.Methods:Human fibrosarcoma HT-1080 cells were treated with different doses of triptolide for 72 h.Then the expression and activity of matrix metalloproteinase(MMP)-2 and -9 were measured and the invasiveness of triptolide-treated HT-1080 cells was compared with that of anti-MMP-9- treated HT-1080 cells.Results:18 nmol/L triptolide inhibited the gene expression and activity of MMP-9,but not those of MMP-2,in HT-1080 cells.In addition,both 18 nmol/L triptolide and 3μg/mL anti-MMP-9 significantly reduced the invasive potential of HT-1080 cells,by about 50%and 35%, respectively,compared with the control.Whereas there was no significant difference between the effect of 18 nmol/L triptolide and that of anti-MMP-9 on invasive potential of HT-1080 cells. Conclusions:These data suggest that triptolide inhibits tumor cell invasion partly by reducing MMP-9 gene expression and activity.

Effects of Triptolide on Cell Proliferation and CXCR4 Expression in Burkitt’s Lymphoma Raji Cells In Vitro

Objective： To investigate the inhibitory effects of triptolide on cell proliferation and CXCR4 expression in Burkitt＇s lymphoma cell line Raji cells. Methods： The effects of triptolide on the growth of Raji cells were studied by 3-（4, 5-Dimethyl-2-thiazolyl）-2, 5-diphenyl-2H-tetrazolium（MTT） assay. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α （rhSDF-1α） in vitro. Results： Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. Triptolide could downregulate the CXCR4 expression on Raji cells in a dose-dependent manner. Furthermore, chemotaxis assays showed that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent. Conclusion： Triptolide could inhibit the proliferation and migration of Raji cells in vitro. The underlying anti-tumor mechanism of triptolide might be related to the anti-proliferative effect and the blockage of SDF-1/CXCR4 axis.

Inhibition of zymosan-induced cytokine and chemokine expression in human corneal fibroblasts by triptolide

AIM：To investigate the effects of triptolide on proinflammatory cytokine and chemokine expression induced by the fungal component zymosan in cultured human corneal fibroblasts（HCFs）.·METHODS：HCFs were cultured in the absence or presence of zymosan or triptolide.The release of interleukin（IL）-6,IL-8,and monocyte chemoattractant protein-1（MCP-1）into culture supernatants was measured with enzyme-linked immunosorbent assays.The cellular abundance of the m RNAs for these proteins was determined by reverse transcription and real-time polymerase chain reaction analysis.The phosphorylation of mitogen-activated protein kinases（MAPKs）and the endogenous nuclear factor-κB（NF-κB）inhibitor IκB-αwas examined by immunoblot analysis.The release of lactate dehydrogenase（LDH）activity from HCFs was measured with a colorimetric assay.·R ESULTS：Triptolide inhibited the zymosan-induced release of IL-6,IL-8,and MCP-1 from HCFs in a concentration-and time-dependent manner.It also inhibited the zymosan-induced up-regulation of IL-6,IL-8,and MCP-1 m RNA abundance in these cells.Furthermore,triptolide attenuated zymosan-induced phosphorylation of the MAPKs extracellular signalregulated kinase（ERK）,c-Jun NH2-terminal kinase（JNK）,and p38 as well as the phosphorylation and degradation of IκB-α.Triptolide did not exhibit cytotoxicity for HCFs.·C ONCLUSION：Triptolide inhibited proinflammatory cytokine and chemokine production by HCFs exposed tozymosan,with this action likely being mediated by suppression of MAPK and NF-κB signaling pathways.This compound might thus be expected to limit the infiltration of inflammatory cells into the cornea associated with fungal infection.

Triptolide-induced Apoptosis by Inactivating Nuclear Factor-kappa B Apoptotic Pathway in Multiple Myeloma in vitro

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B（NF-κB） and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.

Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation

Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.

Effects of triptolide on hippocampal microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer＇s disease

The principal pathology of Alzheimer＇s disease includes neuronal extracellular deposition of amyloid-beta peptides and formation of senile pl aques, which in turn induce neuroinflammation in the brain. Triptolide, a natural extract from the vine-like herb Tripterygium wilfordii Hook F, has potent anti-inflammatory and immunosuppressive efficacy. Therefore, we determined if triptolide can inhibit activation and proliferation of microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer＇s disease. We used 1 or 5 μg/kg/d triptolide to treat APP/PS1 double transgenic mice （aged 4-4.5 months） for 45 days. Unbiased stereology analysis found that triptolide dose-dependent- ly reduced the total number of microglial cells, and transformed microglial cells into the resting state. Further, triptolide （5 μg/kg/d） also reduced the total number of hippocampal astrocytes. Our in vivo test results indicate that triptolide suppresses activation and proliferation of microglial cells and astrocytes in the hippocampus of APP/PS 1 double transgenic mice with Alzheimer＇s disease.

Effect of Triptolide on Expression of Oxidative Carbonyl Protein in Renal Cortex of Rats with Diabetic Nephropathy

The traditional Chinese medicine（Tripterygium wilfordii Hook.f., TWH） has been clinically used to treat primary and secondary renal diseases and proteinuria for nearly 40 years. However, there is a rare literature about the effect of triptolide（the main active ingredient of TWH） on the expression of oxidative carbonyl protein（OCP） in diabetic nephropathy（DN）. This study aimed to provide experimental evidence for triptolide treatment on DN through its effect on the expression of OCP, in order to investigate the effects of triptolide on the expression of OCP in rats with DN. Sixty SD rats were randomly divided into five groups： control group, high-dose triptolide（Th） group, low-dose triptolide（Tl） group, DN model group, and positive control（benazepril） group. The DN model was established using streptozotocin. Urinary protein excretion, fasting blood glucose（FBG）, superoxide dismutase（SOD） in renal homogenate, malondialdehyde（MDA） in renal homogenate and renal nitrotyrosine by immunohistochemistry, and the expression of OCP by oxyblotimmune blotting were detected. In the DN model group, rat urinary protein excretion and renal MDA were significantly increased, while renal SOD significantly decreased and nitrotyrosine expression was obviously upregulated in the kidney. After triptolide treatment, 24-h urinary protein excretion（61.96±19.00 vs. 18.32±4.78 mg/day, P〈0.001）, renal MDA（8.09±0.79 vs. 5.45±0.68 nmol/L, P〈0.001）, and nitrotyrosine expression were decreased. Furthermore, renal OCP significantly decreased, while renal SOD（82.50±19.10 vs. 124.00±20.52 U/L, P〈0.001） was elevated. This study revealed that triptolide can down-regulate the expression of OCP in the renal cortex of DN rats.

Effects of Triptolide on the Expression and Activity of NF-κB in Synovium of Collagen-induced Arthritis Rats

Summary： The expression and activity of NF-kB in the synovium of collagen-induced arthritis （CIA） rats was detected in order to investigate the possible therapeutic effects of triptolide on rheumatoid arthritis （RA）. The experimental Wistar rat model of CIA was set up by intradermal injection of emulsion of bovine collagen 11 and the successful rate of setting-up models was evaluated by arthritis index （AI）. Rats were grouped randomly into three groups： normal, model and treatment group. The expression of TNF-α and IL-6 in synovial fluid was detected by ELISA, and the expression and activity of NF kB in synovium by immunohistochemistry method and by electrophoretic mobility shift assay （EMSA） respectively. As compared with normal group, the expression of TNF a and IL-6 in synovia （P〈0. 05）, and the expression and activity of NF-kB （P〈0.05） in synovium were increased in model group. There was statistical difference in above-mentioned indexes between model group and treatment group. Triptolide may play a protective role in IRA via downregulating the expression and activity of NF-kB in synovium.

Triptolide Inhibits Expression of Inflammatory Cytokines and Proliferation of Fibroblast-like Synoviocytes Induced by IL-6/sIL-6R-Mediated JAK2/STAT3 Signaling Pathway

Triptolide,a component of the Chinese herb Tripterygium wilfordii Hook F,has been proved to be effective in the treatment of rheumatoid arthritis(RA).However,its underlying mechanisms on RA have not yet been well established.We observed the inhibitory effect of triptolide on the expression of inflammatory cytokines and proliferation of fibroblast-like synoviocytes(FLS)induced by the complex of interleukin-6(IL-6)and the soluble form of the IL-6 receptor(sIL-6R).Furthermore,to clarify the underlying mechanisms,we treated FLS with the Janus-activated kinase 2(JAK2)inhibitor/signal transducer and activator of transcription 3(STAT3)activation blocker AZD1480.In this study,immunohistochemical staining was used to identify vimentin(+)and CD68(−)in FLS.The FLS proliferation was measured by cell proliferation assay,and the cell cycles were analyzed by flow cytometry.Furthermore,ELISA was used to detect the expression of the inflammatory factors in culture solution.The expression levels of p-JAK2,JAK2,p-STAT3 and STAT3 were investigated through Western blotting analysis.The results showed that IL-6/sIL-6R significantly increased the cell proliferation and expression of inflammatory cytokines,including IL-6,interleukin-1β(IL-1β)and vascular endothelial growth factor(VEGF).Triptolide or AZD1480 inhibited the cell proliferation and inflammatory cytokine expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3.The study suggested that the physiological effects of triptolide on RA were due to its contribution to the inhibition of the inflammatory cytokine expression and FLS proliferation by suppressing the JAK2/STAT3 signaling pathway.It may provide an innovative insight into the effect of triptolide in preventing RA pathogenesis.