Inhibitory Effect Of Triptolide Research Articles

Triptolide attenuates cardiac remodeling by inhibiting pyroptosis and EndMT via modulating USP14/Keap1/Nrf2 pathway

BackgroundCardiac remodeling is a common pathological feature in many cardiac diseases, characterized by cardiac hypertrophy and fibrosis. Triptolide (TP) is a natural compound derived from Tripterygium wilfordii Hook F. However, the related mechanism of it in cardiac remodeling has not been fully understood. Methods and resultsTransverse aortic constriction (TAC)-induced cardiac hypertrophic mouse model and angiotensin II (Ang II)-induced cardiomyocytes hypertrophic model were performed. Firstly, the results indicate that TP can improve cardiac function, decreased cardiomyocyte surface area and fibrosis area, as well as lowered the protein expressions of brain natriuretic peptide (BNP), β-major histocompatibility complex (β-MHC), type I and III collagen (Col I and III). Secondly, TP suppressed cardiac pyroptosis, and decreased the levels of Interleukin-1β (IL-1β), Interleukin-18 (IL-18) by Enzyme-linked immunosorbent assay (ELISA), and pyroptosis-associated proteins. Furthermore, TP enhanced the expressions of Nuclear factor erythroid 2-related factor 2 (Nrf2) and Heme oxygenase 1 (HO-1). Interestingly, when Nrf2 was silenced by siRNA, TP lost its properties of reducing pyroptosis and cardiac hypertrophy. In addition, in the Transforming Growth Factor β1 (TGF-β1)-induced primary human coronary artery endothelial cells (HCAEC) model, TP was found to inhibit the process of endothelial-to-mesenchymal transition (EndMT), characterized by the loss of endothelial-specific markers and the gain of mesenchymal markers. This was accompanied by a suppression of Slug, Snail, and Twist expression. Meanwhile, the inhibitory effect of TP on EndMT was weakened when Nrf2 was silenced by siRNA. Lastly, potential targets of TP were identified through network pharmacology analysis, and found that Ubiquitin-Specific Protease 14 (USP14) was one of them. Simultaneously, the data indicated that decrease the upregulation of USP14 and Kelch-like ECH-Associated Protein 1 (Keap1) caused by cardiac remodeling. However, Keap1 was decreased and Nrf2 was increased when USP14 was silenced. Furthermore, CoIP analysis showed that USP14 directly interacts with Keap1. ConclusionTP can observably reduce pyroptosis and EndMT by targeting the USP14/Keap1/Nrf2 pathway, thereby significantly attenuating cardiac remodeling.

MO449: Polycystins are Upregulated in Fibrotic Kidneys and Promote Renal Fibrosis

Abstract BACKGROUND AND AIMS Mutations in PKD1 or PKD2 gene, which encodes polycystin-1 or polycystin-2, cause autosomal polycystic kidney disease (ADPKD). The development of ADPKD is associated with the progression of renal fibrosis. Whether renal fibrosis in ADPKD is a direct effect of PKD mutation or a consequence of cyst growth-induced tubular obstruction is currently unknown. Polycystin-2 has been identified as a direct target of triptolide and we used triptolide as a probe to study the role of polycystin-2 in renal fibrosis. METHOD Unilateral ureteral obstruction (UUO), unilateral ischemia-reperfusion injury (UIRI) and aristolochic acid nephropathy mouse models were established. Mice were treated with vehicle or triptolide. Western blotting and Masson's trichrome staining were performed to evaluate renal fibrosis. Moreover, human kidney 2 (HK2) cells and normal rat kidney 49F (NRK-49F) cells were used as in vitro models. RESULTS Here we showed that polycystin-2 is up-regulated in these three mouse models and tightly correlated with the expression of collagen-I in a time-dependent manner. Treatment with triptolide inhibited the expression of polycystin-2 and pro-fibrotic markers in UUO and UIRI models. Moreover, triptolide dose-dependently inhibited the expression of polycystin-2 and pro-fibrotic markers in rat renal fibroblasts or in TGF-β stimulated human renal epithelial (HK2) cells. Knockdown of PKD2 reduced the expression of pro-fibrotic markers in TGF-β stimulated or unstimulated HK2 cells. Moreover, we showed that knockdown of PKD2 attenuated the inhibitory effect of triptolide on the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Finally, using Pkd2 conditional knockout mice, we showed that heterozygous or homozygous deletion of Pkd2 reduced the expression of pro-fibrotic markers in UUO kidneys. Polycystin-1 was also up-regulated in three renal fibrotic models and conditional deletion of Pkd1 reduced the expression of pro-fibrotic markers in UUO or folic acid-induced fibrotic kidneys. Furthermore, conditional knockout of the methyltransferase EZH2 attenuated the anti-fibrotic responses induced by PKD1 deletion in UUO kidneys. CONCLUSION Polycystins are pro-fibrotic proteins in injured kidneys suggesting that renal fibrosis in ADPKD kidneys is not a direct effect of PKD mutation.

Triptolide inhibits JAK2/STAT3 signaling and induces lethal autophagy through ROS generation in cisplatin‑resistant SKOV3/DDP ovarian cancer cells.

Advanced and recurrent ovarian cancer has a poor prognosis and is frequently resistant to numerous therapeutics; thus, safe and effective drugs are needed to combat this disease. Previous studies have demonstrated that triptolide (TPL) exhibits anticancer and sensitization effects against cisplatin (DDP)-resistant ovarian cancer both in vitro and in vivo by inducing apoptosis; however, the involvement of autophagy induced by TPL in resistant ovarian carcinoma remains unclear. In the present study, the results revealed that TPL induced autophagy to facilitate SKOV3/DDP ovarian cancer cell death. The xenograft experiment revealed that the autophagy inhibitor CQ significantly reduced TPL-mediated chemosensitization and tumor growth inhibition. Mechanically, TPL-induced autophagy in SKOV3/DDP cells was associated with the induction of ROS generation and inhibition of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription-3 (STAT3) pathway. The inhibitory effect of TPL on the JAK2/STAT3 pathway could be restored in the presence of the antioxidant NAC. Furthermore, it was further determined that TPL disrupted the interaction between Mcl-1 and Beclin1, which was prevented by the JAK2/STAT3 signaling activator IL-6. Overall, the present results revealed a novel molecular mechanism whereby TPL induced lethal autophagy through the ROS-JAK2/STAT3 signaling cascade in SKOV3/DDP cells. The present study has provided the groundwork for future application of TPL in the treatment of ovarian cancer.

Antitumor properties of triptolide: phenotype regulation of macrophage differentiation

ABSTRACTTumor-associated macrophages (TAMs), which generally exhibit an M2-like phenotype, play a critical role in tumor development. Triptolide exerts a unique bioactive spectrum of anticancer activities. The aim of this study was to determine whether triptolide has any effect on the activation of TAMs and the production of tumor-promoting mediators. ICR-1 mice with azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumors and BALB/c mice co-inoculated with 4T1 cells and M2-polarized RAW264.7 cells were used to examine whether the inhibitory effect of triptolide on tumor progression was mediated by the targeting of TAMs. Real-time PCR, Western blot, immunofluorescence staining, and flow cytometry assays were performed to determine the expression of cell surface markers and cytokine production. The results showed that triptolide inhibited macrophage differentiation toward the M2 phenotype and abolished M2 macrophage-mediated tumor progression. Furthermore, triptolide inhibited the expression of M2 markers, such as CD206, Arginase 1, and CD204, and inhibited the secretion of anti-inflammatory cytokines. Thus our study indicated that triptolide selectively inhibited the functions of M2-polarized macrophages and TAMs, and this inhibitory effect of triptolide on TAM viability, differentiation, and cytokine production might elucidate the major mechanisms underlying its antitumor activity. Our findings provide important information for the potential clinical application of triptolide in cancer therapy.

Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a.

BackgroundGinsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells.MethodsCCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3β in MLTC-1 cells.ResultsTP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3β was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3β. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3β.ConclusionThese results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.

Triptolide Induces Glioma Cell Autophagy and Apoptosis via Upregulating the ROS/JNK and Downregulating the Akt/mTOR Signaling Pathways.

Apoptosis and autophagy are the two prominent forms of developmental cell death, and researches have shown that crosstalk exists between these two processes. A prior study demonstrated that triptolide inhibited the proliferation of malignant glioma cells. However, whether apoptosis and autophagy participate in the inhibitory effect of triptolide in glioma cells has not been clarified. In the present study, we demonstrated that triptolide potently inhibited the growth of glioma cells by inducing cell cycle arrest at the G2/M phase. Additionally, the treatment with triptolide induced apoptosis and autophagy in various glioma cell lines. Triptolide-induced autophagy may have tumor-supporting effects. Autophagy and apoptosis could cross-inhibit each other in glioma cells treated with triptolide. Moreover, we found that triptolide induced ROS production and JNK activation and inhibited the activity of Akt and mTOR. Finally, we demonstrated that triptolide suppressed tumor growth in an orthotopic xenograft glioma model. Collectively, these data indicated that triptolide induced G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK and blocking the Akt/mTOR signaling pathways in glioma cells. Triptolide may be a potential anti-tumor drug targeting gliomas.

Triptolide prevents proliferation and migration of Esophageal Squamous Cell Cancer via MAPK/ERK signaling pathway

Triptolide, the component of traditional Chinese herb, has been used as an inflammatory medicine and reported to be anti-tumor for various cancers recently. However, the effect of triptolide on Esophageal Squamous Cell Cancer (ESCC) has not yet been elucidated. In the study, we found that triptolide significantly inhibited cell proliferation, invasion, migration and survivability of ESCC cells. Moreover, we observed that triptolide induced ESCC cell cycle arrest at the G1/S phase and apoptosis through cyclin D1-CDK4/6 regulation and caspases activation. In addition, we revealed that triptolide regulates cell apoptosis and metastasis by p53 and mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, respectively. Meanwhile, the inhibitory effect of triptolide on ESCC was validated in mouse xenograft model. So, we propose that triptolide may be a candidate drug for ESCC.

Effects of triptolide on pharmacokinetics of fenofibrate in rats and its potential mechanism

Triptolide and fenofibrate are often used together for the treatment of nephrotic syndrome in Chinese clinics.This study investigates the effects of triptolide on the pharmacokinetics of fenofibrate in rats and it potential mechanism.The pharmacokinetics of fenofibrate (20 mg/kg) with or without triptolide pretreatment (2 mg/kg/day for seven days) were investigated. Additionally, the inhibitory effects of triptolide on the metabolic stability of fenofibrate were investigated using rat liver microsome incubation systems.The results indicated that the Cmax (35.34 ± 7.52 vs. 30.43 ± 6.45 μg/mL), t1/2 (6.17 ± 1.15 vs. 4.90 ± 0.82 h) and AUC(0–t) (468.12 ± 35.84 vs. 416.35 ± 32.68 mg h L−1) of fenofibric acid decreased significantly (p < .05). The Tmax of fenofibric acid increased significantly (p < .05) from 5.12 ± 0.36 to 6.07 ± 0.68 h. Additionally, the metabolic stability of fenofibrate was prolonged from 35.8 ± 6.2 to 48.6 ± 7.5 min (p < .05) with the pretreatment of triptolide.In conclusion, these results indicated that triptolide could affect the pharmacokinetics of fenofibric acid, possibly by inhibiting the metabolism of fenofibrate in rat liver when they were co-administered.

Enhanced antitumor effect via combination of triptolide with 5-fluorouracil in pancreatic cancer

Background: Pancreatic cancer is one of the most aggressive types of cancer, and lack of effective treatment results with a very low 6-month survival rate. This study explores the enhancement of therapeutic effect on human pancreatic cancer cell line (AsPC-1) via combination of triptolide (TPL) and 5-fluorouracil (5-FU). Methods: Cell proliferation was measured by sulforhodamine B (SRB), apoptotic cells were assessed by flow cytometry and western blot for cleaved caspase-3 and vimentin to explore the role of vimentin protein in pancreatic cancer cell line. In vivo , AsPC-1 xenografts were treated by TPL, 5-FU and combination respectively and tumor samples were tested by western blot. Results: The results showed that TPL and 5-FU had the effects on AsPC-1 cell line with the dose dependence individually. The combination of TPL and 5-FU enhanced the cytotoxicity significantly compared to 5-FU or TPL alone. Combination index (CI) indicated the effect of TPL combined with 5-FU was highly synergistic. Furthermore, pancreatic cancer cells treated with TPL plus 5-FU exhibited increased apoptosis and higher levels of pro-apoptotic proteins including caspase-3 activity compared to cells treated with TPL or 5-FU alone. In the mechanism study, high vimentin expression was involved in the enhanced apoptosis induced by TPL plus 5-FU. It indicated that the vimentin expression was inhibited by TPL plus 5-FU significantly. In vivo , the tumor growth of AsPC-1 xenografts was much slower in the group treated with TPL plus 5-FU compared to the group treated with TPL or 5-FU alone. Conclusions: (I) TPL has a potent therapeutic effect on pancreatic cancer cell lines; (II) the combination of TPL with 5-FU enhances the therapeutic effect in vitro and in vivo TPL showed high synergistic effect with 5-FU; (III) the inhibitory effect of TPL plus 5-FU on pancreatic cancer is mediated by the induction of apoptosis and TPL enhanced the apoptosis via inhibition of vimentin expression.

Effects of triptolide on pharmacokinetics of amlodipine in rats by using LC–MS/MS

Context: Triptolide and amlodipine are often simultaneously used for reducing urine protein excretion after renal transplantation in China clinics.Objective: This study investigated the effects of triptolide on the pharmacokinetics of amlodipine in male Sprague–Dawley rats.Materials and methods: The pharmacokinetics of amlodipine (1 mg/kg) with or without triptolide pre-treatment (2 mg/kg/day for seven days) were investigated using a sensitive and reliable LC–MS/MS method. Additionally, the inhibitory effects of triptolide on the metabolic stability of amlodipine were investigated using rat liver microsome incubation systems.Results: The results indicated that when the rats were pre-treated with triptolide, the Cmax of amlodipine increased from 13.78 ± 3.57 to 19.96 ± 4.56 ng/mL (p < 0.05), the Tmax increased from 4.04 ± 1.15 to 5.89 ± 1.64 h (p < 0.05), and the AUC0–t increased by approximately 104% (p < 0.05), which suggested that the pharmacokinetic behaviour of amlodipine was affected after oral co-administration of triptolide. Additionally, the metabolic half-life was prolonged from 22.5 ± 4.26 to 36.8 ± 6.37 min (p < 0.05) with the pre-treatment of triptolide.Conclusions: In conclusion, these results indicated that triptolide could affect the pharmacokinetics of amlodipine, possibly by inhibiting the metabolism of amlodipine in rat liver when they are co-administered.

Triptolide Suppresses Alkali Burn-Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression.

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti-inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical-induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real-time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn-induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose-dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn-induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348-1355, 2017. © 2017 Wiley Periodicals, Inc.

Triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer.

Triptolide has been shown to exhibit anticancer activity. However, its mechanism of action is not clearly defined. Herein we report a novel signaling pathway, MDM2/Akt, is involved in the anticancer mechanism of triptolide. We observed that triptolide inhibits MDM2 expression in human breast cancer cells with either wild-type or mutant p53. This MDM2 inhibition resulted in decreased Akt activation. More specifically, triptolide interfered with the interaction between MDM2 and the transcription factor REST to increase expression of the regulatory subunit of PI3-kinase p85 and consequently inhibit Akt activation. We further showed that, regardless of p53 status, triptolide inhibited proliferation, induced apoptosis, and caused G1 phase cell cycle arrest. Triptolide also enhanced the cytotoxic effect of doxorubicin. MDM2 inhibition plays a causative role in these effects. The inhibitory effect of triptolide on MDM2-mediated Akt activation was eliminated with MDM2 overexpression. MDM2-overexpressing tumor cells, in turn, were less susceptible to the anticancer and chemosensitization effects of triptolide than control cells. Triptolide also exhibited anticancer and chemosensitization effects in nude mouse xenograft model. When it was administered to tumor-bearing nude mice, triptolide inhibited tumor growth and enhanced the antitumor effects of doxorubicin. In summary, triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer. Our study helps to elucidate the p53-independent regulatory function of MDM2 in Akt signaling, offering a novel view of the mechanism by which triptolide functions as an anticancer agent.

Inhibitory Effects of Triptolide on Human Liver Cytochrome P450 Enzymes and P-Glycoprotein.

Triptolide is an active component derived from Tripterygium wilfordii and it possesses numerous pharmacological activities. However, it remains unclear how triptolide influences the activity of human liver cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp). In this study, the inhibitory effects of triptolide on the eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs), and the effects of triptolide on the activity of P-gp were investigated using a rhodamine-123 uptake assay. The results showed that triptolide inhibited the activity of CYP1A2 and CYP3A4, with 50 % inhibitory concentration (IC50) values of 14.18 and 8.36μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that triptolide was not only a non-competitive inhibitor of CYP1A2, but also a competitive inhibitor of CYP3A4, with inhibition constant (K i) values of 7.32 and 5.67μM, respectively. In addition, triptolide is a time-dependent inhibitor for CYP1A2, and the concentration at 50 % maximum inactivation (K I) and maximum inactivation (K inact) values were 286.5μM and 0.024min-1, respectively. The rhodamine-123 uptake assay showed that triptolide could not affect the activity of P-gp. The in vitro studies of triptolide with CYP isoforms and P-gp indicate that triptolide has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2 and CYP3A4. Further clinical studies are needed to evaluate the significance of this interaction.

Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated that triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells.

Inhibitory effects of triptolide on titanium particle-induced osteolysis and receptor activator of nuclear factor-κB ligand-mediated osteoclast differentiation.

We examined the effects of triptolide on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and on titanium (Ti) particle-induced osteolysis. To examine the effect of triptolide on osteoclast differentiation, bone marrow macrophages (BMMs) were treated with 100ng/mL of RANKL and 30ng/mL of macrophage-colony stimulating factor, or co-cultured with osteoblasts stimulated with 10nM vitamin D3 and 1μM prostaglandin E2 in the presence or absence of triptolide (2.8-14nM). Osteoclast differentiation and activation were assessed using tartrate-resistant acid phosphatase staining, reverse transcriptase-polymerase chain reaction analysis to determine differentiation marker gene expression and pit formation assays. To examine the effect of triptolide on wear debris-induced osteolysis, titanium (Ti) particles were injected into the calvaria of ICR mice. Then, the mice were divided into three groups and were orally administered vehicle, or 16 or 32μg/kg/day triptolide for tendays, followed by histomorphometric analysis. Triptolide suppressed RANKL-mediated osteoclast differentiation of BMMs in a dose-dependent manner. In a co-culture system, osteoblasts treated with triptolide could not induce osteoclast differentiation of BMMs, which was accompanied by down-regulation of RANKL and up-regulation of osteoprotegrin. Moreover, triptolide significantly inhibited bone resorption, and expression of the bone resorption marker genes. RANKL-induced activation of p38, ERK, and JNK was substantially inhibited by triptolide. Further, in a Ti-induced mouse calvarial erosion model, mice perorally administrated with triptolide showed significant attenuation of Ti-mediated osteolysis. Our data indicated that triptolide had an anti-osteoclastic effect and significantly suppressed wear debris-induced osteolysis in mice.

Effect of triptolide on aromatase activity in human placental microsomes and human placental JEG-3 cells

Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F, engages in multiple pharmacological activities. However, it interferes with the synthesis of sex steroid hormones. This was proven in the present study of male and female subjects with severe reproductive toxicities related to abnormal androgen or estrogen levels. The inhibitory action of triptolide on aromatase, a terminal enzyme responsible for the formation of estrogens from androgens, was determined in vitro to investigate its role in endocrine secretion. Triptolides were incubated with human placental microsomes for 20 min at concentrations of 10, 20, 30, and 40 nM. At 20 nM, triptolide significantly inhibited aromatase, as shown by [3H]2O assay, and its concentration at 50% inhibition (IC50) was determined as approximately 35 nM. Furthermore, the inhibitory effect of triptolide on aromatase was observed in human placental choriocarcinoma (JEG-3) cell lines. Treatment with 10 nM triptolide significantly inhibited the aromatase of JEG-3 cells and gave an IC50 value of approximately 17 nM. Under these concentrations, decreased levels of mRNA and protein expression of aromatase in JEG-3 cells were observed using Western blot analysis and quantitative real-time reverse transcription polymerase chain reaction assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test revealed that treatment with triptolide for 24 h under these concentrations did not trigger evident cell death. These findings characterize the molecular mechanisms of triptolide on the regulation of sex steroid hormones.

Enhanced antitumor effect via combination of triptolide with 5-fluorouracil in pancreatic cancer cell lines.

352 Background: Pancreatic cancer is one of the most aggressive types of cancer, and lack of effective treatment results in a very low 6-month survival rate. This study aims to explore the enhancement of therapeutic effect on human pancreatic cancer cell lines (AsPC-1, MiaPaCa-2 and PANC-1) via combination of triptolide (TPL),derived from the herb Tripterygium wilfordii, and 5-fluorouracil (5-FU). Methods: Cell proliferation was measured by MTT, apoptotic cells were assessed by flow cytometry and western blot for cleaved caspase-8, 9, 3 and PARP. To explore the role of nuclear factor kappaB (NF-kB) activity in pancreatic cancer cell lines, AsPC-1/IkBaM and PANC-1/IkBaM cells (NF-kB activity of AsPC-1 and PANC-1 cells was silenced by IkB-a mutation) were treated with TPL plus 5-FU. NF-kB activity was determined by electrophoretic mobility shift assay. Results: TPL demonstrated toxicity on three pancreatic cancer cell lines with the IC50 of 25-40 nM. The combination of TPL (IC30 concentration) and 5-FU enhanced the cytotoxicity significantly compared to not only 5-FU alone but also Gemcitabine (the first line drug for advanced pancreatic cancer) alone. Combination index (CI) indicated the effect of TPL plus 5-FU was highly synergistic. Furthermore, pancreatic cancer cells treated with TPL plus 5-FU exhibited increased apoptosis, as evidenced by stronger Annexin V/ PI staining, higher levels of pro-apoptotic proteins including cleaved caspases and activated PARP compared to cells treated with TPL or 5-FU or Gemcitabine alone. In the mechanism study, AsPC-1/IkBaM and PANC-1/IkBaM cells showed more resistance to enhanced apoptosis induced by TPL plus 5-FU compared to wild-type cells. It indicated the enhanced effect of TPL was related with NF-kB activity. Conclusions: 1) TPL has a potent therapeutic effect on pancreatic cancer cell lines; 2) the combination of TPL with 5-FU enhances the therapeutic effect which is more powerful than Gemcitabine in vitro, low concentration of TPL showed high synergistic effect with 5-FU; 3) the inhibitory effect of TPL plus 5-FU on pancreatic cancer is mediated by the induction of apoptosis and TPL enhanced the apoptosis via inhibition of NF-kB activity.

Inhibitory effect of triptolide on interleukin-18 and its receptor in rheumatoid arthritis synovial fibroblasts

To determine the effects of triptolide (TP) on the expression of interleukin-18 (IL-18) and its receptor in phorbol 12-myristate 13-acetate (PMA)-stimulated rheumatoid arthritis synovial fibroblasts (RASF). RASF were obtained from the synovial tissue of patients with RA. RASF were pretreated with TP (0~100 ng/ml) for 2 h before stimulation with PMA (50 ng/ml). The bioactivity of IL-18 in the supernatant was detected based on IFN-gamma secretion from IL-18-responding human myelomonocytic KG-1 cells. IL-18 level was analyzed by ELISA. In situ expression of IL-18Ralpha was determined by immunofluorescence assay. To estimate the protein and mRNA expression of IL-18 and IL-18Ralpha in RASF, western blot and quantitative RT-PCR were performed. Nuclear factor-kappaB (NF-kappaB) activity in the whole-cell extract of treated RASF was also measured using an ELISA-based method. TP effectively inhibited the bioactivity of IL-18 in PMA-stimulated RASF. The expression of IL-18 and IL-18R at protein and gene levels was reduced by TP. NF-kappaB activity in PMA-stimulated RASF was profoundly suppressed by TP. These effects showed a high correlation with TP concentration (0~100 ng/ml). TP effectively inhibited the expression of IL-18 and its receptor in PMA-stimulated RASF. These results suggest a mechanism of TP in RA therapy.

Triptolide inhibits NF-κB activation and reduces injury of donor lung induced by ischemia/reperfusion

To investigate the protective effect of triptolide (TRI) on ischemia/reperfusion-induced injury of transplanted rabbit lungs and to investigate the mechanisms underlying the actions of TRI. We established the rabbit lung transplantation model and studied lung injury induced by ischemia/reperfusion and the inhibitory effect of TRI on NF-kappaB. The severity of lung injury was determined by a gradual decline in PvO2, the degree of lung edema, the increase in the myeloperoxidase (MPO) activity, and the ultrastructural changes of transplanted lungs. The activation of NF-kappaB was measured by immunohistochemistry. The increase in intercellular adhesion molecule-1 (ICAM-1), which is the target gene of NF-kappaB, was evaluated by ELISA. After reperfusion, there was a gradual decline in the PvO2 level in the control group (group I). The level of PvO2 in the group treated with lipopolysaccharide (group II) was significantly decreased, whereas that of the group treated with TRI (group III) was markedly improved (P<0.01). In group III, the activity of MPO was downregulated, and the pulmonary edema did not become severe and the ultrastructure of the donor lung remained normal. The activity of NF-kappaB and the expression of ICAM-1 was significantly increased in the donor lungs. TRI blocked NF-kappaB activation and ICAM-1 expression. The effects of TRI on reducing injury to donor lungs induced by ischemia/reperfusion may possibly be mediated by inhibiting the activity of NF-kappaB and the expression of the NF-kappaB target gene ICAM-1. Thus, TRI could be used in lung transplantations for improving the function of donor lungs.

Triptolide suppresses interleukin-1β-induced human β-defensin-2 mRNA expression through inhibition of transcriptional activation of NF-κB in A549 cells

The immunosuppressive effect of triptolide has been associated with suppression of T-cell activation. However, the immunosuppressive effects of triptolide on innate immunity in the epithelial barrier remain to be elucidated. Human beta-defensin (HBD)-2 is an inducible antimicrobial peptide and plays an important role in the innate immunity. We have previously demonstrated that IL-1beta induced HBD-2 mRNA expression in A549 cells through activation of nuclear factor-kappaB (NF-kappaB) transcriptional factor as well as p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), or phosphatidylinositol-3-kinase (PI3K). In this study, we investigated effects of triptolide on IL-1beta-induced HBD-2 mRNA expression in A549 cells. Triptolide inhibited IL-1beta-induced HBD-2 mRNA expression in a dose-dependent manner. Addition of triptolide did not suppress activation of p38 MAPK, JNK, or PI3K in response to IL-1beta. Triptolide inhibited IL-1beta-induced MAPK phosphatase-1 expression at the transcriptional level and resulted in sustained phosphorylation of JNK or p38 MAPK, explaining the little effect of triptolide on IL-1beta-induced phosphorylation of these kinases. Although triptolide partially suppressed IL-1beta-mediated degradation of IkappaB-alpha and nuclear translocation of p65 NF-kappaB, triptolide potently inhibited NF-kappaB promoter-driven luciferase activity in A549 cells. These results collectively suggest that the inhibitory effect of triptolide on IL-1beta-induced HBD-2 mRNA expression in A549 cells seems to be at least in part mediated through nuclear inhibition of NF-kappaB transcriptional activity, but not inhibition of p38 MAPK, JNK, or PI3K. This inhibition may explain the ability of triptolide to diminish innate immune response.