Triptolide Treatment Research Articles

Triptolide decreases rheumatoid arthritis fibroblast-like synoviocyte proliferation, invasion, inflammation and presents a therapeutic effect in collagen-induced arthritis rats via inactivating lncRNA RP11-83J16.1 mediated URI1 and β-catenin signaling

ObjectiveOur previous study observed that long non-coding RNA (lncRNA) RP11-83J16.1 promoted rheumatoid arthritis (RA)-fibroblast-like synoviocyte (RA-FLS) proliferation, invasion and inflammation, which was downregulated by triptolide treatment. Therefore, the present study aimed to further investigate the mechanism and interaction between triptolide and lncRNA RP11-83J16.1 in RA treatment in vitro and in vivo. MethodsRA-FLS was isolated and treated by different concentration of triptolide and lncRNA RP11-83J16.1 overexpression plasmid. Furthermore, collagen-induced arthritis (CIA) rat model was constructed followed by triptolide and lncRNA RP11-83J16.1 overexpression plasmid treatment. ResultsTriptolide inhibited RA-FLS viability and lncRNA RP11-83J16.1 expression in a dose-dependent manner. Afterward, triptolide treatment inhibited RA-FLS proliferation, invasion, levels of inflammatory markers (TNF-α, IL-1β, IL-6, MMP-3, and MMP-9), inactivated lncRNA RP11-83J16.1, URI1 and β-catenin signaling, but promoted apoptosis. However, lncRNA RP11-83J16.1 overexpression weakened the effects of triptolide on regulating RA-FLS cell behaviors, URI1 signaling and β-catenin signaling. In CIA model, triptolide decreased arthritis score, hyperproliferation of synovial cells, inflammation infiltration of synovial tissue, inflammatory markers (TNF-α, IL-1β, IL-6, MMP-3, and MMP-9), inactivated lncRNA RP11-83J16.1, URI1 and β-catenin signaling, but increased cell apoptosis rate of synovial tissue. Nevertheless, lncRNA RP11-83J16.1 curtailed the treatment effect of triptolide in CIA model. ConclusionTriptolide decreases RA-FLS proliferation, invasion, inflammation and presents a therapeutic effect in CIA model via inactivating lncRNA RP11-83J16.1 mediated URI1 and β-catenin signaling.

Triptolide promotes the apoptosis and attenuates the inflammation of fibroblast-like synoviocytes in rheumatoid arthritis by down-regulating lncRNA ENST00000619282.

Rheumatoid arthritis (RA), recognized as a common chronic autoimmune disease, is characterized by the excessive proliferation and inflammatory infiltration of fibroblast-like synoviocytes (FLS). In this study, our purpose is to elucidate the mechanisms of triptolide (TPL) in the treatment of RA by regulating the long non-coding RNA (lncRNA) ENST00000619282, which promoted apoptosis and reduced inflammatory infiltration of FLS in RA (RA-FLS). RA-FLS was treated with different concentrations of TPL at different time points. CCK-8 assay, ELISA, RT-qPCR, immunofluorescence, TUNEL assay, and the transmission electron microscopy were used to measure the changes of cell viability, apoptosis, and the release of inflammatory cytokines. Next, the involvement of ENST00000619282 in TPL-mediated protection against RA was explored. ENST00000619282 expression was significantly increased in the peripheral blood mononuclear cells (PBMCs) of RA patients. ENST0000061928 expression in RA PBMCs was positively associated with ESR, RF, CCP, and DAS28, while TPL treatment led to a downregulation of ENST00000619282. In addition, ENST00000619282 was significantly increased in RA-FLS. Furthermore, overexpression of ENST00000619282 elevated the levels of pro-apoptotic and pro-inflammatory factors, while reduced the levels of anti-apoptotic proteins and antiinflammatory factors. Besides, TPL treatment could reverse these effects by ENST00000619282 overexpression. The anti-RA potential of TPL might be achieved by downregulating ENST00000619282, thereby promoting apoptosis, and reducing the inflammatory response in RA.

Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-\u0399\u0399

BackgroundPyroptosis is a lytic cell death form executed by gasdermins family proteins. Induction of tumor pyroptosis promotes anti-tumor immunity and is a potential cancer treatment strategy. Triptolide (TPL) is a natural product isolated from the traditional Chinese herb which possesses potent anti-tumor activity in human cancers. However, its role in pyroptosis remains to be elucidated.MethodsCell survival was measured by colony formation assay. Cell apoptosis was determined by Annexin V assay. Pyroptosis was evaluated by morphological features and release of interleukin 1β and lactate dehydrogenase A (LDHA). Immunofluorescence staining was employed to measure subcellular localization of proteins. Tumorigenicity was assessed by a xenograft tumor model. Expression levels of mRNAs or proteins were determined by qPCR or western blot assay, respectively.ResultsTriptolide eliminates head and neck cancer cells through inducing gasdermin E (GSDME) mediated pyroptosis. Silencing GSDME attenuates the cytotoxicity of TPL against cancer cells. TPL treatment suppresses expression of c-myc and mitochondrial hexokinase II (HK-II) in cancer cells, leading to activation of the BAD/BAX-caspase 3 cascade and cleavage of GSDME by active caspase 3. Silencing HK-II sensitizes cancer cells to TPL induced pyroptosis, whereas enforced expression of HK-II prevents TPL induced pyroptosis. Mechanistically, HK-II prevents mitochondrial translocation of BAD, BAX proteins and activation of caspase 3, thus attenuating cleavage of GSDME and pyroptosis upon TPL treatment. Furthermore, TPL treatment suppresses NRF2/SLC7A11 (also known as xCT) axis and induces reactive oxygen species (ROS) accumulation, regardless of the status of GSDME. Combination of TPL with erastin, an inhibitor of SLC7A11, exerts robust synergistic effect in suppression of tumor survival in vitro and in a nude mice model.ConclusionsThis study not only provides a new paradigm of TPL in cancer therapy, but also highlights a crucial role of mitochondrial HK-II in linking glucose metabolism with pyroptosis.

Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS mice

BackgroundAlveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PurposeThe aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. Methods50 μl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 μg/kg, 10 μg/kg, 50 μg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 μg/kg, 10 μg/kg, 50 μg/kg) were intraperitoneally injected or 10 μg/50 μl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. ResultsLPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/β and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. ConclusionsTP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

Celastrol and Triptolide Suppress Stemness in Triple Negative Breast Cancer: Notch as a Therapeutic Target for Stem Cells.

Triple negative breast cancer (TNBC) is observed in ~15% of breast cancers and results in poor survival and increased distant metastases. Within the tumor are present a small portion of cancer stem cells that drive tumorigenesis and metastasis. In this study, we aimed to elucidate whether the two natural compounds, celastrol and triptolide, inhibit stemness in TNBC. MDA-MB-231, BT20, and a patient-derived primary cells (PD-TNBC) were used in the study. Mammosphere assay was performed to assess the stemness. Both celastrol and triptolide treatment suppressed mammosphere formation. Furthermore, the compound suppressed expression of cancer stem cell marker proteins DCLK1, ALDH1, and CD133. Notch signaling plays a critical role in stem cells renewal. Both celastrol or triptolide reduced Notch -1 activation and expression of its downstream target proteins HES-1 and HEY-1. However, when NICD 1 was ectopically overexpressed in the cells, it partially rescued proliferation and mammosphere formation of the cells, supporting the role of notch signaling. Together, these data demonstrate that targeting stem cells and the notch signaling pathway may be an effective strategy for curtailing TNBC progression.

Triptolide suppresses the growth and metastasis of non-small cell lung cancer by inhibiting \u03b2-catenin-mediated epithelial\u2013mesenchymal transition

Non-small cell lung cancer (NSCLC) is characterized by a high incidence of metastasis and poor survival. As epithelial–mesenchymal transition (EMT) is well recognized as a major factor initiating tumor metastasis, developing EMT inhibitor could be a feasible treatment for metastatic NSCLC. Recent studies show that triptolide isolated from Tripterygium wilfordii Hook F attenuated the migration and invasion of breast cancer, colon carcinoma, and ovarian cancer cells, and EMT played important roles in this process. In the present study we investigated the effect of triptolide on the migration and invasion of NSCLC cell lines. We showed that triptolide (0.5, 1.0, 2.0 nM) concentration-dependently inhibited the migration and invasion of NCI-H1299 cells. Triptolide treatment concentration-dependently suppressed EMT in NCI-H1299 cells, evidenced by significantly elevated E-cadherin expression and reduced expression of ZEB1, vimentin, and slug. Furthermore, triptolide treatment suppressed β-catenin expression in NCI-H1299 and NCI-H460 cells, overexpression of β-catenin antagonized triptolide-caused inhibition on EMT, whereas knockout of β-catenin enhanced the inhibitory effect of triptolide on EMT. Administration of triptolide (0.75, 1.5 mg/kg per day, ip, every 2 days) for 18 days in NCI-H1299 xenograft mice dose-dependently suppressed the tumor growth, restrained EMT, and decreased lung metastasis, as evidence by significantly decreased expression of mesenchymal markers, increased expression of epithelial markers as well as reduced number of pulmonary lung metastatic foci. These results demonstrate that triptolide suppresses NSCLC metastasis by targeting EMT via reducing β-catenin expression. Our study implies that triptolide may be developed as a potential agent for the therapy of NSCLC metastasis.

Triptolide Modulates the Expression of Inflammation-Associated lncRNA-PACER and lincRNA-p21 in Mycobacterium tuberculosis-Infected Monocyte-Derived Macrophages.

Triptolide is a diterpene triepoxide, which performs its biological activities via mechanisms including induction of apoptosis, targeting of pro-inflammatory cytokines, and reshaping of the epigenetic landscape of target cells. However, the targeting of long non-coding RNAs (lncRNAs) by triptolide has not yet been investigated, despite their emerging roles as key epigenetic regulators of inflammation and immune cell function during Mycobacterium tuberculosis (Mtb) infection. Hence, we investigated whether triptolide targets inflammation-associated lncRNA-PACER and lincRNA-p21 and how this targeting associates with Mtb killing within monocyte-derived macrophages (MDMs).Using RT-qPCR, we found that triptolide induced the expression of lincRNA-p21 but inhibited the expression of lncRNA-PACER in resting MDMs in a dose- and time-dependent manner. Moreover, Mtb infection induced the expression of lincRNA-p21 and lncRNA-PACER, and exposure to triptolide before or after Mtb infection led to further increase of Mtb-induced expression of these lncRNAs in MDMs. We further found that contrary to lncRNA-PACER, triptolide time- and dose-dependently upregulated Ptgs-2, which is a proximal gene regulated by lncRNA-PACER. Also, low-concentration triptolide inhibited the expression of cytokine IL-6, a known target of lincRNA-p21. Mtb infection induced the expression of IL-6 and Ptgs-2, and triptolide treatment further increased IL-6 but decreased Ptgs-2 expression in Mtb-infected MDMs. The inverse relation between the expression of these lncRNAs and their target genes is concordant with the conception that these lncRNAs mediate, at least partially, the cytotoxic and/or anti-inflammatory activities of triptolide in both resting and activated MDMs. Using the CFU count method, we found that triptolide decreased the intracellular growth of Mtb HN878. The alamarBlue assay showed that this decreased Mtb HN878 growth was not as a result of direct targeting of Mtb HN878 by triptolide, but rather evoking MDMs’ intracellular killing mechanisms which we speculate could include triptolide-induced enhancement of MDMs’ effector killing functions mediated by lncRNA-PACER and lincRNA-p21. Altogether, these results provide proof of the modulation of lncRNA-PACER and lincRNA-p21 expression by triptolide, and a possible link between these lncRNAs, the enhancement of MDMs’ effector killing functions and the intracellular Mtb-killing activities of triptolide. These findings prompt for further investigation of the precise contribution of these lncRNAs to triptolide-induced activities in MDMs.

A Triptolide Loaded HER2-Targeted Nano-Drug Delivery System Significantly Suppressed the Proliferation of HER2-Positive and BRAF Mutant Colon Cancer.

BackgroundColon cancer (CRC) was a malignant tumor and there were about 25% of patients with tumor metastasis at diagnosis stage. Chemotherapeutic agents for metastatic CRC patients were with great side effects and the clinical treatment results of advanced CRC were still not satisfactory. Human epidermal growth factor receptor 2 (HER2) is overexpressed in some CRC patients and is an effective target for CRC patient treatment. Anti-HER2 therapy had a beneficial role in the treatment of HER2-positive metastatic CRC with fewer side effects. CRC patients with BRAF mutations were resistant to HER2 antibodies treatment. Therefore, there was an urgent need to develop new therapeutic agents.MethodsHER2 targeted nanoparticles (TPLNP) drug delivery system loading triptolide (TPL) were prepared and identified. The effects of TPLNP and free TPL on cell viability, targeting and cell cycle progression on HT29 (BRAF mutation) with HER2 overexpression, were evaluated by Cell Counting Kit-8 (CCK8), Fluorescence Activating Cell Sorter (FACS) and immunofluorescence methods, respectively. The anti-tumor efficacies of TPLNP were evaluated in subcutaneous xenograft model of colon cancer and the survival rate, tumor volume, liver and kidney indexes of tumor-bearing mice were measured.ResultsTPLNP was small in nanosize (73.4±5.2nm) with narrow size distribution (PDI=0.15±0.02) and favorable zeta potential (pH=9.6, zeta potential: −57.3±6.69mV; pH=7.0, zeta potential: −28.7±5.1mV; pH=5.6, zeta potential: −21.1±4.73mV). Comparing with free TPL treatment group, TPLNP developed stranger colon cancer-killing efficiency in a dose- and time-dependent manner detected with CCK8 method; achieved good in vitro colon cancer targeting detected with flow cytometry and immunofluorescence experiments; enhanced more HT29-HER2 apoptosis and induced more cell cycle arrested in G1-S phase detected with FACS in vitro. As for in vivo antitumor response, TPLNP remarkably inhibited the growth of colon cancer in the colon cancer xenograft model, significantly improved the survival rate and did not exhibit significant liver and kidney toxicity in contrast with free TPL in vivo.ConclusionTPLNP was effectively against colon cancer with HER2 overexpression and BRAF mutation in pre-clinical models. In summary, the TPLNP appeared to be a promising treatment option for CRC in clinical application based on improved efficacy and the favorable safety profile.

The antioxidant effect of triptolide contributes to the therapy in a collagen-induced arthritis rat model

ABSTRACT Background As a chronic autoimmune disease, rheumatoid arthritis (RA) is related to oxidative stress, which may lead to the occurrence and persistence of inflammation in RA. The purpose of this study is to evaluate the potential antioxidant effect of triptolide in collagen-induced arthritis (CIA) rat model. Methods We examined the severity of arthritis, levels of local and systemic oxidative stress, periarticular bone erosion and weight of organs in CIA rats treated with triptolide. Results We found that triptolide decreased the paw thickness and clinical arthritis score, significantly. The mRNA expression and activity of myeloperoxidase and inducible nitric oxide synthase were remarkably decreased in the paws of the CIA rats after triptolide treatment. Triptolide significantly inhibited the levels of nitrite and nitrate in serum, as well as the urinary level of dityrosine. Triptolide treatment also markedly increased bone volume of tibia, but suppressed epiphyseal plate thickness of both femur and tibia. In addition, there was no significant difference in the weight of organs after the therapy, except decreased spleen weight. Conclusions These results suggested that the local and systemic oxidative stress was enhanced in the CIA rats and the therapeutic dose of triptolide had a definite antioxidant effect.

Therapeutic Potential of Triptolide as an Anti-Inflammatory Agent in Dextran Sulfate Sodium-Induced Murine Experimental Colitis.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), is a group of chronic and incurable inflammatory diseases involving the gastrointestinal tract. In this study, we investigated the anti-inflammatory effects of triptolide in a dextran sulfate sodium (DSS)-induced mouse colitis model and LPS-activated macrophages and explored the specific molecular mechanism(s). In mice, triptolide treatment showed significant relief and protection against colitis, and it markedly reduced the inflammatory responses of human monocytes and mouse macrophages. Pharmacological analysis and weighted gene co-expression network analysis (WGCNA) suggested that PDE4B may be an important potential targeting molecule for IBD. Exploration of the specific mechanism of action indicated that triptolide reduced the production of ROS, inhibited macrophage infiltration and M1-type polarization by activating the NRF2/HO-1 signaling pathway, and inhibited the PDE4B/AKT/NF-κB signaling cascade, which may help weaken the intestinal inflammatory response. Our findings laid a theoretical foundation for triptolide as a treatment for IBD and revealed PDE4B as a target molecule, thus providing new ideas for the treatment of IBD.

Low-Dose Triptolide Promotes MDM2 Inhibitor Nutlin3a to Induce Acute Myeloid Leukemia Cell Death Via p53-Dependent and -Independent Mechanisms

The tumor suppressor p53 is central to hematopoietic stem cell function. Loss of p53 function is associated with poor prognosis of acute myeloid leukemia (AML). P53 negative regulator MDM2 is frequently overexpressed in AML, thus it becomes an attractive therapeutic target for the treatment of AML with wild-type p53 (Wt-p53). Targeting MDM2 to restore p53 activity has been assessed in AML, but clinical outcomes are less promising. Notably, p53-mutated AML still lacks effective treatment approaches. Therefore, our study strived to explore potent therapeutics to target AML with p53 wild-type and mutations. Triptolide (TPL), an ancient herbal medicine, has been used to treat immune disorders for centuries. Accumulating evidence including ours have proven that low dose of TPL potentiates the efficacy of chemotherapies in a broad range of tumor types. In this study, we evaluated the anti-leukemic efficacy of combining low dose TPL with Nutlin3a, a MDM2 inhibitor, against AML with p53 wild-type and mutations. We found that low-dose TPL synergized with Nutlin3a to induce mitochondrial apoptosis in AML cells with Wt-p53 bothin vitroandin vivo. Underlying mechanism for the synergy showed that Nutlin3a upregulated pro-apoptosis factors (e.g. PUMA, p21), while low-dose TPL suppressed MDM2 transcription and reduced anti-apoptosis proteins (e.g. XIAP, Mcl-1) in Wt-p53 AML cells. Interestingly, we also observed that TPL but not Nutlin3a induced cell death in p53 shRNA knockdown, p53 deficiency cell lines (e.g. THP-1, HL-60) and primary samples. Thus, the p53-independent role of TPL was consequently focused. Our RNA-Seq analysis identified 621 differential expression genes (DEGs) after TPL treatment. These genes, including MYC, ATF4 and 4EBP1, were enriched in intrinsic apoptosis pathway responding to ER stress and thus we concluded that Triptolide played its p53 independent role via MYC-ATF4 axis. Collectively, these findings suggest that coadministration of Nutlin3a with low dose TPL would benefit for AML patients. Disclosures Carter: AstraZeneca:Research Funding;Syndax:Research Funding;Ascentage:Research Funding;Amgen:Research Funding. OffLabel Disclosure: Triptolide (TPL), an ancient herbal medicine, is applying to treat immune disorders.

Downregulation of miR-200a Protects Mouse Leydig Cells Against Triptolide by Triggering Autophagy.

BackgroundMicroRNAs play important roles in testicular development and spermatogenesis. Previous research has indicated that the level of miR-200a was significantly upregulated in patients with different spermatogenic impairments. However, the mechanism by which miR-200a regulated spermatogenic impairments remains unclear.MethodsLeydig cells were treated with triptolide (TP) to mimic spermatogenic impairments. CCK-8 and flow cytometry were used to detect the proliferation and apoptosis in Leydig cells, respectively. In addition, Western blot assay was used to examine ATG7, ATG5, p62 protein levels in MLTC-1 cells.ResultsTP dose-dependently upregulated the expression of miR-200a in MLTC-1 cells. In addition, TP inhibited the proliferation of MLTC-1 cells via inducing apoptosis and oxidative stress; however, these phenomena were notably reversed by miR-200a antagomir. Furthermore, luciferase reporter assay identified that ATG7 was the direct binding target of miR-200a. TP treatment markedly inhibited the activation of autophagy in MLTC-1 cells via inhibition of ATG7. Conversely, downregulation of miR-200a significantly induced autophagy in TP-treated MLTC-1 cells by activation of ATG7. Meanwhile, the cell protective effects of miR-200a against TP were reversed by autophagy inhibitor 3MA, indicating that autophagy plays an important role.ConclusionThese results indicated that downregulation of miR-200a could protect MLTC-1 cells against TP by inducing autophagy. Therefore, miR-200a might serve as a new therapeutic target for the treatment of male hypogonadism.

Influence of a combination of triptolide and ferulic acid on the activities of CYP450 enzymes and oxidative stress in HaCaT cells.

Topical administration of triptolide (TP) is effective in the treatment of rheumatoid arthritis (RA), but it can also induce skin irritation. Previous studies have used data mining strategies to analyze the application of Tripterygium wilfordii in the treatment of RA and have shown that TP and ferulic acid (FA) can be used in combination due to their component compatibility. The aims of the present study were to investigate the mechanisms underlying the effects of TP treatment and to identify its effects on metabolism and oxidative damage in the skin. MTT assay results suggested that the HaCaT cell survival rate was significantly increased when the compatibility ratio of TP to FA was 1:100. Moreover, the combination of TP with FA (TP + FA) did not significantly affect the activities of the cytochrome P40 (CYP) enzymes CYP family 1 subfamily A member 2 (CYP1A2), CYP2E1 and CYP3A4, when used as a ‘cocktail’. It was found that TP + FA significantly decreased the production levels of reactive oxygen species (ROS), superoxide dismutase and malondialdehyde in HaCaT cells, while significantly increasing levels of glutathione and catalase. In addition, TP + FA significantly increased nuclear factor erythroid 2-related factor 2 protein expression, compared with TP alone. Thus, the present results indicated that the underlying mechanism of TP + FA efficacy may be related to decreased ROS production level in HaCaT cells, increased production levels of key antioxidant factors and increased antioxidant activity of the epidermis, all of which were correlated with a protective effect against oxidative damage.

Effect of Triptolide on Temporal Expression of Cell Cycle Regulators During Cardiac Hypertrophy.

Adult mammalian cardiomyocytes may reenter the cell cycle and cause cardiac hypertrophy. Triptolide (TP) can regulate the expressions of various cell cycle regulators in cancer cells. However, its effects on cell cycle regulators during myocardial hypertrophy and mechanism are unclear. This study was designed to explore the profile of cell cycle of cardiomyocytes and the temporal expression of their regulators during cardiac hypertrophy, as well as the effects of TP. The hypertrophy models employed were neonatal rat ventricular myocytes (NRVMs) stimulated with angiotensin II (Ang II) for scheduled times (from 5 min to 48 h) in vitro and mice treated with isoprenaline (Iso) for from 1 to 21 days, respectively. TP was used in vitro at 1 μg/L and in vivo at 10 μg/kg. NRVMs were analyzed using flow cytometry to detect the cell cycle, and the expression levels of mRNA and protein of various cell cycle regulators were determined using real-time PCR and Western blot. It was found NRVM numbers in phases S and G2 increased, while that in the G1 phase decreased significantly after Ang II stimulation. The mRNA expression levels of p21 and p27 increased soon after stimulation, and thereafter, mRNA expression levels of all cell cycle factors showed a decreasing trend and reached their lowest levels in 1–3 h, except for cyclin-dependent kinase 1 (CDK1) and CDK4 mRNA. The mRNA expression levels of CDK1, p21, and p27 increased markedly after stimulation with Ang II for 24–48 h. In myocardium tissue, CDK and cyclin expression levels peaked in 3–7 days, followed by a decreasing trend, while those of p21 and p27 mRNA remained at a high level on day 21. Expression levels of all protein were consistent with the results of mRNA in NRVMs or mice. The influence of Ang II or Iso on protein expression was more obvious than that on mRNA. TP treatment effectively prevented the imbalance in the expression of cell cycle regulators in the hypertrophy model group. In Conclusion, an imbalance in the expression of cell cycle regulators occurs during cardiac hypertrophy, and triptolide corrects these abnormal expression levels and attenuates cardiac hypertrophy.

Healing Effects of Single-Dose Triptolide in Rats with Severe Acute Pancreatitis

Aim: Severe acute pancreatitis (SAP) carries high morbidity and mortality risk. If the proinflammatory response phase of SAP cannot be controlled, it may result in multiorgan failure (MOF). Nuclear factor-kappa B (NF-κB) activation plays an important role in the development of MOF. In this study, it was aimed to investigate the healing effects of triptolide, an anti-inflammatory and immunosuppressive agent in rats with SAP.Material and Methods: A total of 20 Wistar-Albino rats were divided into two groups as the SAP and triptolide treatment (TT) groups. SAP was induced by intraperitoneal injection of cerulean (50 mg/kg) in both groups. TT group was administered a single dose (0.2 mg/kg) triptolide 24 hour after the induction of SAP. Serum ALT, AST, GGT, Lipase, Glucose, ALP and amylase levels and pancreatic tissue samples were examined.Results: Serum glucose and amylase levels were found to be significantly lower in the TT group (p=0.011 and p=0.035, respectively). There was no significant difference between the groups in terms of other biochemical parameters. Pancreatic edema, acinar cell degeneration, fat necrosis, intrapancreatic&perivascular inflammation, inflammation in the peripancreatic fat tissue were common histopathological findings in both groups. There was no significant difference between the groups in terms of histopathologic changes.Conclusion: Cerulein-induced pancreatitis is a successful method for experimental SAP. The healing effects of single-dose triptolide treatment are not evident in the early phase of SAP. The therapeutic effects of triptolide on inflammatory and oxidative stress were not significantly approved by histopathological and biochemical parameters by the pancreatic tissue.

Triptolide protects human retinal pigment epithelial ARPE-19 cells against high glucose-induced cell injury by regulation of miR-29b/PTEN

Oxidative stress and inflammation are necessary pathogenic factors contributing to the aetiology of diabetic retinopathy (DR). Triptolide (TPL) is derived from the traditional Chinese herb lei gong teng with anti-inflammatory, immunosuppressive and antitumor activities. This article was developed to examine the effect of TPL on DR. ARPE-19 cells were pre-treated with TPL and then stimulated by high glucose (HG). We found that TPL treatment enhanced cell viability, decreased apoptosis and ROS production in HG-treated RPE cells. MiR-29b was low-expressed in HG-treated cells, but TPL raised its expression. In addition, the protective activity of TPL towards ARPE-19 cells was attenuated when miR-29b was reduced. By utilising bioinformatics evaluation, PTEN was predicted as a downstream target of miR-29b. Also, TPL obstructed PI3K/AKT signalling pathways in HG-treated ARPE-19 Cells. Taken together, TPL secured ARPE-19 cells from HG-induced oxidative damage via regulating miR-29b/PTEN axis.

Triptolide-induced apoptosis in non-small cell lung cancer via a novel miR204-5p/Caveolin-1/Akt-mediated pathway.

Lung cancer is one of the most prevalent malignancies world-wide with non-small cell lung cancer (NSCLC) comprising nearly 80% of all cases. Unfortunately, many lung cancer patients are diagnosed at advanced stages of the disease with an associated poor prognosis. Recently, the Chinese herb root extract Triptolide/Minnelide (TL) has shown significant promise as a therapeutic agent for NSCLC treatment both in vitro and in vivo. The aim of this study was to investigate the underlying mechanism(s) of action regarding TL-induced cytotoxicity in NSCLC. We demonstrate that triptolide treatment of A549 and H460 NSCLC cells decreases Caveolin-1 (CAV-1) mRNA/protein expression, resulting in activation of the Akt/Bcl-2-mediated mitochondrial apoptosis pathway. CAV-1 down-regulation was triggered by Micro-RNA 204-5p (miR204-5p) up-regulation and could be significantly blocked by pre-treatment with both Sirt-1/Sirt-3 specific siRNA and SIRT-1/SIRT-3 enzyme inhibitors, EX-527 and nicotinamide. Overall, our results provide evidence for a novel mechanism by which TL exerts its cytotoxic effects on NSCLC via CAV-1 down-regulation. Furthermore, these findings demonstrate a pivotal role for TL induction of the Akt/Bax pathway in apoptosis of human lung cancer.

Triptolide enhances lipolysis of adipocytes by enhancing ATGL transcription via upregulation of p53.

Lipolysis is an essential physiological activity of adipocytes. The Patatin Like Phospholipase Domain Containing 2 (PNPLA2) gene encodes the enzyme adipose triglyceride lipase (ATGL) responsible for triglyceride hydrolysis, the first step in lipolysis. In this study, we investigated the potential of triptolide (TP), a natural plant extract, to induce weight loss by examining its effect on ATGL expression. We found that long- and short-term TP administration reduced body weight and fat weight and increased heat production in brown adipose tissue in wild-type C57BL/6 mice. In 3T3-L1 fibroblasts and porcine adipocytes, TP treatment reduced the number of lipid droplets as determined by Oil Red O and BODIPY staining, with concomitant increases in free fatty acid and triglyceride levels in the culture medium. Combined treatment with TP and p53 inhibitor reversed these lipolytic effects. We next amplified the ATGL promoter region and identified conserved p53 binding sites in the sequence by in silico analysis. The results of the dual-luciferase reporter assay using a construct containing the ATGL promoter harboring the p53 binding site showed that p53 induces ATGL promoter activity and consequently, ATGL transcription. These results demonstrate that TP has therapeutic value as an anti-obesity agent and acts by promoting lipolysis via upregulation of p53 and ATGL transcription.

Triptolide Inhibits the Proliferation of HaCaT Cells Induced by IL22 via Upregulating miR-181b-5p.

BackgroundEvidence has been shown that triptolide was effective in the treatment of psoriasis; however, the mechanisms remain poorly understood. Thus, this study aimed to investigate the role of triptolide on the proliferation and differentiation of HaCaT cells which are treated with IL22 to mimic abnormal proliferation/differentiation in keratinocyte of psoriasis.Materials and MethodsHaCaT cells were transfected with miR-181b-5p antagomir for 24 h, and then exposed to 10 μM Triptolide for 24 h, following by 100 ng/mL of IL22 for 24 h. In addition, the proliferation and cell cycle distribution in HaCaT cells were assessed by immunofluorescence or flow cytometry assays, respectively.ResultsTriptolide obviously upregulated the level of miR-181b-5p in HaCaT cells. In addition, triptolide significantly inhibited IL22-induced proliferation of HaCaT cells via inducing cell cycle arrest. Moreover, IL22 markedly inhibited the differentiation of HaCaT cells, and this phenomenon was reversed by triptolide treatment. In contrast, the effects of triptolide on the proliferation and differentiation in IL22-stimulated HaCaT cells were notably reversed by miR-181b-5p antagomir. Moreover, dual-luciferase assay showed that E2F5 was the direct target of miR-181b-5p in HaCaT cells. Meanwhile, upregulation of miR-181b-5p obviously decreased the level of E2F5 in HaCaT cells.ConclusionIn this study, we found that triptolide could inhibit the proliferation and promote the differentiation in IL22-stimulated keratinocytes via upregulating miR-181b-5p. These data indicated that triptolide may be a potential agent for the treatment of psoriasis.

Triptolide impairs genome integrity by directly blocking the enzymatic activity of DNA-PKcs in human cells

Triptolide is a multi-functional natural small molecular compound extracted from a traditional Chinese medicinal herb. Triptolide and its derivatives exhibit cytotoxicity through inducing DNA damage, therefore increasing sensitivity to DNA-damage based chemotherapy or radiotherapy in different types of cells. However, the regulatory mechanism of genotoxicity by triptolide, and the loss of genome integrity induced by triptolide are not fully understood. Here, we measured the effects of triptolide on genome integrity in a human fibroblast line HCA2-hTERT using the neutral comet assay. We demonstrated that treating cells with triptolide induced genomic instability in HCA2-hTERT cells. Furthermore, we observed the accumulation of γH2AX foci in triptolide treated cells than control cells at 24 h post ionizing radiation. Further mechanistic studies indicated that triptolide inhibited the enzymatic activity of DNA-PKcs, the critical nonhomologous end joining factor. In vitro kinase activity assays showed that triptolide suppressed the kinase activity of DNA-PKcs and molecular docking also predicted a potential interaction between triptolide and DNA-PKcs. As a consequence, we found that triptolide treatment enhanced the interaction between DNA-PKcs and KU80 and hampered the following recruitment of 53BP1. Altogether, our finding provides a new perspective about the toxicity of triptolide in non-cancer cells and highlights the necessity of taking genome effects of triptolide and its derivatives into consideration in the future clinical and research applications.