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Background: A balanced protein homeostasis network helps cholangiocarcinoma (CCA) maintain their oncogenic growth, and disrupting proteostasis therapeutically will induce proteotoxic stress. Phosphatase and tensin homolog (PTEN) have been reported to be involved in proteostasis, and PTEN-associated pathways are commonly altered in CCA. Celastrol, a triterpene from plants, exhibits cytotoxic effects in various types of cancer. However, the underlying mechanisms remain unclear., Purpose: We investigated the therapeutic effect of celastrol in CCA and identified the molecular characteristics of tumors that were sensitive to celastrol. The target of celastrol was explored. We then evaluated the candidate combination therapeutic strategy to increase the effectiveness of celastrol in celastrol-insensitive CCA tumors., Methods: Various CCA cells were categorized as either celastrol-sensitive or celastrol-insensitive based on their response to celastrol. The molecular characteristics of cells from different groups were determined by RNA-seq. PTEN status and its role in proteasome activity in CCA cells were investigated. The CMAP analysis, molecular docking, and functional assay were performed to explore the effect of celastrol on proteasome activities. The correlation between PTEN status and clinical outcomes, as well as proteasomal activity, were measured in CCA patients. The synergistic therapeutic effect of autophagy inhibitors on celastrol-insensitive CCA cells were measured., Results: Diverse responses to celastrol were observed in CCA cells. PTEN expression varied among different CCA cells, and its status could impact cell sensitivity to celastrol: PTEN high tumor cells were resistant to celastrol, while PTEN low cells were more sensitive. Celastrol induced proteasomal dysregulation in CCA cells by directly targeting PSMB5. Cells with low PTEN status transcriptionally promoted proteasome subunit expression in an AKT-dependent manner, making these cells more reliant on proteasomal activities to maintain proteostasis. This caused the PTEN low CCA cells sensitive to celastrol. A negative correlation was found between PTEN levels and the proteasome signature in CCA patients. Moreover, celastrol treatment could induce autophagy in PTEN high CCA cells. Disrupting the autophagic pathway in PTEN high CCA cells enhanced the cytotoxic effect of celastrol., Conclusion: PTEN status in CCA cells determines their sensitivity to celastrol, and autophagy inhibitors could enhance the anti-tumor effect in PTEN high CCA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background: Chronic myeloid leukemia (CML) is driven primarily by the constitutively active BCR-ABL fusion oncoprotein. Although the development of tyrosine kinase inhibitors has markedly improved the prognosis of CML patients, it remains a significant challenge to overcome drug-resistant mutations, such as the T315I mutation of BCR-ABL, and achieve treatment-free remission in the clinic., Purpose: The identification of new intervention targets beyond BCR-ABL could provide new perspectives for future research and therapeutic intervention. A network pharmacology analysis was conducted to identify the most promising natural product with anti-CML activity. Celastrol was selected for further analysis to gain insights into its mechanism of action (MoA), with the aim of identifying potential new intervention targets for BCR-ABL T315I-mutant CML., Methods: Transcriptomic and proteomic analyses were conducted to systematically investigate the molecular MoA of celastrol in K562 T315I cells. To identify the target proteins of celastrol, mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) was carried out, followed by validations with genetic knockdown and overexpression, cell proliferation assay, comet assay, Western blotting, celastrol probe-based in situ labeling and pull-down assay, molecular docking, and biolayer interferometry., Results: Our multi-omics analyses revealed that celastrol primarily induces DNA damage accumulation and the unfolded protein response in K562 T315I cells. Among the twelve most potential celastrol targets, experimental evidence demonstrated that the direct interaction of celastrol with YY1 and HMCES increases the levels of DNA damage, leading to cell death., Conclusion: This study represents the first investigation utilizing a proteome-wide label-free target deconvolution approach, MS-CETSA, to identify the protein targets of celastrol. This study also develops a new systems pharmacology strategy. The findings provide new insights into the multifaceted mechanisms of celastrol and, more importantly, highlight the potential of targeting proteins in DNA damage and repair pathways, particularly YY1 and HMCES, to combat drug-resistant CML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Celastrol (Cel), extracted from Tripterygium wilfordii Hook , is a potential antiobesity drug, except for its adverse reactions in clinic. In the present study, we synthesized a promising celastrol-chitosan conjugate (Cel-CS1K) and evaluated its antiobesity effect and biological safety in diet-induced obese mice. Cel-CS1K showed higher drug loading (over 10 wt %), good solubility (18-19 mg/mL) in water, slower peak time ( T max = 4 h), and clearance ( T 1/2 = 8.97 h) in rats. Cel-CS1K effectively attenuated the cytotoxicity, celastrol-induced apoptosis, and fat accumulation of hepatocytes. Cel-CS1K reduced body weight and dietary amount same as the free Cel but with lower toxicity in blood, liver, and testis. Cel-CS1K improved the glucose homeostasis, HDL-C level, insulin sensitivity, and leptin sensitivity, while it significantly reduced the gene expression levels of LDL-C, TG, and TC in obese mice. Furthermore, the adipose-related gene expression levels provided evidence in support of a role for Cel-CS1K in losing weight by the multimode regulation. Overall, Cel-CS1K provides a translatable therapeutic strategy for the treatment of diet-induced obese humans.

Tripterygium glycosides (TG) is extracted from the roots of Chinese herbal medicine named Tripterygium wilfordii Hook F (TwHF). TG tablets are the representative TwHF-based agents with anti-inflammatory and immunomodulatory activities for treating rheumatoid arthritis. Although the curative effect of TG is remarkable, the clinical application is limited by a variety of organ toxicity. One of the most serious side-effects induced by TG is damage of the male reproductive system and the toxic mechanism is still not fully elucidated. TG-induced testicular injury was observed in male mice by treated with different concentrations of TG. The results showed that TG induced a significant decrease in testicular index. Pathological observation showed that spermatogenic cells were obviously shed, arranged loosely, and the spermatogenic epithelium was thin compared with control mice. In addition, the toxic effect of TG on mouse spermatogonia GC-1 cells was investigated. The results displayed that TG induced significant cytotoxicity in mouse GC-1 cells. To explore the potential toxic components that triggered testicular injury, the effects of 8 main components of TG on the viability of GC-1 cells were detected. The results showed that celastrol was the most toxic component of TG to GC-1 cells. Western blot analysis showed that LC3-II and the ratio of LC3-II/LC3-I were significantly increased and the expression level of p62 were decreased in both TG and celastrol treated cells, which indicated the significant activation of autophagy in spermatogonia cells. Therefore, autophagy plays an important role in the testicular injury induced by TG, and inhibition of autophagy is expected to reduce the testicular toxicity of TG., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Tripterygium wilfordii has been widely used for the treatment of rheumatoid arthritis, which is frequently accompanied by severe gastrointestinal damage. The molecular mechanism underlying the gastrointestinal injury of Tripterygium wilfordii are yet to be elucidated., Methods: Transmission electron microscopy, and pathological and biochemical analyses were applied to assess intestinal bleeding. Metabolic changes in the serum and intestine were determined by metabolomics. In vivo (time-dependent effect and dose-response) and in vitro (double luciferase reporter gene system, DRATs, molecular docking, HepG2 cells and small intestinal organoids) studies were used to identify the inhibitory role of celastrol on intestinal farnesoid X receptor (FXR) signaling. Fxr-knockout mice and FXR inhibitors and agonists were used to evaluate the role of FXR in the intestinal bleeding induced by Tripterygium wilfordii., Results: Co-treatment with triptolide + celastrol (from Tripterygium wilfordii) induced intestinal bleeding in mice. Metabolomic analysis indicated that celastrol suppressed intestinal FXR signaling, and further molecular studies revealed that celastrol was a novel intestinal FXR antagonist. In Fxr-knockout mice or the wild-type mice pre-treated with pharmacological inhibitors of FXR, triptolide alone could activate the duodenal JNK pathway and induce intestinal bleeding, which recapitulated the pathogenic features obtained by co-treatment with triptolide and celastrol. Lastly, intestinal bleeding induced by co-treatment with triptolide and celastrol could be effectively attenuated by the FXR or gut-restricted FXR agonist through downregulation of the duodenal JNK pathway., Conclusions: The synergistic effect between triptolide and celastrol contributed to the gastrointestinal injury induced by Tripterygium wilfordii via dysregulation of the FXR-JNK axis, suggesting that celastrol should be included in the quality standards system for evaluation of Tripterygium wilfordii preparations. Determining the mechanism of the FXR-JNK axis in intestinal bleeding could aid in the identification of additional therapeutic targets for the treatment of gastrointestinal hemorrhage diseases. This study also provides a new standard for the quality assessment of Tripterygium wilfordii used in the treatment of gastrointestinal disorders., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest, (Copyright © 2023. Published by Elsevier GmbH.)

Ethnopharmacological Relevance: Tripterygium wilfordii tablets (TWT) is widely used to treat autoimmune diseases such as rheumatoid arthritis. Celastrol, one main active ingredient in TWT, has been shown to produce a variety of beneficial effects, including anti-inflammatory, anti-obesity, anti-cancer, and immunomodulatory. However, whether TWT could protect against Concanavalin A (Con A)-induced hepatitis remains unclear., The Aim of the Study: This study aims to investigate the protective effect of TWT against Con A-induced hepatitis and elucidate the underlying mechanism., Materials and Methods: Metabolomic analysis, pathological analysis, biochemical analysis, qPCR and Western blot analysis and the Pxr-null mice were used in this study., Results: The results indicated that TWT and its active ingredient celastrol could protect against Con A-induced acute hepatitis. Plasma metabolomics analysis revealed that metabolic perturbations related to bile acid and fatty acid metabolism induced by Con A were reversed by celastrol. The level of itaconate in the liver was increased by celastrol and speculated as an active endogenous compound mediating the protective effect of celastrol. Administration of 4-octanyl itaconate (4-OI) as a cell-permeable itaconate mimicker was found to attenuate Con A-induced liver injury through activation of the pregnane X receptor (PXR) and enhancement of the transcription factor EB (TFEB)-mediated autophagy., Conclusions: Celastrol increased itaconate and 4-OI promoted activation of TFEB-mediated lysosomal autophagy to protect against Con A-induced liver injury in a PXR-dependent manner. Our study reported a protective effect of celastrol against Con A-induced AIH via an increased production of itaconate and upregulation of TFEB. The results highlighted that PXR and TFEB-mediated lysosomal autophagic pathway may offer promising therapeutic target for the treatment of autoimmune hepatitis., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Six undescribed abietane-type diterpenoids (tripterydinoids A-F) and five undescribed oleanane-type triterpenoids (tripterytrinoids A-E) were obtained and determined from the stem and branch of Tripterygium wilfordii Hook. f. (Celastraceae). Tripterydinoids A-C possessed the abietane-type diterpenoid skeleton with rare 8, 9-epoxy ring. The structures of undescribed compounds were established by extensive spectroscopic studies [HRESIMS, 1D/2D-NMR and electronic circular dichroism (ECD) calculation]. The absolute configurations of tripterydinoids A, B, E and tripterytrinoid A were defined by X-ray crystallographic analyses. Bioactivity screening indicated that tripterydinoids A-C exhibited potent inhibitory effects against NO release in LPS-activated RAW 264.7 macrophages with IC 50 values of 6.93, 4.46 and 2.98 μM, respectively. Meanwhile, tripterydinoids A-D and tripterytrinoids B, C showed moderate and selective cytotoxicities against five human tumor cell lines (A375, Huh7, MCF-7, HCT-116 and NCI-H460)., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We describe the potential anti coronavirus disease 2019 (COVID-19) action of the methide quinone inhibitor, celastrol. The related methide quinone dexamethasone is, so far, among COVID-19 medications perhaps the most effective drug for patients with severe symptoms. We observe a parallel redox biology behavior between the antioxidant action of celastrol when scavenging the superoxide radical, and the adduct formation of celastrol with the main COVID-19 protease. The related molecular mechanism is envisioned using molecular mechanics and dynamics calculations. It proposes a covalent bond between the S(Cys145) amino acid thiolate and the celastrol A ring, assisted by proton transfers by His164 and His41 amino acids, and a π interaction from Met49 to the celastrol B ring. Specifically, celastrol possesses two moieties that are able to independently scavenge the superoxide radical: the carboxylic framework located at ring E, and the methide-quinone ring A. The latter captures the superoxide electron, releasing molecular oxygen, and is the feature of interest that correlates with the mechanism of COVID-19 inhibition. This unusual scavenging of the superoxide radical is described using density functional theory (DFT) methods, and is supported experimentally by cyclic voltammetry and X-ray diffraction.

Celastrol and triptolide, chemical compounds isolated from Tripterygium wilfordii hook (also known as thunder god vine), are effective against rheumatoid arthritis (RA). Celastrol targets numerous signaling pathways involving NF‑κB, endoplasmic reticulum Ca2+‑ATPase, myeloid differentiation factor 2, toll‑like receptor 4, pro‑inflammatory chemokines, DNA damage, cell cycle arrest and apoptosis. Triptolide, inhibits NF‑κB, the receptor activator of NF‑κB (RANK)/RANK ligand/osteoprotegerin signaling pathway, cyclooxygenase‑2, matrix metalloproteases and cytokines. The present review examined the chemistry and bioavailability of celastrol and triptolide, and their molecular targets in treating RA. Clinical studies have demonstrated that T. wilfordii has several promising bioactivities, but its multi‑target toxicity has restricted its application. Thus, dosage control and structural modification of T. wilfordii are required to reduce the toxicity. In this review, future directions for research into these promising natural products are discussed.

Celastrol, a principal bioactive ingredient of Tripterygium wilfordii Hook F, has gained extensive exploration due to its unique structure features and multiple promising biological activities. This review will focus on the structural modifications, structure-activity relationships, pharmacology, and toxicology of celastrol in the past ten years. We hope this review would be helpful to get a better grasp of the progresses in the field and provide constructive suggestions for the further study of celastrol., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The extract of the medicinal plant, Tripterygium wilfordii Hook. f. (TW), has been used in the treatment of diverse autoimmune diseases, including rheumatoid arthritis. However, the high frequency of toxic side effects has limited its clinical use. In order to reduce toxicity without losing the therapeutic benefit, the pharmacological activity and toxicity of four compounds (T-96, triptolide, neotripterifordin, and tripterifordin) from TW were evaluated. The current study revealed that these compounds interfere with the IL-1β signaling pathway, which stimulates the secretion of pro-inflammatory cytokines (IL-6) in primary rheumatoid arthritis synovial fibroblasts (RASFs). These compounds inhibit IL-6 production, and among these, T-96 was the most effective. Moreover, T-96 blocks activation of NF-kappa B and p38 and ameliorates the joint destruction and the clinical signs of the disease in adjuvant-induced arthritic rats. These data suggest that among the four compounds of the TW, T-96 possesses highest anti-rheumatoid arthritis activity though inhibiting IL-1-mediated inflammatory signaling pathways.

Psoriasis is a severe disfiguring skin disease affecting approximately 3% of people worldwide and negatively affecting their daily lives. The pathogenesis of psoriasis is complicated, and typical therapeutic strategies for psoriasis mainly focus on anti-inflammation. Considering the side effects, withdrawal rebound, high cost, and many other disadvantages of existing treatments, we developed a new topical therapeutic formulation consisting of niosomes loaded with celastrol, a triterpenoid extracted from Tripterygium. Celastrol niosomes were prepared by the thin film hydration method and probe sonication. The niosomes were composed of Span 20, Span 60, and cholesterol at a weight ratio of 3:1:1. The particle size of the niosomes was approximately 147 nm, with yield of up to 90%. Celastrol niosomes showed improved in vitro permeation ability compared to the raw drug. In our in vivo study, celastrol niosomes effectively alleviated erythema and scaling on the dorsal skin of psoriasis mouse models. Spleen weight and the levels of cytokines, including IL-22, IL-23, and IL-17, decreased after the treatment, indicating the high therapeutic potential of this formulation for psoriasis. In conclusion, encapsulation of celastrol by niosomes increased the water-solubility and permeation of celastrol into the skin, significantly improving its anti-psoriasis activity in mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cancer resistance to chemotherapy is closely related to tumor heterogeneity, i.e., the existence of distinct subpopulations of cancer cells in a tumor mass. An important role is assigned to cancer stem cells (CSCs), a small subset of cancer cells with high tumorigenic potential and capacity of self-renewal and differentiation. These properties of CSCs are sustained by the ability of those cells to maintain a low intracellular reactive oxygen species (ROS) levels, via upregulation of ROS scavenging systems. However, the accumulation of ROS over a critical threshold disturbs CSCs-redox homeostasis causing severe cytotoxic consequences. In the present study, we investigated the capacity of celastrol, a natural pentacyclic triterpenoid, to induce the formation of ROS and, consequently, cell death of the colon cancer cells with acquired resistant to cytotoxic drugs (LOVO/DX cell line). LOVO/DX cells express several important stem-like cell features, including a higher frequency of side population (SP) cells, higher expression of multidrug resistant proteins, overexpression of CSC-specific cell surface marker (CD44), increased expression of DNA repair gene (PARP1), and low intracellular ROS level. We found that celastrol, at higher concentrations (above 1  μ M), significantly increased ROS amount in LOVO/DX cells at both cytoplasmic and mitochondrial levels. This prooxidant activity was associated with the induction of DNA double-strand breaks (DSBs) and apoptotic/necrotic cell death, as well as with inhibition of cell proliferation by S phase cell cycle arrest. Coincubation with NAC, a ROS scavenger, completely reversed the above effects. In summary, our results provide evidence that celastrol exhibits effective cytotoxic effects via ROS-dependent mechanisms on drug-resistant colon cancer cells. These findings strongly suggest the potential of celastrol to effectively kill cancer stem-like cells, and thus, it is a promising agent to treat severe, resistant to conventional therapy, colon cancers., Competing Interests: The authors declare no conflicts of interest.

Celastrol and triptolide, as the two main bio-activity ingredients in Tripterygium wilfordii, have wide anticancer pharmacological potency, as well as anti-inflammatory and immunosuppression effects. However, they have potential hepatotoxicity and underlying mechanisms of them-induced toxicity mediated by hepatic CYP450s have not been well delineated. In the present study, we accessed the toxic effects and possible mechanism of celastrol and triptolide on primary rat hepatocytes. Models of subdued/enhanced activity of CYP450 enzymes in primary rat hepatocytes were also constructed to evaluate the relationship between the two ingredients and CYP450s. LC-MS/MS was used to establish a detection method of the amount of triptolide in rat hepatocytes. As the results, cell viability, biochemical index, and mitochondrial membrane potential indicated that celastrol and triptolide had toxic potencies on hepatocytes. Moreover, the toxic effects were enhanced when the compounds combined with 1-aminobenzotriazole (enzyme inhibitor) while they were mitigated when combined with phenobarbital (an enzyme inducer). Meanwhile, celastrol could affect the amount of triptolide in the cell. We therefore put forward that increase of triptolide in the cell might be one of the main causes of hepatotoxicity caused by Tripterygium wilfordii .

A critical function of the PTEN-induced kinase 1 (PINK1)-Parkin pathway is to mediate the clearing of unhealthy or damaged mitochondria via mitophagy. Loss of either PINK1 or Parkin protein expression is associated with Parkinson's disease. Here, using a high-throughput screening approach along with recombinant protein expression and kinase, immunoblotting, and immunofluorescence live-cell imaging assays, we report that celastrol, a pentacyclic triterpenoid isolated from extracts of the medicinal plant Tripterygium wilfordii , blocks recruitment pof Parkin to mitochondria, preventing mitophagy in response to mitochondrial depolarization induced by carbonyl cyanide m -chlorophenylhydrazone or to gamitrinib-induced inhibition of mitochondrial heat shock protein 90 (HSP90). Celastrol's effect on mitophagy was independent of its known role in microtubule disruption. Instead, we show that celastrol suppresses Parkin recruitment by inactivating PINK1 and preventing it from phosphorylating Parkin and also ubiquitin. We also observed that PINK1 directly and strongly associates with TOM20, a component of the translocase of outer mitochondrial membrane (TOM) machinery and relatively weak binding to another TOM subunit, TOM70. Moreover, celastrol disrupted binding between PINK1 and TOM20 both in vitro and in vivo but did not affect binding between TOM20 and TOM70. Using native gel analysis, we also show that celastrol disrupts PINK1 complex formation upon mitochondrial depolarization and sequesters PINK1 to high-molecular-weight protein aggregates. These results reveal that celastrol regulates the mitochondrial quality control pathway by interfering with PINK1-TOM20 binding., (© 2019 Zhang et al.)

The purpose of this study was to investigate the renal protective effect of celastrol on diabetic rats. Furthermore, the mechanism of its action was discussed whether it was related to MAPK/NF-κB signaling pathway. There were a total of 36 rats. Six rats were randomly chosen as the control group. The remaining 30 rats were given 1% streptozotocin intraperitoneal injection (50 mg/kg) and were randomly divided into five groups: the model control group, the low-dose celastrol group, the high-dose celastrol group, the Tripterygium wilfordii polyglycosides group, and the MAPK/NF-κB inhibitor group. After 4 weeks of continuous administration, 24-hr urine volume, urinary protein, blood urea nitrogen, and serum creatinine content were observed, and hematoxylin-eosin (HE) staining of the kidney and liver were evaluated. p38MAPK was designated by immunohistochemical method, and NF-κB p65 in renal tissue was detected by western blotting. Our results showed that celastrol could not only reduce contents of creatinine and urea nitrogen in blood but also reduce excretion of urinary protein in diabetic rats, improve renal pathological injury, and down-regulate the expression of p38MAPK and NF-κB p65. In conclusion, celastrol could protect kidney of diabetic rats by regulating the signal pathway of MAPK/NF-κB, inhibiting inflammation and delaying renal injury., (© 2019 John Wiley & Sons, Ltd.)

Tripterygium wilfordii Hook F. (TWHF), a traditional Chinese medicine, has been widely used to treat autoimmune and inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus and dermatomyositis in China. Recently, studies have demonstrated that the bioactive components of TWHF have effective therapeutic potential for neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and Multiple Sclerosis. In this paper, we summarize the research progress of triptolide and celastrol (the two major TWHF components) as well as their analogues in the treatment of neurodegenerative diseases. In addition, we review and discuss the molecular mechanisms and structure features of those two bioactive TWHF components, highlighting their therapeutic promise in neurodegenerative diseases.

Two polymorphs of celastrol were discovered and fully characterized by X-ray powder diffraction, thermogravimetry analysis, and differential scanning calorimetry. The single-crystal structures of form I and the isostructural solvate of form II were disclosed by single-crystal X-ray diffraction. The apparent solubility and wettability of both the crystalline forms were determined. It was found that surfactant can significantly improve the solubility of celastrol up to more than 10 4 times. Tween 80 and sodium dodecyl sulfate largely improved the wettability of the 2 crystals. Form I shows better wettability than form II in all the buffer solutions with polymers and surfactants. Compared with form II, form I exhibits higher solubility in carboxymethylcellulose and polyvinylpyrrolidone media but much lower solubility in tween 80 and sodium dodecyl sulfate solutions. An investigation of wettability and solubility mechanisms was fully explored, and a hypothesis was proposed to understand the abnormal solubility differences., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

In recent years, a large amount of clinical and experimental data has shown that M2-like polarized tumor-associated macrophages (TAMs) play an important role in cancer metastasis. Therefore, TAMs, especially M2-like TAMs is a promising target for anti-tumor metastasis therapy. Here, we found that celastrol dose-dependently suppressed IL-13 induced CD206 expression both in RAW264.7 and in primary macrophages. Consistently, celastrol also inhibited the expression of M2-like specific genes, including MRC1, Arg1, Fizz1, Mgl2 and CD11c. Further, by the employment of 4T1 breast cancer model, we found that celastrol significantly prevented cancer metastasis in vivo. Mechanistically, celastrol completely ameliorated STAT6 phosphorylation, which is the key signal molecule responsible for M2 polarization. Our research puts forward a new application of celastrol in anti-cancer metastasis, by intervening M2-like polarization through inhibiting STAT6., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The respiratory disease pulmonary fibrosis (PF), which is characterized by scar formation throughout the lung, imposes a serious health burden. No effective drug without side effects has been proven to prevent this fatal lung disease. In this context, this study was undertaken to elucidate the protective effect of celastrol, a quinine methide pentacyclic triterpenoid from a Chinese medicinal plant 'thunder god vine' against bleomycin (BLM)-induced PF. We also attempted to study how the cytokine transforming growth factor-β (TGF-β) stimulates fibrosis through the induction of epithelial-mesenchymal transition (EMT) and the role of celastrol in regulating EMT. TGF-β (5 ng/ml) was administered to human alveolar epithelial adenocarcinoma A549 cells to induce fibrotic response in cells. Induction of EMT was analysed in cells through morphological analysis and expression of epithelial and mesenchymal markers by Western blotting. Bleomycin at a concentration of 3 U/Kg b.w was used to induce fibrosis in adult male rat lungs. Celastrol (5 mg/kg b.w) was given to rats twice a week after BLM administration for a period of 28 days. Western blot and immunofluorescence analyses were performed with lung tissue sample to find out the potential of celastrol in regulating EMT during the progression of fibrosis. TGF-β induces EMT in A549 cells as demonstrated by changes in epithelial cell morphology and expression of epithelial and mesenchymal marker proteins. The expressions of epithelial marker proteins E-cadherin and claudin were found to be reduced in the BLM-induced group of rats. Expression of mesenchymal markers, such as N-cadherin, snail, slug, vimentin and β-catenin, was enhanced in BLM-induced rat lungs. Celastrol reverts these cellular changes in rat lungs, and it was found that celastrol regulates EMT through the inhibition of heat shock protein 90 (HSP 90). Together, the results indicate that EMT is a crucial phenomenon for the progression of fibrosis, and celastrol provides protection against PF through the regulation of EMT., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The synthesis of celastrol analogues containing amino acid ester at the C(29) position and their evaluation for cytotoxic activities in vitro were reported. The MTT test showed that a set of derivatives with lower IC 50 values than that of the positive control group cisplatin and the parent compound celastrol, which exhibited greater antiproliferative activities. The most potent title compounds 2a and 2e exhibited cytotoxic activities in vitro against HeLa and A549 cell lines with IC 50 values of 0.371 and 0.237 μm, 0.235 and 0.109 μm, respectively. The apoptosis assay demonstrated that 2a and 2e can induces of A549 cell apoptosis in low concentrations. These results showed that 2a and 2e may be promising for further research as antitumor agents., (© 2018 Wiley-VHCA AG, Zurich, Switzerland.)

In our ongoing search for bioactive constituents, a new sesquiterpene polyol ester, named triptersinine U (1), together with five known triterpenes (2-6) and seven sesquiterpene pyridine alkaloids (7-13), were isolated from the roots of Tripterygium wilfordii Hook. f. Their chemical structures were elucidated using extensive spectroscopic analyses, including 1D and 2D NMR, and HRESIMS, as well as comparison with previously reported data. Cytotoxic activities of all compounds 1-13 were evaluated against six human tumor cell lines (HepG2, Hep3B, Bcap37, U251, MCF-7 and A549) using the MTT in vitro assay. The results showed that triterpenes exhibited moderate cytotoxic activities toward the tested cell lines.

Leukemia cells aberrantly overexpress senescence-suppression telomerase reverse transcriptase (TERT) and down-regulate key senescence-promoting genes to escape complete senescence, resulting in immortalization and malignant progression. Accordingly, induction of complete senescence is a sensible strategy for anti-leukemia therapy. However, effective senescence-based anti-leukemia drugs with low toxicity are currently lacking. In this study, we found that triptonide (chemical name diterpene triepoxide), a small molecule derived from the herb Tripterygium wilfordii Hook, strongly induced complete senescence in cultured acute myeloid leukemia (AML) cell lines, and potently inhibited growth and colony formation of U937 and HL-60 AML cell line with IC 50 values of 7.5 and 12nM, respectively. Strikingly, triptonide (4mg/kg) nearly completely suppressed human leukemia cell tumorigenicity (>99%) without obvious toxicity in a mouse xenograft model. Mechanistic studies showed that triptonide induced senescence followed by apoptosis mainly by suppressing transcription of TERT and oncogenic c-Myc, while concomitantly promoting transcription of senescence-promoting genes p16 and p21 and the pro-apoptotic gene encoding DNA damage-inducible transcript 3. These effects of triptonide are mediated by selective mitogen-activated protein kinase kinase-3/p38 signaling pathway activation. Our study provides a conceptual framework for inducing complete senescence as an effective anti-leukemia therapeutic strategy through a "multiple-hits" model and supports further development of triptonide as an anti-cancer agent., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Celastrol has potential application for the treatment of prostate cancer. However it causes autophagy as a protective response in prostate and other types of cancers, thus unveiling the underlying mechanisms may benefit its future application. In the present study, we demonstrate that the miR-17-92a cluster plays a negative role in celastrol induced-autophagy. Dissection of miR-17-92a cluster revealed the role of miR-17 seed family (miR-20a and miR-17) in autophagy inhibition in the context of prostate cancer cells. Autophagy-related gene ATG7 was validated as a target of miR-17 seed family by dual-luciferase assay and qPCR. Celastrol induced autophagy was inhibited by miR-20a or miR-17, while the inhibitory effects were rescued in the presence of pcDNA-ATG7 lacking 3' UTR, demonstrating that these two members target ATG7 to inhibit celastrol-induced autophagy. As celastrol degrades androgen receptor (AR), a key transcription factor in prostate cancer cells, we further investigated whether AR affected miR-17-92a expression in prostate cancer cells. AR binding sites were found in the promoter and two introns of miR-17-92a. In addition, higher expression levels of miR-17-92a were observed in AR positive cells compared with AR negative cells. Ectopic expression of AR could enhance the expression of miR-17-92a cluster in AR-negative prostate cancer cells while knockdown of AR decreased miR-17-92a expression in AR-positive cells, demonstrating the regulation of AR on miR-17-92a transcription. In summary, our results demonstrate that celastrol downregulates AR and its target miR-17-92a, leading to autophagy induction in prostate cancer cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Three new triterpenoids, triregelolides A, B (1, 2), and triregeloic acid (3), were isolated from the stems of Tripterygium regelii along with twenty known triterpene analogues (4-23). The structures of three new compounds were identified by analyzing their NMR spectroscopic and HRESIMS data. Compounds 4, 7, 8, 10, 13, 14, 17, 21-23 were isolated from T. regelii for the first time. Compounds 3, 5, 6, 8, 9, 10, 14 and 16 showed inhibitory effects on the proliferation of human breast cancer cells MCF-7 by 24.1%, 69.6%, 72.8%, 21.6%, 23.1%, 43.3%, 25.5% and 23.5% (p<0.05) at a concentration of 10μM, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Celastrol is an important bioactive triterpenoid in traditional Chinese medicinal plant, Tripterygium wilfordii. Methyl Jasmonate (MJ) is a common plant hormone which can regulate the secondary metabolism in higher plants. In this study, the mevalonate (MVA) pathway genes in T. wilfordii were firstly cloned. The suspension cells of T. wilfordii were elicited by MJ, and the expressions of MVA pathway genes were all enhanced in different levels ranging from 2.13 to 22.33 times of that at 0 h. The expressions were also enhanced compared with the CK group separately. The accumulation of celastrol in the suspension cells after the treatment was quantified and co-analyzed with the genes expression levels. The production of celastrol was significantly increased to 0.742 mg g(-1) after MJ treatment in 288 h which is consistent with the genes expressions. The results provide plenty of gene information for the biosynthesis of terpenoids in T. wilfordii and a viable way to improve the accumulation of celastrol in T. wilfordii suspension cells.

To establish a LC-MS/MS method for determination of tripterine in Beagle plasma and study its pharmacokinetics after oral administration of tripterygium tablet. Plasma samples were extracted with dichloromethane and separated on a Phenomenex Luna C₈ (2.0 mm×50 mm, 3 μm) column with methanol-acetonitrile isopropanol(1∶1)-1‰formic acid (15∶55 ∶30) as the mobile phase. Tripterine ([M+H] ⁺, m/z 451.3/201.1) and internal standard prednisolone ([M+H] ⁺, m/z 361.1/147.1) were monitored in multiple reaction monitoring (MRM). The concentration-time curves were simulated by drug and statistic software 1.0 and the pharmacokinetic parameters were calculated. There was a good linear relationship between peak area ratio and concentration of tripterine and internal standard prednisolone within range of 0.680 0-136.0 μg•L⁻¹. The limit of quantitation was 0.680 0 μg•L⁻¹ and the intra- and inter-day precision was within 6.15%. The absolute recovery rate was between 50.42% to 51.65%. The concentration-time curves were consistent with the one-compartment model(w=1/cc). The main pharmacokinetic parameters after a single dose were as follows： Cmax (35.64±9.540) μg •L⁻¹,Tmax(2.62±0.69) h,T1/2(2.93±0.29) h, CL (0.308±0.056) L•kg⁻¹•h⁻¹, AUC0-12 (131.16±31.94) μg•L•h⁻¹, AUC0-∞ (142.83±37.57) μg•L•h⁻¹. The established LC-MS/MS method was proved to be sensitive, accurate and convenient, suitable for the pharmacokinetic study of Tripterygium tablet in Beagle dogs., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Tibial dyschondroplasia (TD) is an important long bone defect of broiler chickens that disturbs the proximal growth plate and is characterized by non-vascularized cartilage, a distended growth plate and lameness. Celastrol, a medicinal root extract from the plant Tripterygium wilfordii, is reported widely as a well-known heat-shock protein 90 (Hsp90) inhibitor. Recently, Hsp90 inhibition in chondrocyte differentiation and growth-plate vascularization were effective in restoring the morphology of the growth plate. The present study was aimed at investigating Hsp90 inhibition in TD using celastrol. The broiler chicks were divided into three groups; Control; TD induced (40 mg/kg thiram) and celastrol treatment. Hsp90, vascular endothelial growth factor and Flk-1 expressions were evaluated by quantitative real-time polymerase chain reaction and the protein levels of Hsp90 were measured by Western blot analysis. Antioxidant enzymes were determined to assess the liver damage caused by thiram and the protective effects of the medicine were evaluated by levels of serum biomarkers. The expression levels of Hsp90 and vascular endothelial growth factor mRNA transcripts were increased while Flk-1 receptor was decreased in TD-affected chicks. Celastrol therapy inhibited Hsp90 mRNA and protein levels and up-regulated the expressions of receptor Flk-1 in TD-affected tibial growth plates significantly (P < 0.05) in addition to rectifying the damaging effects of thiram on the liver by decreasing the levels of aspartate aminotransferase, alanine aminotransferase and malondialdehyde and correcting the oxidative imbalance. In conclusion, administering celastrol to dyschondroplastic chicks prevented un-vascularized growth plate, lameness and reinstated angiogenesis. Celastrol may be efficacious for the treatment of TD through the inhibition of Hsp90 expression and limiting the liver damage caused by thiram in broiler chickens.

Marek's disease (MD), a highly infectious lymphoproliferative disease in chickens, is caused by a cell-associated oncogenic herpesvirus, Marek's disease virus (MDV). MSB-1 is a MD-derived lymphoblastoid cell line and can induce tumors when inoculated into susceptible chickens. Betulinic acid, which is present as one of the major effective components in many traditional Chinese medicines, has recently been reported to inhibit growth of cancer cells and employed as a potential anticancer agent. Tripterine, a major active compound extracted from the Chinese herb Tripterygium wilfordii Hook F, has now also shown anti-tumor activities in various cancers. The aim of this study was to investigate the synergistic growth-inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism. Viability of MSB-1 cells was assessed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) method. Cell apoptotic analysis was performed by fluorescence detection. NF-κB transcription activity was detected by measuring luciferase activity. Western blotting was used to analyze the expression of p65, IκB and Meq. Our results showed that the proliferation in the combination group was significantly decreased as compared with that of monotherapy using betulinic acid or tripterine, accompanied by an induction of apoptosis, inhibition of NF-κB transcriptional activity and its targeting oncogenic gene Meq. The results suggest that the combination of betulinic acid and tripterine at lower concentration may produce a synergistic inhibitive effect on MSB-1 cells that warrants further investigation for its potential clinical applications.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease. Necroptosis plays an important role in the pathogenesis of UC. Celastrol, a triterpene from the root bark of the Chinese medicinal plant Tripterygium wilfordii, has been reported to have anti-oxidant and anti-inflammatory activities in colitis. It is not known, however, how celastrol exerts its beneficial effects. The aim of this study is to investigate the effects and possible mechanism of celastrol in UC. Colitis was induced in mice by administration of 5% dextran sulfate sodium (DSS) in drinking water for 4days. Celastrol was administered intraperitoneally (1mg/kg) for 7days after colitis was induced. Our results showed that celastrol treatment ameliorated the severity of colitis, decreased the level of interleukin (IL)-1β, IL-6 and myeloperoxidase (MPO) and upregulated the level of E-cadherin in colitis mice. Moreover, the TUNEL staining and cleaved caspase-3 immunohistochemistry staining proved decreased necrotic cell death after celastrol treatment. On the mechanism, decreased level of necroptosis factors RIP3 and MLKL, and increased level of active caspase-8 were detected after celastrol treatment. Taken together, our results demonstrated that celastrol exerted beneficial effects in colitis treatment via suppressing the RIP3/MLKL necroptosis pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Triptolide and celastrol are two main active compounds isolated from Thunder God Vine with the potent anticancer activity. However, the anticancer effect of triptolide in combination with celastrol is still unknown. In the present study, we demonstrated that the combination of triptolide with celastrol synergistically induced cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increased intracellular ROS accumulation in cancer cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine dramatically blocked the apoptosis induced by co-treatment with triptolide and celastrol. Treatment with celastrol alone led to the decreased expressions of HSP90 client proteins including survivin, AKT, EGFR, which was enhanced by the addition of triptolide. Additionally, the celastrol-induced expression of HSP70 and HSP27 was abrogated by triptolide. In the nude mice with xenograft tumors, the lower-dose combination of triptolide with celastrol significantly inhibited the growth of tumors without obvious toxicity. Overall, triptolide in combination with celastrol showed outstanding synergistic anticancer effect in vitro and in vivo, suggesting that this beneficial combination may offer a promising treatment option for cancer patients.

Ethnopharmacological Relevance: Tripterygium wilfordii (lei gong teng; Thunder of God Vine), which belongs to the Celastraceae family, has long been used in traditional Chinese medicine to treat inflammation and rheumatoid arthritis. Celastrol is a bioactive compound isolated from T. wilfordii., Aim of the Study: We investigated whether celastrol suppressed binding of lipopolysaccharides (LPS) to myeloid differentiation factor 2 (MD2), thereby downregulating Toll-like receptor4 (TLR4) activation in mouse primary macrophages., Materials and Methods: Cytokine expression was determined by polymerase chain reaction analysis and enzyme-linked immunosorbent assay in bone marrow-derived primary macrophages (BMDMs). The kinase activity of tank-binding kinase 1 (TBK1) was examined by a luciferase reporter assay and an in vitro kinase assay. LPS binding to MD2 was examined by an in vitro binding assay and confocal microscopy analysis., Results: Celastrol reduced LPS-induced expression of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-1β, at both the mRNA and protein levels in BMDMs. Celastrol suppressed LPS binding to MD2, as shown by the in vitro binding assay, whereas it did not inhibit TBK1. In addition, co-localization of LPS with MD2 in BMDMs was blocked by celastrol. The inhibitory effects of celastrol on LPS binding to MD2 were reversed by thiol donors (N-acetyl-L-cysteine and dithiothreitol), suggesting that the thiol reactivity of celastrol contributes to its inhibitory effects on TLR4 activation in macrophages., Conclusion: Our results demonstrate that celastrol suppresses TLR4 activation through the inhibition of LPS binding to the TLR4/MD2 complex. These results provide a novel mechanism of action by which celastrol contributes to the anti-inflammatory activity of T. wilfordii., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Inflammation plays a vital role in the pathogenesis in lupus nephritis (LN), which is largely attributable to the activation of nuclear factor kappa B (NF-κB) signal pathway. NF-κB up-regulates pro-inflammatory mediators, such as TNF-α, cyclo-oxygenase-2 (COX-2) and ICAM-1, and promotes macrophage infiltration into renal tissue, further inducing the progression of LN. Over the past 30 years, research has demonstrated that Tripterygium wilfordii Hook F (TWHF) possesses potent anti-inflammatory and immunosuppressive activities, and that demethylzeylasteral (T-96), an extract of TWHF, may be one of the responsible compounds. Here, we investigate the pharmacodynamic role and therapeutic mechanism by which T-96 suppresses inflammation and reduces renal pathology in the lupus-prone MRL/lpr mice., Methods: Forty-eight MRL/lpr mice were equally randomly divided into 6 groups (1.2, 0.6 or 0.3 mg/10 g T-96, 0.022 pills/10 g kang lang chuang san (one of Traditional Chinese herb as positive control), 0.125 mg/10 g prednisone and 0.1 ml/10 g normal saline as the LN disease control group). Also, eight WT C57BL/6 mice were used as normal control. After treatment by gavage with 0.10 ml/10 g/day volumes for 8 weeks, all mice were sacrificed and renal tissues were collected. The amount of 24 h proteinuria and the levels of anti-dsDNA antibody in serum were assessed respectively at weeks 0, 4 and 8. Inflammation, cytokines and NF-κB levels were assessed by histological examinations, immunohistochemical analyses and Western blot analyses., Results: In comparison with untreated MRL/lpr mice, mice treated with 1.2 and 0.6 mg/10 g of T-96 showed a significant improvement in 24 h proteinuria and the levels of anti-dsDNA antibody in serum. In addition, T-96 reduced the secretion of pro-inflammatory mediators such as TNF-α, COX-2 and ICAM-1, and the infiltration of macrophages in renal tissue. Moreover, T-96 significantly suppressed phosphorylations of cytoplasmic IKK and nuclear p65., Conclusion: This study suggests that T-96 exhibits reno-protective effects in LN accompanied by inhibiting the activation of NF-κB, reducing the downstream pro-inflammatory mediators and thus restricting macrophage infiltration. Because of these potent properties, T-96 should be considered as a promising therapeutic drug for LN.

IL-37 is an anti-inflammatory cytokine that was only recently identified, and it is highly expressed in tissues from patients with inflammatory and autoimmune diseases. Inflammatory cytokines and inflammatory stimuli can induce the upregulation of IL-37. However, it has not been reported whether anti-inflammatory medications induce the expression of IL-37. In this work, we uncovered, for the first time, that two main bioactive components, triptolide and triptonide, from the herb Tripterygium wilfordii Hook f. (TwHF), which possess anti-inflammatory activity, upregulate the expression of IL-37, and this expression was suppressed by ERK1/2 and p38 MAPK inhibitors. Overall, our research demonstrated, for the first time, that anti-inflammatory active components (triptolide and triptonide) upregulated the expression of IL-37 most likely via activation of the ERK1/2 and p38 MAPK pathways.Cellular & Molecular Immunology advance online publication, 6 October 2014; doi:10.1038/cmi.2014.92.

Celastrol is a natural terpenoid isolated from Tripterygium wilfordii, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Here, we investigated whether celastrol induces apoptosis on hepatocellular carcinoma Bel-7402 cells and further explored the underlying molecular mechanisms. Celastrol caused a dose- and time-dependent growth inhibition and apoptosis of Bel-7402 cells. It increased apoptosis through the up-regulation of Bax and the down-regulation of Bcl-2 in Bel-7402 cells. Moreover, celastrol induced the release of cytochrome c and increased the activation of caspase-3 and caspase-9, suggesting that celastrol-induced apoptosis was related to the mitochondrial pathway. These results indicated that celastrol could induce apoptosis in Bel-7402 cells, which may be associated with the activation of the mitochondria-mediated pathway.

Suberoylanilide hydroxamic acid (SAHA) is a promising histone deacetylase (HDAC) inhibitor approved by the US Food and Drug Administration (FDA) and whose clinical application for solid tumours is partially limited by decreased susceptibility in cancer cells due to nuclear factor (NF)-κB activation. As an NF-κB inhibitor, celastrol exhibits potent anticancer effects but has failed to enter clinical trials due to its toxicity. In this report, we demonstrated that the combination of celastrol and SAHA exerted substantial synergistic efficacy against human cancer cells in vitro and in vivo accompanied by enhanced caspase-mediated apoptosis. This drug combination inhibited the activation of NF-κB caused by SAHA monotherapy and consequently led to increased apoptosis in cancer cells. Interestingly, E-cadherin was dramatically downregulated in celastrol-resistant cancer cells, and E-cadherin expression was closely related to decreased sensitivity to celastrol. However, our combination treatment significantly augmented the expression of E-cadherin, suggesting that mutual mechanisms contributed to the synergistic anticancer activity. Furthermore, the enhanced anticancer efficacy of celastrol combined with SAHA was validated in a human lung cancer 95-D xenograft model without increased toxicity. Taken together, our data demonstrated the synergistic anticancer effects of celastrol and SAHA due to their reciprocal sensitisation, which was simultaneously regulated by NF-κB and E-cadherin; thus, the combination of celastrol and SAHA was superior to other combination regimens that rely on a single mechanism. Our findings not only open new opportunities for the clinical development of SAHA but should also motivate the clinical investigation of celastrol, which has been hampered by its toxicity., (© 2014 UICC.)

Celastrol, a major active ingredient of Chinese herb Tripterygium wilfordii Hook. f. (thunder god vine), has exhibited a broad spectrum of pharmacological activities, including anti-inflammation, anti-cancer and immunosuppression. In the present study, we used animal models of inflammatory pain and neuropathic pain, generated by carrageenan injection and spared nerve injury (SNI), respectively, to evaluate the effect of celastrol and to address the mechanisms underlying pain processing. Intraperitoneal (i.p.) injection of celastrol produced a dose-dependent inhibition of carrageenan-induced edema and allodynia. Real-time PCR analysis showed that celastrol (0.3 mg/kg, i.p.) significantly reduced mRNA expressions of inflammatory cytokines, TNF-α, IL-6, IL-1β, in carrageenan-injected mice. In SNI mice, pain behavior studies showed that celastrol (1 mg/kg, i.p.) effectively prevented the hypersensitivity of mechanical nociceptive response on the third day post-surgery and the seventh day post-surgery. Furthermore, the anti-hyperalgesic effects of celastrol in carrageenan-injected mice and SNI mice were reversed by SR144528 (1 mg/kg, i.p.), a specific cannabinoid receptor-2 (CB2) receptor antagonist, but not by SR141716 (1 mg/kg, i.p.), a specific cannabinoid receptor-1 (CB1) receptor antagonist. Taken together, our results demonstrate the analgesia effects of celastrol through CB2 signaling and propose the potential of exploiting celastrol as a novel candidate for pain relief.

The present study was the first time to investigate the effects of celastrol, derived from Trypterygium wilfordii Hook F. ("Thunder of God Vine"), a traditional Chinese medicine plant, on the metabolism of model probe substrates of CYP isoforms, CYP1A2, CYP2C11, CYP2D6, CYP2E1 and CYP3A2, which are important in the metabolism of a variety of xenobiotics. The effects of celastrol on CYP1A2 (phenacetin O-deethylase), CYP2C11 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A2 (testosterone 6β-hydroxylase) activities were investigated using rat liver microsomes. HPLC-DAD was used to measure the model substrates and metabolites. Inhibition of rat CYP isoforms (IC50) by celastrol in potency order was CYP2C11 (10.2 μM)>CYP3A2 (23.2 μM)>CYP1A2 (52.8 μM)>CYP2E1 (74.2 μM)>CYP2D6 (76.4 μM). Enzyme kinetic studies showed that the celastrol was not only a competitive inhibitor of CYP1A2 and 2C11, but also a mixed-type inhibitor of CYP3A2, with Ki of 39.2 μM, 7.05 μM and 14.2 μM, respectively. The data indicate that celastrol inhibited the metabolism of CYP1A2, 2C and 3A substrates in rat liver in vitro with a different mode of inhibition. These in vitro studies of celastrol with CYP isoforms may be helpful for the development and application of celastrol as a promising anti-cancer agent. Further systematic studies in humans in vitro and in vivo are needed to identify the interactions of celastrol with cytochrome P450s., (© 2013.)

Context: Celastrol, a natural compound derived from the herb Tripterygium wilfordii, is known to have anticancer activity, but is not soluble in water., Objective: Formation of celastrol liposomes, to avoid the use of toxic solubilising agents., Materials and Methods: Two different formulations of PEGylated celastrol liposomes were fabricated. Liposomal characteristics and serum stability were determined using dynamic light scattering. Drug entrapment efficacy and drug release were measured spectrophotometrically. Cellular internalisation and anticancer activity was measured in prostate cancer cells., Results: Liposomal celastrol displayed efficient serum stability, cellular internalisation and anticancer activity, comparable to that of the free drug reconstituted in dimethyl sulfoxide., Discussion and Conclusion: Liposomal celastrol can decrease the viability of prostate cancer cells, while eliminating the need for toxic solubilising agents.

Celastrol is one of the principal active ingredients of Tripterygium wilfordii Hook.f., a toxic Chinese medical herb traditionally prescribed for controlling pain and inhibiting inflammation in various chronic inflammatory diseases, including rheumatoid arthritis (RA). Resistance to apoptosis of fibroblast-like synoviocytes is considered a major characteristic of RA. In this study, we test celastrol's cytotoxic effect and potential mechanisms in human rheumatoid synovial fibroblasts (RA-FLS). In the cytotoxic assay, we found that celastrol dose-dependently decreased RA-FLS viability and increased LDH release. The apoptotic nuclear morphology was observed after celastrol treatment as determined by DAPI fluorescence staining. Flow cytometry analysis with PI and Annexin V revealed that celastrol induced RA-FLS cell cycle arrest in the G2/M phase and apoptosis. Furthermore, celastrol dramatically increased expression of Bax/Bcl-2, proteolytic cleavage of Caspase-3, -9, PARP, and decreased expression of FasR. In addition, celastrol treatment resulted in DNA damage. Collectively, we concluded that celastrol inhibits RA-FLS proliferation by inducing DNA damage, cell cycle arrest, and apoptosis in vitro, which might provide data for its application in RA treatment.

The commercial preparation named “Tripterygium glycosides” prepared by column chromatography has been used for the treatment of inflammatory and autoimmune diseases with significant efficacy but concurrent toxicity. The aim of this study was to reduce the toxicity of Tripterygium extracts, using cytotoxicity and anti-inflammatory activity of the three principal active components of Tripterygium wilfordii Hook. F. (TWHF)as guiding parameters. Column chromatography was replaced by sodium carbonate extraction for removing the acidic compounds and enriching epoxyditerpenoids and alkaloids in the extract. Results showed that the therapeutic index (IC50/EC50) on murine macrophage Raw 264.7 cells and rat mesangial HBZY-1 cells of the extract prepared by sodium carbonate extraction was significantly higher than that of Tripterygium glycosides(0.8 and 5.2 vs. 0.3 and 2.6, p < 0.05), while its cytotoxicity on human liver HL7702 cells was significantly lower (14.5 ± 1.4 vs. 6.8 ± 0.9, p < 0.05). Further acute oral toxicity experiments showed that the LD50 value of this extract was 1,210 mg/kg compared to 257 mg/kg for Tripterygium glycosides. All the above results suggest that Tripterygium extract prepared by sodium carbonate extraction may represent a potentially optimal source of medicine with good therapeutic index.

Aim: This study investigates the capacity of a potent anti-inflammatory nanomedicine, celastrol, incorporated into poly(amidoamine) dendrimers, to inhibit endotoxin-mediated signaling in microglia., Materials & Methods: Celastrol was incorporated into amino (Cel/G4-NH(2)) and hydroxyl (Cel/G4-OH) terminus poly(amidoamine) (G4) dendrimers. Cell viability, release of nitric oxide, IL-6, TNF-α and activation of MAPK (e.g., p38 and JNK) and NF-κB were assessed in endotoxin (i.e., lipopolysaccharide) stimulated microglial cells., Results: G4-OH and G4-NH(2) increased celastrol aqueous solubility by seven- and 12-fold, respectively. G4-OH and Cel/G4-OH suppressed lipopolysaccharide-mediated release of proinflammatory mediators, such as nitric oxide and IL-6, but not TNF-α, without reducing microglial cell viability, while Cel/G4-NH(2) potentiated cytotoxicity and cytokine release. Blockade of proinflammatory signaling was accompanied by attenuation of p38 MAPK activation., Conclusion: This study supports the potential use of poly(amidoamine) dendrimers for effective anti-inflammatory therapy in the chronically inflamed CNS.

Celastrol, a quinone methide triterpene isolated from Tripterygium wilfordii Hook F., has various biochemical and pharmacological activities, and is now being developed as a promising anti-tumor agent. Inhibitory activity of compounds towards UDP-glucuronosyltransferase (UGT) is an important cause of clinical drug-drug interactions and herb-drug interactions. The aim of the present study is to investigate the inhibition of celastrol towards two important UDP-glucuronosyltransferase (UGT) isoforms UGT1A6 and UGT2B7. Recombinant UGT isoforms and non-specific substrate 4-methylumbelliferone (4-MU) were used. The results showed that celastrol strongly inhibited the UGT1A6 and 2B7-mediated 4-MU glucuronidation reaction, with 0.9 ± 0.1% and 1.8 ± 0.2% residual 4-MU glucuronidation activity at 100 μM of celastrol, respectively. Furthermore, inhibition kinetic study (Dixon plot and Lineweaver-Burk plot) demonstrated that celastrol noncompetitively inhibited the UGT1A1-mediated 4-MU glucuronidation, and competitively inhibited UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameters (Ki) were calculated to be 0.49 μM and 0.045 μM for UGT1A6 and UGT2B7, respectively. At the therapeutic concentration of celastrol for anti-tumor utilization, the possibility of celastrol-drug interaction and celastrol-containing herbs-drug interaction were strongly indicated. However, given the complicated nature of herbs, these results should be viewed with more caution.

Two new sesquiterpene pyridine alkaloids hypoglaunines E (1) and F (2) and a new triterpenoid saponin hypoglaside A (3), together with a known diterpenoid glucoside11-O-β-D-glucopyranosyl neotriptophenolide (4) and two known triterpenoids 23-noroxopristimerol(5) and 2,3-dihydroxy-6-oxo-D:A-froedo-24 nor-1,3,5(10),7-oleanatetraen-29-oic acid (6), have been isolated from the root barks of Tripterygiumhypoglaucum. Their structures were elucidated by NMR spectroscopy and MS analysis.Compounds 1–3 were screened for their cytotoxic activities against five cancer celllines, but all of them were inactive.

Celastrol, an important natural product and Hsp90 inhibitor with a wide range of biological and medical activities and broad use as a biological probe, acts by an as yet undetermined mode of action. It is known to undergo Michael additions with biological sulfur nucleophiles. Here it is demonstrated that nucleophiles add to the pharmacophore of celastrol in a remarkable stereospecific manner. Extensive characterization of the addition products has been obtained using NMR spectrometry, nuclear Overhauser effects, and density functional theory to determine facial selectivity and gain insight into the orbital interactions of the reactive centers. This stereospecificity of celastrol may be important to its protein target selectivity., (© 2011 American Chemical Society)

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Identification of active constituents and their molecular targets from traditional medicine is an enormous opportunity for modern pharmacology. Celastrol is one such compound that was originally identified from traditional Chinese medicine (Thunder of God Vine) almost three decades ago and generally used for the treatment of inflammatory and auto-immune diseases. Celastrol has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. The anti-inflammatory effects of this triterpene have been demonstrated in animal models of different inflammatory diseases, including arthritis, Alzheimer's disease, asthma, and systemic lupus erythematosus. This triterpene has also been found to inhibit the proliferation of a variety of tumor cells and suppress tumor initiation, promotion and metastasis in various cancer models in vivo. Celastrol's ability to modulate the expression of pro-inflammatory cytokines, MHC II, HO-1, iNOS, NF-κB, Notch-1, AKT/mTOR, CXCR4, TRAIL receptors DR4 and DR5, CHOP, JNK, VEGF, adhesion molecules, proteasome activity, topoisomerase II, potassium channels, and heat shock response has been reported. This review describes the various molecular targets of celastrol, cellular responses to celastrol, and animal studies with celastrol in cancer and other inflammatory disorders., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Celastrol, a natural biologically active compound isolated from Tripterygium wilfordii Hook F root extracts, has been shown to possess antitumor properties and therefore, is an interesting candidate for the development of novel chemotherapeutic cancer agents. In this study, we have demonstrated that Celastrol is a potent inhibitor of hypoxia-induced angiogenic and metastatic activity as shown by a decrease in the proliferation of both endothelial and cancer cells, blocking of migration as well as of tube formation of endothelial cells, and by inhibition of cancer cell invasion under hypoxic conditions. Moreover, Celastrol decreased hypoxia-inducible factor-1α (HIF-1α) mRNA levels under both normoxia and hypoxia and inhibited hypoxia-induced accumulation of nuclear HIF-1α protein. Meanwhile, inhibition of nuclear HIF-1α protein levels were accompanied by a reduction in the transcriptional activity of HIF-1α target genes, including VEGF. In addition, the inhibitory effect of Celastrol on HIF-1α protein was partly due to its suppression of HSP90 activity. We conclude that Celastrol regulates HIF-1α at multiple levels that may together or individually contribute to its antitumor activity against hypoxia-induced angiogenesis and metastasis.

The aim of the present study was to evaluate the underlying apoptotic mechanisms of celastrol, a major biologically active component of Tripterygium regelii, in human breast adenocarcinoma MCF-7 cells. Celastrol was isolated from T. regelii chloroform extract by silica gel column chromatography, and its chemical structure was identified via (1)H NMR and (13)C NMR. Celastrol significantly inhibited cell growth in dose- and time-dependent manners. Celastrol induced sub-G1 DNA accumulation, formation of apoptotic bodies, nuclear condensation, and a DNA ladder in MCF-7 cells. Celastrol triggered the activation of caspase family proteins. Celastrol caused activation of caspase-7, -8, and -9, PARP cleavage, caspase-8-mediated bid cleavage, and release of cytochrome c and AIF. In addition, celastrol decreased the expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein. These results suggest that celastrol inhibits the proliferation of MCF-7 cells through induction of apoptosis, which is mediated by a mitochondrial-dependent caspase pathway., (Copyright © 2010 Elsevier Ltd. All rights reserved.)