Celastrol-Induced Nur77 Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial Ubiquitination and Autophagy.

Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.

Celastrol alleviates atopic dermatitis by regulating Ezrin-mediated mitochondrial fission and fusion.

Celastrol, a bioactive molecule extracted from the plant Tripterygium wilfordii Hook F., possesses anti-inflammatory, anti-obesity and anti-tumour properties. Despite its efficacy in improving erythema and scaling in psoriatic mice, the specific therapeutic mechanism of celastrol in atopic dermatitis (AD) remains unknown. This study aims to examine the role and mechanism of celastrol in AD using TNF-α-stimulated HaCaT cells and DNCB-induced Balb/c mice as in vitro and in vivo AD models, respectively. Celastrol was found to inhibit the increased epidermal thickness, reduce spleen and lymph node weights, attenuate inflammatory cell infiltration and mast cell degranulation and decrease thymic stromal lymphopoietin (TSLP) as well as various inflammatory factors (IL-4, IL-13, TNF-α, IL-5, IL-31, IL-33, IgE, TSLP, IL-17, IL-23, IL-1ß, CCL11 and CCL17) in AD mice. Additionally, celastrol inhibited Ezrin phosphorylation at Thr567, restored mitochondrial network structure, promoted translocation of Drp1 to the cytoplasm and reduced TNF-α-induced cellular reactive oxygen species (ROS), mitochondrial ROS (mtROS) and mitochondrial membrane potential (MMP) production. Interestingly, Mdivi-1 (a mitochondrial fission inhibitor) and Ezrin-specific siRNAs lowered inflammatory factor levels and restored mitochondrial reticular formation, as well as ROS, mtROS and MMP production. Co-immunoprecipitation revealed that Ezrin interacted with Drp1. Knocking down Ezrin reduced mitochondrial fission protein Drp1 phosphorylation and Fis1 expression while increasing the expression of fusion proteins Mfn1 and Mfn2. The regulation of mitochondrial fission and fusion by Ezrin was confirmed. Overall, celastrol may alleviate AD by regulating Ezrin-mediated mitochondrial fission and fusion, which may become a novel therapeutic reagent for alleviating AD.

Celastrol inhibits platelet function and thrombus formation.

INTRODUCTION: Celastrol is an active pentacyclic triterpenoid extracted from Tripterygium wilfordii and has anti-inflammatory and anti-tumor properties. Whether Celastrol modulates platelet function remains unknown. Our study investigated its role in platelet function and thrombosis. METHODS: Human platelets were isolated and incubated with Celastrol (0, 1, 3 and 5 µM) at 37 °C for 1 h to measure platelet aggregation, granules release, spreading, thrombin-induced clot retraction and intracellular calcium mobilization. Additionally, Celastrol (2 mg/kg) was intraperitoneally administrated into mice to evaluate hemostasis and thrombosis in vivo. RESULTS: Celastrol treatment significantly decreased platelet aggregation and secretion of dense or alpha granules induced by collagen-related peptide (CRP) or thrombin in a dose-dependent manner. Additionally, Celastrol-treated platelets showed a dramatically reduced spreading activity and decreased clot retraction. Moreover, Celastrol administration prolonged tail bleeding time and inhibited formation of arterial/venous thrombosis. Furthermore, Celastrol significantly reduced calcium mobilization. CONCLUSION: Celastrol inhibits platelet function and venous/arterial thrombosis, implying that it might be utilized for treating thrombotic diseases.

Synthesis of novel hydrophilic celastrol nanoformulation by entrapment within calcium phosphate nanoparticle and study of its antioxidant activity against neurotoxin-induced damage in human neuroblastoma cells.

Celastrol, a pentacyclic triterpenoid found in Chinese herb Tripterygium wilfordii, is considered as one of the top-five natural medicinal compounds with high antioxidant property. However, celastrol has poor aqueous solubility and thereby low bioavailability, restricting its clinical application as drug. To overcome this problem, we nanonized celastrol by entrapping it within hydrophilic nanocarrier - calcium phosphate nanoparticle. The synthesized calcium phosphate celastrol nanoparticle (CPCN) had average size of 35 nm, spherical shape, significant stability with (-) 37 mV zeta potential, celastrol entrapment efficiency around 75 % and low celastrol release kinetics spanning over 7 days, as measured by different techniques like FESEM, AFM, DLS, and spectrophotometry. Studies on the antioxidant potency of CPCN by flow cytometry and fluorescence microscopy depicted that the toxicity developed in human neuroblastoma cells SH-SY5Y by treatment with the selective neurotoxin MPP+ iodide (N-Methyl-4-phenylpyridinium iodide) got reduced by pretreatment of the cells with CPCN. Determination of cellular ROS content, depolarization level of mitochondrial membrane potential, cell cycle analysis and nuclear damage in MPP+-exposed cells demonstrated that CPCN had about 65 % more antioxidant efficacy over that of bulk celastrol. Thus, the nanonization process transformed hydrophobic celastrol into hydrophilic CPCN, having high potentiality to be developed as an effective antioxidant drug.

Celastrol inhibits angiogenesis and the biological processes of MDA-MB-231 cells via the DEGS1/S1P signaling pathway.

Celastrol (Cel) shows potent antitumor activity in various experimental models. This study examined the relationship between Cel's antivascular and antitumor effects and sphingolipids. CCK-8 assay, transwell assay, Matrigel, PCR-array/RT-PCR/western blotting/immunohistochemistry assay, ELISA and HE staining were used to detect cell proliferation, migration and invasion, adhesion and angiogenesis, mRNA and protein expression, S1P production and tumor morphology. The results showed that Cel could inhibit proliferation, migration or invasion, adhesion and angiogenesis of human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 cells by downregulating the expression of degenerative spermatocyte homolog 1 (DEGS1). Transfection experiments showed that downregulation of DEGS1 inhibited the above processes and sphingosine-1-phosphate (S1P) production of HUVECs and MDA-MB-231 cells, while upregulation of DEGS1 had the opposite effects. Coculture experiments showed that HUVECs could promote proliferation, migration and invasion of MDA-MB-231 cells through S1P/sphingosine-1-phosphate receptor (S1PR) signaling pathway, while Cel inhibited these processes in MDA-MB-231 cells induced by HUVECs. Animal experiments showed that Cel could inhibit tumor growth in nude mice. Western blotting, immunohistochemistry and ELISA assay showed that Cel downregulated the expression of DEGS1, CD146, S1PR1-3 and S1P production. These data confirm that DEGS1/S1P signaling pathway may be related to the antivascular and antitumor effects of cel.

The anti-cancer mechanism of Celastrol by targeting JAK2/STAT3 signaling pathway in gastric and ovarian cancer.

BACKGROUND: Celastrol is a natural triterpene exhibiting significant and extensive antitumor activity in a wide range of cancer. Due to unfavorable toxicity profile and undefined mechanism, Celastrol's application in clinical cancer therapy remains limited. Herein, we elucidate the pharmacological mechanism of Celastrol's anticancer effects, with a focus on STAT3 signaling pathway in cancers with high incidence of metastasis. METHODS: The safety profile of Celastrol were assessed in mice. In vitro analysis was performed in gastric cancer and ovarian cancer to assess the cytotoxicity, induction of reactive oxygen species (ROS) of Celastrol using STAT3 knockout cancer cells. Effects of Celastrol on STAT3 activation and transcription activity, JAK2/STAT3 signaling protein expression were assessed. Additionally, proteomic contrastive analysis was performed to explore the molecular association of Celastrol with STAT3 deletion in cancer cells. RESULTS: Celastrol has no obvious toxic effect at 1.5 mg/kg/day in a 15 days' administration. Celastrol inhibits tumor growth and increases ROS in a STAT3 dependent manner in gastric and ovarian cancer celllines. On molecular level, it downregulates IL-6 level and inhibits the JAK2/STAT3 signaling pathway by suppressing STAT3' activation and transcription activity. Proteomic contrastive analysis suggests a similar cellular mechanism of action between Celastrol and STAT3 deletion on regulating cancer progression pathways related to migration and invasion. CONCLUSION: Our research elucidates the anti-cancer mechanism of Celastrol through targeting the JAK2/STAT3 signaling pathway in cancer with high incidence of metastasis. This study provides a solid theoretical basis for the application of Celastrol in cancer therapy.

Celastrol attenuates the invasion and migration and augments the anticancer effects of olaparib in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is a leading malignancy among men globally, with rising incidence rates emphasizing the critical need for better detection and therapeutic approaches. The roles of HSP90AB1 and PARP1 in prostate cancer cells suggest potential targets for enhancing treatment efficacy. METHODS: This study investigated the overexpression of HSP90AB1 and PARP1 in prostate cancer cells and the impact of HSP90AB1 knockdown on the sensitivity of these cells to the PARP inhibitor olaparib. We also explored the combined effect of olaparib and celastrol, an HSP90 inhibitor, on the clonogenic survival, migration, proliferation, and overall viability of prostate cancer cells, alongside the modulation of the PI3K/AKT pathway. An in vivo PC3 xenograft mouse model was used to assess the antitumor effects of the combined treatment. RESULTS: Our findings revealed significant overexpression of HSP90AB1 and PARP1 in prostate cancer cells. Knockdown of HSP90AB1 increased cell sensitivity to olaparib. The combination of olaparib and celastrol significantly reduced prostate cancer cell survival, migration, proliferation, and enhanced cumulative DNA damage. Celastrol also downregulated the PI3K/AKT pathway, increasing cell susceptibility to olaparib. In vivo experiments demonstrated that celastrol and olaparib together exerted strong antitumor effects. CONCLUSIONS: The study indicates that targeting both HSP90AB1 and PARP1 presents a promising therapeutic strategy for prostate cancer. The synergistic combination of celastrol and olaparib enhances the efficacy of treatment against prostate cancer, offering a potent approach to combat this disease.

Chemoproteomics-based profiling reveals potential antimalarial mechanism of Celastrol by disrupting spermidine and protein synthesis.

BACKGROUND: Malaria remains a global health burden, and the emergence and increasing spread of drug resistance to current antimalarials poses a major challenge to malaria control. There is an urgent need to find new drugs or strategies to alleviate this predicament. Celastrol (Cel) is an extensively studied natural bioactive compound that has shown potentially promising antimalarial activity, but its antimalarial mechanism remains largely elusive. METHODS: We first established the Plasmodium berghei ANKA-infected C57BL/6 mouse model and systematically evaluated the antimalarial effects of Cel in conjunction with in vitro culture of Plasmodium falciparum. The potential antimalarial targets of Cel were then identified using a Cel activity probe based on the activity-based protein profiling (ABPP) technology. Subsequently, the antimalarial mechanism was analyzed by integrating with proteomics and transcriptomics. The binding of Cel to the identified key target proteins was verified by a series of biochemical experiments and functional assays. RESULTS: The results of the pharmacodynamic assay showed that Cel has favorable antimalarial activity both in vivo and in vitro. The ABPP-based target profiling showed that Cel can bind to a number of proteins in the parasite. Among the 31 identified potential target proteins of Cel, PfSpdsyn and PfEGF1-α were verified to be two critical target proteins, suggesting the role of Cel in interfering with the de novo synthesis of spermidine and proteins of the parasite, thus exerting its antimalarial effects. CONCLUSIONS: In conclusion, this study reports for the first time the potential antimalarial targets and mechanism of action of Cel using the ABPP strategy. Our work not only support the expansion of Cel as a potential antimalarial agent or adjuvant, but also establishes the necessary theoretical basis for the development of potential antimalarial drugs with pentacyclic triterpenoid structures, as represented by Cel. Video Abstract.

Targeting epigenetic and post-translational modifications regulating pyroptosis for the treatment of inflammatory diseases.

Inflammatory diseases, including infectious diseases, diabetes-related diseases, arthritis-related diseases, neurological diseases, digestive diseases, and tumor, continue to threaten human health and impose a significant financial burden despite advancements in clinical treatment. Pyroptosis, a pro-inflammatory programmed cell death pathway, plays an important role in the regulation of inflammation. Moderate pyroptosis contributes to the activation of native immunity, whereas excessive pyroptosis is associated with the occurrence and progression of inflammation. Pyroptosis is complicated and tightly controlled by various factors. Accumulating evidence has confirmed that epigenetic modifications and post-translational modifications (PTMs) play vital roles in the regulation of pyroptosis. Epigenetic modifications, which include DNA methylation and histone modifications (such as methylation and acetylation), and post-translational modifications (such as ubiquitination, phosphorylation, and acetylation) precisely manipulate gene expression and protein functions at the transcriptional and post-translational levels, respectively. In this review, we summarize the major pathways of pyroptosis and focus on the regulatory roles and mechanisms of epigenetic and post-translational modifications of pyroptotic components. We also illustrate these within pyroptosis-associated inflammatory diseases. In addition, we discuss the effects of novel therapeutic strategies targeting epigenetic and post-translational modifications on pyroptosis, and provide prospective insight into the regulation of pyroptosis for the treatment of inflammatory diseases.

ROS-responsive celastrol-nanomedicine alleviates inflammation for dry eye disease.

Dry eye disease (DED) is a major global eye disease leading to severe eye discomfort and even vision impairment. The incidence of DED has been gradually increasing with the high frequency of use of electronic devices. It has been demonstrated that celastrol (Cel) has excellent therapeutic efficacy in ocular disorders. However, the poor water solubility and short half-life of Cel limit its further therapeutic applications. In this work, a reactive oxygen species (ROS) sensitive polymeric micelle was fabricated for Cel delivery. The micelles improve the solubility of Cel, and the resulting Cel loaded micelles exhibit an enhanced intervention effect for DED. Thein vitroresults demonstrated that Cel-nanomedicine had a marked ROS responsive release behavior. The results ofin vitroandin vivoexperiments demonstrated that Cel has excellent biological activities to alleviate inflammation in DED by inhibiting TLR4 signaling activation and reducing pro-inflammatory cytokine expression. Therefore, the Cel nanomedicine can effectively eliminate ocular inflammation, promote corneal epithelial repair, and restore the number of goblet cells and tear secretion, providing a new option for the treatment of DED.

Celastrol-Ligustrazine compound proven to be a novel drug candidate for idiopathic pulmonary fibrosis by intervening in the TGF-ß1 mediated pathways-an experimental in vitro and vivo study.

Idiopathic Pulmonary Fibrosis (IPF) is a disease characterized by pulmonary interstitial fibrosis and collagen proliferation, currently lacking effective therapeutic options. The combined use of Celastrol and Ligustrazine has been proved to synergistically improve the pathological processes of inflammation and fibrosis. In earlier studies, we designed and synthesized a Celastrol-Ligustrazine compound CL-001, though its role in IPF remains unclear. Here, the effects and mechanisms of CL-001 in bleomycin (BLM)-induced IPF were investigated. In vivo, CL-001 significantly improved lung function, reduced pulmonary inflammation, and decreased collagen deposition, thereby preventing the progression of IPF. In vitro, CL-001 concurrently inhibited both Smad-dependent and Smad-independent pathways, thereby suppressing TGF-ß1-induced epithelial-mesenchymal transition (EMT) and epithelial cell migration. This inhibitory effect was superior to that of Celastrol or Ligustrazine administered alone. Additionally, CL-001 significantly increased the level of apoptosis and promoted the expression of apoptosis-related proteins (Caspase-8 and PARP), ultimately leading to widespread apoptosis in activated lung epithelial cells. In summary, CL-001 exhibits excellent anti-IPF effects both in vitro and in vivo, suggesting its potential as a novel candidate drug for IPF, warranting further development.

Enzyme/ROS dual-sensitive nanoplatform with on-demand Celastrol release capacity for enhanced ulcerative colitis therapy by ROS scavenging, microbiota rebalancing, inflammation alleviating.

BACKGROUND: The oral administration of drugs for treating ulcerative colitis (UC) is hindered by several factors, including inadequate gastrointestinal stability, insufficient accumulation in colonic lesions, and uncontrolled drug release. METHODS: A multiple sensitive nano-delivery system comprising ß-cyclodextrin (CD) and 4-(hydroxymethyl)phenylboronic acid (PAPE) with enzyme/reactive oxygen species (ROS) sensitivity was developed to load celastrol (Cel) as a comprehensive treatment for UC. RESULTS: Owing to the positive charge in the site of inflamed colonic mucosa, the negatively charged nanomedicine (Cel/NPs) could efficiently accumulate. Expectedly, Cel/NPs showed excellent localization ability to colon in vitro and in vivo tests. The elevated concentration of ROS and intestinal enzymes in the colon microenvironment quickly break the CD, resulting in Cel release partially to rebalance microbiota and recover the intestinal barrier. The accompanying cellular internalization of residual Cel/NPs, along with the high concentration of cellular ROS to trigger Cel burst release, could decrease the expression of inflammatory cytokines, inhibit colonic cell apoptosis, promote the macrophage polarization, scavenge ROS, and regulate the TLR4/NF-κB signaling pathway, which certified that Cel/NPs possessed a notably anti-UC therapy outcome. CONCLUSIONS: We provide a promising strategy for addressing UC symptoms via an enzyme/ROS-sensitive oral platform capable of releasing drugs on demand.

Celastrol reduces lung inflammation induced by multiwalled carbon nanotubes in mice via NF-κb-signaling pathway.

Multiwalled carbon nanotubes (MWCNTs) have numerous applications in the field of carbon nanomaterials. However, the associated toxicity concerns have increased significantly because of their widespread use. The inhalation of MWCNTs can lead to nanoparticle deposition in the lung tissue, causing inflammation and health risks. In this study, celastrol, a natural plant medicine with potent anti-inflammatory properties, effectively reduced the number of inflammatory cells, including white blood cells, neutrophils, and lymphocytes, and levels of inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, in mice lungs exposed to MWCNTs. Moreover, celastrol inhibited the activation of the NF-κB-signaling pathway. This study confirmed these findings by demonstrating comparable reductions in inflammation upon exposure to MWCNTs in mice with the deletion of NF-κB (P50-/-). These results indicate the utility of celastrol as a promising pharmacological agent for preventing MWCNT-induced lung tissue inflammation.

Celastrol enhances the viability of random-pattern skin flaps by regulating autophagy through the AMPK-mTOR-TFEB axis.

In reconstructive and plastic surgery, random-pattern skin flaps (RPSF) are often used to correct defects. However, their clinical usefulness is limited due to their susceptibility to necrosis, especially on the distal side of the RPSF. This study validates the protective effect of celastrol (CEL) on flap viability and explores in terms of underlying mechanisms of action. The viability of different groups of RPSF was evaluated by survival zone analysis, laser doppler blood flow, and histological analysis. The effects of CEL on flap angiogenesis, apoptosis, oxidative stress, and autophagy were evaluated by Western blot, immunohistochemistry, and immunofluorescence assays. Finally, its mechanistic aspects were explored by autophagy inhibitor and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. On the seventh day after surgery, the survival area size, blood supply, and microvessel count of RPSF were augmented following the administration of CEL. Additionally, CEL stimulated angiogenesis, suppressed apoptosis, and lowered oxidative stress levels immediately after elevated autophagy in ischemic regions; These effects can be reversed using the autophagy inhibitor chloroquine (CQ). Specifically, CQ has been observed to counteract the protective impact of CEL on the RPSF. Moreover, it has also been discovered that CEL triggers the AMPK-mTOR-TFEB axis activation in the area affected by ischemia. In CEL-treated skin flaps, AMPK inhibitors were demonstrated to suppress the AMPK-mTOR-TFEB axis and reduce autophagy levels. This investigation suggests that CEL benefits the survival of RPSF by augmenting angiogenesis and impeding oxidative stress and apoptosis. The results are credited to increased autophagy, made possible by the AMPK-mTOR-TFEB axis activation.

Structure-Activity Relationship of Oleanane-Type Pentacyclic Triterpenoids on Nuclear Factor κB Activation and Intracellular Trafficking and N-Linked Glycosylation of Intercellular Adhesion Molecule-1.

In our previous study, two oleanane-type pentacyclic triterpenoids (oleanolic acid and maslinic acid) were reported to affect the N-glycosylation and intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1). The present study was aimed at investigating the structure-activity relationship of 13 oleanane-type natural triterpenoids with respect to the nuclear factor κB (NF-κB) signaling pathway and the expression, intracellular trafficking, and N-glycosylation of the ICAM-1 protein in human lung adenocarcinoma A549 cells. Hederagenin, echinocystic acid, erythrodiol, and maslinic acid, which all possess two hydroxyl groups, decreased the viability of A549 cells. Celastrol and pristimerin, both of which possess an α,ß-unsaturated carbonyl group, decreased cell viability but more strongly inhibited the interleukin-1α-induced NF-κB signaling pathway. Oleanolic acid, moronic acid, and glycyrrhetinic acid interfered with N-glycosylation without affecting the cell surface expression of the ICAM-1 protein. In contrast, α-boswellic acid and maslinic acid interfered with the N-glycosylation of the ICAM-1 protein, which resulted in the accumulation of high-mannose-type N-glycans. Among the oleanane-type triterpenoids tested, α-boswellic acid and maslinic acid uniquely interfered with the intracellular trafficking and N-glycosylation of glycoproteins.

Celastrol Pyrazine Derivative Alleviates Silicosis Progression via Inducing ROS-Mediated Apoptosis in Activated Fibroblasts.

Silicosis is a complex occupational disease without recognized effective treatment. Celastrol, a natural product, has shown antioxidant, anti-inflammatory, and anti-fibrotic activities, but the narrow therapeutic window and high toxicity severely limit its clinical application. Through structural optimization, we have identified a highly efficient and low-toxicity celastrol derivative, CEL-07. In this study, we systematically investigated the therapeutic potential and underlying mechanisms of CEL-07 in silicosis fibrosis. By constructing a silicosis mouse model and analyzing with HE, Masson, Sirius Red, and immunohistochemical staining, CEL-07 significantly prevented the progress of inflammation and fibrosis, and it effectively improved the lung respiratory function of silicosis mice. Additionally, CEL-07 markedly suppressed the expression of inflammatory factors (IL-6, IL-1α, TNF-α, and TNF-ß) and fibrotic factors (α-SMA, collagen I, and collagen III), and promoted apoptosis of fibroblasts by increasing ROS accumulation. Moreover, bioinformatics analysis combined with experimental validation revealed that CEL-07 inhibited the pathways associated with inflammation (PI3K-AKT and JAK2-STAT3) and the expression of apoptosis-related proteins. Overall, these results suggest that CEL-07 may serve as a potential candidate for the treatment of silicosis.

[Central inflammatory mechanism of celastrol in intervention of obesity-depression comorbidity in mice from amygdala-dorsal raphe nucleus].

This study aims to further elucidate the efficacy targets of celastrol(CEL) intervention in central inflammation in mice with obesity-depression comorbiditiy, based on the differential mRNA expression in the amygdala(AMY) and dorsal raphe nucleus(DRN) after CEL intervention. C57BL/6J mice were randomly divided into a normal diet group(Chow), a obesity-depression comorbidity(COM) group, and low-, medium-, and high-dose CEL groups(CEL-L, CEL-M, CEL-H, 0.5, 1.0, 2.0 mg·kg~(-1)). The Chow group received a normal diet, while the COM group and CEL-L, CEL-M, CEL-H groups received a high-fat diet combined with chronic stress from wet bedding. After 10 weeks of feeding, the mice were orally administered CEL for three weeks. Subsequently, the AMY and DRN of mice in the Chow, COM, and CEL-H groups were subjected to transcriptome analysis, and the intersection of target differentially expressed genes in both nuclei was visualized using a Venn diagram. The intersected genes were then imported into STRING for protein-protein interaction(PPI) analysis, and Gene Ontology(GO) analysis was performed using DAVID to identify the core targets regulated by CEL in the AMY and DRN. Independent samples were subjected to quantitative real-time PCR(qPCR) to validate the intersection genes. The results revealed that the common genes regulated by CEL in the AMY and DRN included chemokine family genes Ccl2, Ccl5, Ccl7, Cxcl10, Cxcr6, and Hsp70 family genes Hspa1a, Hspa1b, as well as Myd88, Il2ra, Irf7, Slc17a8, Drd2, Parp9, and Nampt. GO analysis showed that the top 5 nodes Ccl2, Cxcl10, Myd88, Ccl5, and Irf7 were all involved in immune-inflammation regulation(P<0.01). The qPCR results from independent samples showed that in the AMY, compared with the results in the Chow group, chemokine family genes, Hsp70, Myd88, Il2ra, Irf7, Slc17a8, Parp9, and Nampt were significantly up-regulated in the COM group, with Drd2 showing a decreasing trend; these pathological changes were significantly improved in the CEL-H group compared to the COM group. In the DRN, compared with the results in the Chow group, chemokine family genes, Hsp70, Myd88, Il2ra, Irf7, Parp9, and Nampt were significantly down-regulated, while Slc17a8 was significantly up-regulated in the COM group; compared with those in the COM group, Cxcr6, Irf7, and Drd2 were significantly up-regulated, while Slc17a8 was significantly down-regulated in the CEL-H group. In both the AMY and DRN, the expression of Irf7 by CEL showed both inhibition and activation in a dose-dependent manner(R~2 were 0.709 8 and 0.917 2, respectively). These findings suggest that CEL can effectively improve neuroinflammation by regulating bidirectional expression of the same target proteins, thereby intervening in the immune activation of the AMY and immune suppression of the DRN in COM mice.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

Heme oxygenase-1 inhibits DENV-induced endothelial hyperpermeability and serves as a potential target against dengue hemorrhagic fever.

Dengue virus (DENV) is a cause of vascular endothelial dysfunction and vascular leakage, which are characterized as hallmarks of dengue hemorrhagic fever or dengue shock syndrome, which become a severe global health emergency with substantial morbidity and mortality. Currently, there are still no promising therapeutics to alleviate the dengue-associated vascular hemorrhage in a clinical setting. In the present study, we first observed that heme oxygenase-1 (HO-1) expression level was highly suppressed in severe DENV-infected patients. In contrast, the overexpression of HO-1 could attenuate DENV-induced pathogenesis, including plasma leakage and thrombocytopenia, in an AG129 mouse model. Our data indicate that overexpression of HO-1 or its metabolite biliverdin can maintain endothelial integrity upon DENV infection in vitro and in vivo. We further characterized the positive regulatory effect of HO-1 on the endothelial adhesion factor vascular endothelial-cadherin to decrease DENV-induced endothelial hyperpermeability. Subsequently, we confirmed that two medicinal plant-derived compounds, andrographolide, and celastrol, widely used as a nutritional or medicinal supplement are useful to attenuate DENV-induced plasma leakage through induction of the HO-1 expression in DENV-infected AG129 mice. In conclusion, our findings reveal that induction of the HO-1 signal pathway is a promising option for the treatment of DENV-induced vascular pathologies.

Celastrol acts as a new histone deacetylase inhibitor to inhibit colorectal cancer cell growth via regulating macrophage polarity.

The tumorigenesis and progression of colorectal cancer are closely related to the tumor microenvironment, especially inflammatory response. Inhibitors of histone deacetylase (HDAC) have been reported as epigenetic regulators of the immune system to treat cancer and inflammatory diseases and our results demonstrated that Celastrol could act as a new HDAC inhibitor. Considering macrophages as important members of the tumor microenvironment, we further found that Celastrol could influence the polarization of macrophages to inhibit colorectal cancer cell growth. Specially, we used the supernatant of HCT116 and SW480 cells to induce Ana-1 cells in vitro and chose the spontaneous colorectal cancer model APCmin/+ mice as an animal model to validate in vivo. The results indicated that Celastrol could reverse the polarization of macrophages from M2 to M1 through impacting the colorectal tumor microenvironment both in vitro and in vivo. Furthermore, using bioinformatics analysis, we found that Celastrol might mechanistically polarize the macrophages through MAPK signaling pathway. In conclusion, our findings identified that Celastrol as a new HDAC inhibitor and suggested that Celastrol could modulate macrophage polarization, thus inhibiting colorectal cancer growth, which may provide some novel therapeutic strategies for colorectal cancer.