Light-promoted stereoselective late-stage difunctionalization and anti-tumor activity of oridonin.

The late-stage difunctionalization of diterpene oridonin by light-promoted direct oxyamination with various O-benzoylhydroxylamines was carried out to afford C16α-N-C17-OBz-oridonin derivatives (1-25) for the first time. Though as a radical reaction, it features high stereoselectivity to only produce C16α-N-C17-OBz-oridonins. The in vitro antiproliferative activity of these C16α-N-C17-OBz-oridonins against the human breast cancer cell lines (MCF-7) was evaluated by MTT assay, showing that most of the synthesized compounds possessed moderate anticancer activity against MCF-7 cell lines superior or similar to the parent compound oridonin. The derivative 25 with a N-methyl-N-(naphthalen-1-ylmethyl) substitution showed better cytotoxicity against MCF-7 cells (IC50 value of 11.75 µM) than oridonin (IC50 value of 17.95 µM).

Design and synthesis of 6,20-epoxy A-ring modified oridonin derivatives with antitumor activity through extrinsic and mitochondrial pathways.

Oridonin is an antitumor ent-kaurane diterpenoid that medicinal chemists have been paying close attention to in recent years. Herein, a novel 6,20-epoxy A-ring modified oridonin derivative 2 was obtained by a 6-step synthesis. A series of 14-O derivatives of 2 (EpskA1-EpskA24) were synthesized to further enhance the activity. Based on their cytotoxicity against MCF-7, A549 and L-02 cells, EpskA9, EpskA10 and EpskA21 were chosen for further screening to obtain a wider antitumor spectrum. Collectively, EpskA21 showed the most potent antiproliferative activity, inhibiting proliferation and migration, and inducing apoptosis and cell cycle arrest in MCF-7 and MIA-PaCa-2 cells. With the help of network pharmacology analysis, apoptosis-related proteins were selected and further tested by western blot assay. The inhibition of PI3K/AKT and an increase in the levels of Bax/Bcl-2 ratio, Cyt-C, cleaved-Caspase-9, cleaved-Caspase-3 and cleaved-PARP was observed, indicating that EpskA21 induced apoptosis through the mitochondrial pathway. Given that an increase in DR5 expression and activated Caspase-8 were also observed, the extrinsic apoptosis pathway might also be related to the antitumor effect.

Effect of Oridonin on Experimental Animal Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a serious disease that occurs in premature and low-birth-weight infants. In recent years, the incidence of BPD has not decreased, and there is no effective treatment for it. Oridonin (Ori) is a traditional Chinese medicine with a wide range of biological activities, especially pharmacological and anti-inflammatory. It is well known that inflammation plays a key role in BPD. However, the therapeutic effect of Ori on BPD has not been studied. Therefore, in the present study, we will observe the anti-inflammatory activity of Ori in an experimental animal model of BPD. Here, we showed that Ori could significantly decrease hyperoxia-induced alveolar injury, inhibit neutrophil recruitment, myeloperoxidase concentrations, and release inflammatory factors in BPD neonatal rats. Taken together, the experimental results suggested that Ori can significantly improve BPD in neonatal rats by inhibiting inflammatory response.

Effects of oridonin on sperm function and the PI3K/PDK1/AKT signaling pathway: Implications for reproductive toxicity.

Oridonin, a natural terpenoid isolated from the leaves of Isodon rubescens (Hemsley) H.Hara, is widely used in oriental medicine for its anticancer properties across various cancer types. Despite its prevalent use, the toxic effects of oridonin on male reproduction, particularly its impact on sperm functions and the mechanisms involved, are not well understood. This study aimed to explore the effects and underlying mechanisms of oridonin on sperm functions. We initially treated Duroc boar spermatozoa with varying concentrations of oridonin (0, 5, 50, 75, 100, and 150 µM) and incubated them to induce capacitation. We then assessed cell viability and several sperm functions, including sperm motility and motion kinematics, capacitation status, and ATP levels. We also analyzed the expression levels of proteins associated with the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (AKT) signaling pathway and phosphotyrosine proteins. Our results indicate that oridonin adversely affects most sperm functions in a dose-dependent manner. We observed significant decreases in AKT, p-AKT (Thr308), phosphatase and tensin homolog (PTEN), p-PDK1, and p-PI3K levels following oridonin treatment, alongside an abnormal increase in phosphotyrosine proteins. These findings suggest that oridonin may disrupt normal levels of tyrosine-phosphorylated proteins by inhibiting the PI3K/PDK1/AKT signaling pathway, which is crucial for cell proliferation, metabolism, and apoptosis, thus potentially harming sperm functions. Consequently, we recommend considering the reproductive toxicity of oridonin when using it as a therapeutic agent.

The water and oridonin sources in the ice ribbons of Isodon rubescens.

To study the source and content change of oridonin in the ice ribbons, the contents of oridonin in the ice ribbons and bleeding sap of Isodon rubescens at different times were determined with RP-HPLC. The paraffin sectioning and electron microscopy imaging were performed to study the transport channel of oridonin in the stem. The results showed that there were abundant xylem rays and perfect pit pairs in the secondary xylem of I. rubescens stems. The oridonin content in the ice ribbons of I. rubescens stems was lower than that in the stem of I. rubescens and even decreased over time. The contents of oridonin in the bleeding sap of I. rubescens stems was equal to that in second-day ice ribbons and was lower than that in first-day ice ribbons. The water in the ice ribbons of I. rubescens stems originated from water absorbed by the roots from soil. This water was transported from the roots of I. rubescens to the stem and then transferred through efficient lateral conducting tissues to the surface of the stem. The oridonin in the phloem and cortex of I. rubescens stems dissolves in water originating from the soil and freezes in the form of ice ribbons below 0 °C.

Oridonin ameliorates renal fibrosis in diabetic nephropathy by inhibiting the Wnt/ß-catenin signaling pathway.

Diabetic nephropathy (DN) is one of the most serious and frequent complications among diabetes patients and presently constitutes vast the cases of end-stage renal disease worldwide. Tubulointerstitial fibrosis is a crucial factor related to the occurrence and progression of DN. Oridonin (Ori) is a diterpenoid derived from rubescens that has diverse pharmacological properties. Our previous study showed that Ori can protect against DN by decreasing the inflammatory response. However, whether Ori can alleviate renal fibrosis in DN remains unknown. Here, we investigated the mechanism through which Ori affects the Wnt/ß-catenin signaling pathway in diabetic rats and human proximal tubular epithelial cells (HK-2) exposed to high glucose (HG) levels. Our results revealed that Ori treatment markedly decreased urinary protein excretion levels, improved renal function and alleviated renal fibrosis in diabetic rats. In vitro, HG treatment increased the migration of HK-2 cells while reducing their viability and proliferation rate, and treatment with Ori reversed these changes. Additionally, the knockdown of ß-catenin arrested cell migration and reduced the expression levels of Wnt/ß-catenin signaling-related molecules (Wnt4, p-GSK3ß and ß-catenin) and fibrosis-related molecules (α-smooth muscle actin, collagen I and fibronectin), and Ori treatment exerted an effect similar to that observed after the knockdown of ß-catenin. Furthermore, the combination of Ori treatment and ß-catenin downregulation exerted more pronounced biological effects than treatment alone. These findings may provide the first line of evidence showing that Ori alleviates fibrosis in DN by inhibiting the Wnt/ß-catenin signaling pathway and thereby reveal a novel therapeutic avenue for treating tubulointerstitial fibrosis.

Oridonin ameliorates ocular surface inflammatory responses by inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway in dry eye.

Chronic inflammation is one of the central drivers in the development of dry eye disease (DED), in which pyroptosis induced by the NLRP3/caspase-1/gasdermin D (GSDMD) pathway plays a key role. This pathway has become a major target for the treatment of a variety of inflammatory disorders. Oridonin (Ori) is a naturally occurring substance with anti-inflammatory properties obtained from Rabdosia rubescens. Whether Ori can exert an anti-inflammatory effect on DED, and its anti-inflammatory mechanism of action, are still unknown. This experiment is intended to investigate the impact of Ori on the hyperosmolarity-induced NLRP3/caspase-1/GSDMD pyroptosis pathway in immortalized human corneal epithelial (HCE-T) cells, as well as its efficacy and mechanism of action on ocular surface injury in DED mice. Our study showed that Ori could inhibit hyperosmotic-induced pyroptosis through the NLRP3/caspase-1/GSDMD pathway in HCE-T cells, and similarly, Ori inhibited the expression of this pathway in DED mice. Moreover, Ori was protective against hyperosmolarity-induced HCE-T cell damage. In addition, we found that the morphology and number of HCE-T cells were altered under culture conditions of various osmolarities. With increasing osmolarity, the proliferation, migration, and healing ability of HCE-T cells decreased significantly, and the expression of N-GSDMD was elevated. In a mouse model of DED, Ori application inhibited the expression of the NLRP3/caspase-1/GSDMD pyroptosis pathway, improved DED signs and injury, decreased corneal sodium fluorescein staining scores, and increased tear volume. Thus, our study suggests that Ori has potential applications for the treatment of DED, provides potential novel therapeutic approaches to treat DED, and provides a theoretical foundation for treating DED using Ori.

Synthesis and Application of a pH-Responsive Functional Metal-Organic Framework: In Vitro Investigation for Delivery of Oridonin in Cancer Therapy.

Oridonin (Ori) is a naturally existing diterpenoid substance that mainly exists in the Chinese medicinal plant Rabdosia rubescens. It was previously found to possess intriguing biological properties; however, the quick clearance from plasma and limited solubility in water restricts its use as a drug. Several metal-organic frameworks (MOFs), having big surfaces and large pores, have recently been considered promising drug transporters. The zeolitic imidazolate framework-8 (ZIF-8), a form of MOF consisting of 2-methylimidazole with zinc ions, is structurally stable under physiologically neutral conditions, while it can degrade at low pH values such as in tumor cells. Herein, a nanosized drug delivery system, Ori@ZIF-8, was successfully designed for encapsulating and transporting oridonin to the tumor site. The drug loading of the prepared Ori@ZIF-8 was 26.78%, and the particles' mean size was 240.5 nm. In vitro, the release of Ori@ZIF-8 exhibited acid sensitivity, with a slow release under neutral conditions and rapid release of the drug under weakly acidic conditions. According to the in vitro anti-tumor experiments, Ori@ZIF-8 produced higher cytotoxicity than free Ori and induced apoptosis in A549 cancer cells. In conclusion, Ori@ZIF-8 could be a potential pH-responsive carrier to accurately release more oridonins at the tumor site.

Integrating Epigenetics, Proteomics, and Metabolomics to Reveal the Involvement of Wnt/ß-Catenin Signaling Pathway in Oridonin-Induced Reproductive Toxicity.

Oridonin is the primary active component in the traditional Chinese medicine Rabdosia rubescens, displaying anti-inflammatory, anti-tumor, and antibacterial effects. It is widely employed in clinical therapy for acute and chronic pharyngitis, tonsillitis, as well as bronchitis. Nevertheless, the clinical application of oridonin is significantly restricted due to its reproductive toxicity, with the exact mechanism remaining unclear. The aim of this study was to investigate the mechanism of oridonin-induced damage to HTR-8/SVneo cells. Through the integration of epigenetics, proteomics, and metabolomics methodologies, the mechanisms of oridonin-induced reproductive toxicity were discovered and confirmed through fluorescence imaging, RT-qPCR, and Western blotting. Experimental findings indicated that oridonin altered m6A levels, gene and protein expression levels, along with metabolite levels within the cells. Additionally, oridonin triggered oxidative stress and mitochondrial damage, leading to a notable decrease in WNT6, ß-catenin, CLDN1, CCND1, and ZO-1 protein levels. This implied that the inhibition of the Wnt/ß-catenin signaling pathway and disruption of tight junction might be attributed to the cytotoxicity induced by oridonin and mitochondrial dysfunction, ultimately resulting in damage to HTR-8/SVneo cells.

Suppression of PERK/eIF2α/CHOP pathway enhances oridonin-induced apoptosis by inhibiting autophagy in Small-Cell lung cancer cells.

Chinese herbs have been used to treat small-cell lung cancer (SCLC) due to their low toxicity and significant efficacy. This study focused on oridonin, a natural compound extracted from Rabdosia rubescens, and aimed to investigate its potential antitumor activity on SCLC and to evaluate the synergistic effect of combining oridonin with other small molecules. In this study, oridonin exhibited a dual effect. At lower concentrations, it suppressed the cell viability of SCLC cells (H1688 and H446). At high concentrations, oridonin induced SCLC cell apoptosis, damaged HBE cells in vitro and compromised the function of the liver and heart in vivo. The lower concentration of oridonin induced autophagy by enhancing the expression of p62 and the LC3B-II/LC3B-I ratio. This phenomenon might be associated with the activation of the protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2α)/growth arrest and DNA damage-inducible gene 153 (CHOP/GAD153) pathway. Therefore, the combined effect of oridonin with GSK2606414 or 3- methyladenine increased apoptosis in SCLC cells and reduced tumor growth. A similar phenomenon was observed after oridonin was combined with p62 or CHOP RNA interference treatment. Simultaneously, the combination of oridonin and GSK2606414 exhibited therapeutic efficacy without manifesting adverse effects. Our findings suggest that oridonin at lower concentrations can induce autophagy by activating the PERK/eIF2α/CHOP signaling pathway. The inhibition of the PERK/eIF2α/CHOP pathway could enhance oridonin therapeutic responses by triggering apoptosis. The novel therapeutic approach of combining oridonin with a PERK inhibitor is promising as a strategy for the treatment of SCLC.

Oridonin, a small molecule inhibitor of cancer stem cell with potent cytotoxicity and differentiation potential.

Cancer stem cells (CSCs) drive malignant tumor progression, recurrence, and metastasis with unique characteristics, including self-renewal and resistance to conventional treatments. Conventional differentiation inducers, although promising, have limited cytotoxicity and may inadvertently enhance CSC stemness. To address these challenges, ongoing efforts are dedicated to developing strategies that can effectively combine both cytotoxicity and differentiation-inducing effects. In this study, we introduce oridonin (Ori), a small molecule with dual differentiation-inducing and cytotoxicity properties capable of eliminating tumor CSCs. We isolated CSCs in B16F10 cells using the Hoechst side population method and assessed the differentiation effect of Ori. Ori's differentiation-inducing effect was further evaluated using human acute promyelocytic leukemia. The cytotoxic potential of Ori against MCF-7 and B16F10 cell lines was assessed through various methods. In vivo anti-tumor and anti-CSC efficacy of Ori was investigated using mouse melanoma and CSCs melanoma models. Safety evaluation included zebrafish embryotoxicity and mouse acute toxicity experiments. As a result, Ori effectively dismantles tumorspheres, inhibits proliferation, and reduces the expression of CSC-specific markers. It induces significant differentiation, especially in the case of NB4. Additionally, Ori upregulates TP53 expression, mitigates the hypoxic tumor microenvironment, suppresses stemness, and inhibits PD-L1 expression, prompting a robust anti-cancer immune response. Ori demonstrates pronounced cytotoxicity, inducing notable pro-apoptotic effects on B16F10 and MCF-7 cells, with specific triggering of mitochondrial apoptosis. Importantly, Ori maintains a commendable biosafety record. The dual-action prowess of Ori not only induces the differentiation of CSCs but also dispatches differentiated and residual tumor cells, effectively thwarting the relentless march of tumor progression.

Oridonin attenuates liver ischemia-reperfusion injury by suppressing PKM2/NLRP3-mediated macrophage pyroptosis.

BACKGROUND: The NOD-like receptor protein 3 (NLRP3) mediated pyroptosis of macrophages is closely associated with liver ischemia reperfusion injury (IRI). As a covalent inhibitor of NLRP3, Oridonin (Ori), has strong anti-inflammasome effect, but its effect and mechanisms for liver IRI are still unknown. METHODS: Mice and liver macrophages were treated with Ori, respectively. Co-IP and LC-MS/MS analysis of the interaction between PKM2 and NLRP3 in macrophages. Liver damage was detected using H&E staining. Pyroptosis was detected by WB, TEM, and ELISA. RESULTS: Ori ameliorated liver macrophage pyroptosis and liver IRI. Mechanistically, Ori inhibited the interaction between pyruvate kinase M2 isoform (PKM2) and NLRP3 in hypoxia/reoxygenation（H/R）-induced macrophages, while the inhibition of PKM2/NLRP3 reduced liver macrophage pyroptosis and liver IRI. CONCLUSION: Ori exerted protective effects on liver IRI via suppressing PKM2/NLRP3-mediated liver macrophage pyroptosis, which might become a potential therapeutic target in the clinic.

Glutathione-depleting Liposome Adjuvant for Augmenting the Efficacy of a Glutathione Covalent Inhibitor Oridonin for Acute Myeloid Leukemia Therapy.

BACKGROUND: Discrepancies in the utilization of reactive oxygen species (ROS) between cancer cells and their normal counterparts constitute a pivotal juncture for the precise treatment of cancer, delineating a noteworthy trajectory in the field of targeted therapies. This phenomenon is particularly conspicuous in the domain of nano-drug precision treatment. Despite substantial strides in employing nanoparticles to disrupt ROS for cancer therapy, current strategies continue to grapple with challenges pertaining to efficacy and specificity. One of the primary hurdles lies in the elevated levels of intracellular glutathione (GSH). Presently, predominant methods to mitigate intracellular GSH involve inhibiting its synthesis or promoting GSH efflux. However, a conspicuous gap remains in the absence of a strategy capable of directly and efficiently clearing GSH. METHODS: We initially elucidated the chemical mechanism underpinning oridonin, a diminutive pharmacological agent demonstrated to perturb reactive oxygen species, through its covalent interaction with glutathione. Subsequently, we employed the incorporation of maleimide-liposomes, renowned for their capacity to disrupt the ROS delivery system, to ameliorate the drug's water solubility and pharmacokinetics, thereby enhancing its ROS-disruptive efficacy. In a pursuit to further refine the targeting for acute myeloid leukemia (AML), we harnessed the maleic imide and thiol reaction mechanism, facilitating the coupling of Toll-like receptor 2 (TLR2) peptides to the liposomes' surface via maleic imide. This strategic approach offers a novel method for the precise removal of GSH, and its enhancement endeavors are directed towards fortifying the precision and efficacy of the drug's impact on AML targets. RESULTS: We demonstrated that this peptide-liposome-small molecule machinery targets AML and consequently induces cell apoptosis both in vitro and in vivo through three disparate mechanisms: (I) Oridonin, as a Michael acceptor molecule, inhibits GSH function through covalent bonding, triggering an initial imbalance of oxidative stress. (II) Maleimide further induces GSH exhaustion, aggravating redox imbalance as a complementary augment with oridonin. (III) Peptide targets TLR2, enhances the directivity and enrichment of oridonin within AML cells. CONCLUSION: The rationally designed nanocomplex provides a ROS drug enhancement and targeted delivery platform, representing a potential solution by disrupting redox balance for AML therapy.

Oridonin exerts anticonvulsant profile and neuroprotective activity in epileptic mice by inhibiting NLRP3-mediated pyroptosis.

BACKGROUND: Epilepsy is a chronic disabling disease poorly controlled by available antiseizure medications. Oridonin, a bioactive alkaloid with anti-inflammatory properties and neuroprotective effects, can inhibit the increased excitability of neurons caused by glutamate accumulation at the cellular level. However, whether oridonin affects neuronal excitability and whether it has antiepileptic potential has not been reported in animal models or clinical studies. METHOD: Pentylenetetrazol was injected into mice to create a model of chronic epilepsy. Seizure severity was assessed using the Racine scale, and the duration and latency of seizures were observed. Abnormal neuronal discharge was detected using electroencephalography, and neuronal excitability was assessed using calcium imaging. Damage to hippocampal neurons was evaluated using Hematoxylin-Eosin and Nissl staining. The expression of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and other pyroptosis-related proteins was determined using western blotting and immunofluorescence. A neuronal pyroptosis model was established using the supernatant of BV2 cells treated with lipopolysaccharide and adenosine triphosphate to stimulate hippocampal neurons. RESULTS: Oridonin (1 and 5 mg/kg) reduced neuronal damage, increased the latency of seizures, and shortened the duration of fully kindled seizures in chronic epilepsy model mice. Oridonin decreased abnormal discharge during epileptic episodes and suppressed increased neuronal excitability. In vitro experiments showed that oridonin alleviated pyroptosis in hippocampal HT22 neurons. CONCLUSION: Oridonin exerts neuroprotective effects by inhibiting pyroptosis through the NLRP3/caspase-1 pathway in chronic epilepsy model mice. It also reduces pyroptosis in hippocampal neurons in vitro, suggesting its potential as a therapy for epilepsy.

Oridonin promotes RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress signaling pathway JNK/Nrf2/HO-1.

The incidence and mortality of breast cancer, the most common malignant tumor among women in the world, are increasing year by year, which greatly threatens women's health. Ferroptosis is an iron and lipid reactive oxygen species (ROS)-dependent process, a novel form of cell death that is distinct from apoptosis and is closely related to the progression of breast cancer. Inducing the occurrence of ferroptosis in tumor cells can effectively block its malignant progress in vivo. Oridonin (ORI), the primary active ingredient extracted from the Chinese herbal medicine Rabdosia rubescens, has been shown to cause glutathione depletion and directly inhibit glutathione peroxidase 4 induced cell death by ferroptosis, but its mechanism of action in breast cancer remains inadequately elucidated. Therefore, we further investigated whether ORI could promote RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress pathway JNK/Nrf2/HO-1. In our study, we assessed cell survival of RSL3 and ORI treatment by MTT assay, and found that co-treatment with RSL3 and ORI inhibited cell proliferation, as evidenced by the cloning assay. To investigate the ability of ORI to promote RSL3-induced ferroptosis in breast cancer cells, we measured levels of ROS, malondialdehyde, glutathione, superoxide dismutase, and Fe2+ content. Lipid peroxidation, ROS, and mitochondrial membrane potential levels induced by co-treatment of ORI with RSL3 were reversed by ferrostatin-1, further confirming that the cell death induced by RSL3 and ORI was ferroptosis rather than other programmed cell death modes. Moreover, RSL3 and ORI co-treatment regulated the JNK/Nrf2/HO-1 axis, as demonstrated by western blotting and target activator validation. Our results showed that ORI could enhance the inhibitory effect of RSL3 on breast cancer cells viability via the induction of ferroptosis. Mechanistically, it potentiated RSL3-induced ferroptosis in breast cancer cells by activating the JNK/Nrf2/HO-1 axis. This study provides a theoretical basis for the application of ORI based on the mechanism of ferroptosis, and provides potential natural drug candidates for cancer prevention and treatment.

Research and Development Progression of Oridonin for Hematological Malignancies Therapy.

Oridonin is a tetracyclic diterpenoid compound extracted from the medicinal herb Isodon and related species. Since 1976, studies have reported the significant anti-tumor activity of oridonin in vivo. Recently, an increasing number of studies have confirmed the anti-tumor effects of oridonin in various types of cancers, and its effect on hematological malignancies stands out. Herein, we have systematically reviewed the anti-- tumor effects of oridonin and its specific mechanisms in hematological malignancies, including the regulation of cancer proteins, activation of intrinsic and extrinsic apoptosis signaling pathways, accumulation of reactive oxygen species (ROS), modulation of chaperone proteins and miRNA expression, combination therapy with chemotherapeutic drugs, and the development of its derivatives. Taken together, oridonin exhibits multiple anti-tumor activities and serves as a multi-target agent, making it worthy of further investigation.

Oridonin inhibited epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling.

Oridonin is the main bioactive component of Rabdosia rubescens, and its anticancer activity has been reported in a variety of cancers. However, the molecular mechanism of oridonin in laryngeal carcinoma remains unclear. In the present study, the cytotoxic effect of oridonin on laryngeal carcinoma Hep-2 and TU212 cell lines were initially detected by modified MTT assay. The results showed that oridonin had a dose-dependent anti-proliferative effect on laryngeal carcinoma Hep-2 and TU212 cells. Next, we found that oridonin significantly inhibited the migration and invasion of human laryngeal carcinoma Hep-2 and TU212 cell lines by wound healing assay and transwell assay. Subsequently, the results of quantitative real-time PCR assay and western blotting assay confirmed that oridonin upregulated the expression of E-cadherin while downregulated the expression of N-cadherin in a concentration-dependent manner at mRNA and protein levels. In addition, phosphorylation levels of liver kinase B1 (p-LKB1) and AMP-activated protein kinase (p-AMPK) were also elevated upon oridonin treatment. To further verify the role of LKB1/AMPK signaling pathway in laryngeal carcinoma, overexpression of LKB1 was constructed by plasmid transfection. The data exhibited that overexpression of LKB1 could further reinforce the increase of E-cadherin level and decrease of N-cadherin level mediated by oridonin. Additionally, AMPK inhibitor compound C could reverse anti-metastatic effect of oridonin on laryngeal carcinoma, and antagonise EMT expression. In contrast, AMPK activator AICAR presented the opposite effect. In conclusion, our study revealed that oridonin could remarkably reverse the epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling pathway, which suggested that oridonin may be a potential candidate for the treatment of laryngeal carcinoma in the future.

Novel Oridonin Analog CYD0682 Inhibits Hepatic Stellate Cell Activation via the Heat Shock Protein 90-Dependent STAT3 Pathway.

INTRODUCTION: Activated hepatic stellate cells (HSCs) are the primary effector cells in hepatic fibrosis, over depositing extracellular matrix (ECM) proteins. Our previous work found oridonin analog CYD0682 attenuates proliferation, Transforming Growth Factor ß (TGFß)-induced signaling, and ECM production in immortalized HSCs. The underlying mechanism behind these reductions is unclear. The Signal Transduction and Activator of Transcription 3 (STAT3) pathway plays a central role in HSC activation and has been found to be overexpressed in models of hepatic injury. In this study, we will examine the effect of CYD0682 on STAT3 signaling. METHODS: Immortalized human (LX-2) and rat (HSC-T6) HSC lines were treated with CYD0682 or Tanespimycin (17-AAG) with or without TGF-ß. Nuclear and cytosolic proteins were extracted. Protein expression was analyzed with Western blot. DNA binding activity was assessed with STAT3 DNA Binding ELISA. Cell viability was assessed with Alamar blue assay. RESULTS: CYD0682 treatment inhibited STAT3 phosphorylation at tyrosine 705 in a dose-dependent manner in LX-2 and HSC-T6 cells. STAT3 DNA binding activity and STAT3 regulated protein c-myc were significantly decreased by CYD0682. Notably, TGFß-induced STAT3 phosphorylation and ECM protein expression were inhibited by CYD0682. STAT3 is reported to be a Heat Shock Protein 90 (HSP90) client protein. Notably, CYD0682 attenuated the expression of endogenous STAT3 and other HSP90 client proteins FAK, IKKα, AKT and CDK9. HSP90 specific inhibitor 17-AAG suppressed endogenous and TGFß-induced STAT3 phosphorylation and ECM protein production. CONCLUSIONS: CYD0682 attenuates endogenous and TGFß-induced STAT3 activation and ECM production via an HSP90 dependent pathway in HSCs. Further study of this pathway may present new targets for therapeutic intervention in hepatic fibrosis.

Antibacterial Mechanisms and Antivirulence Activities of Oridonin against Pathogenic Aeromonas hydrophila AS 1.1801.

Aeromonas hydrophila, a Gram-negative bacterium widely found in freshwater environments, acts as a common conditional pathogen affecting humans, livestock, and aquatic animals. In this study, the impact of oridonin, an ent-kaurane diterpenoid compound derived from Rabdosia rubescens, on the virulence factors of A. hydrophila AS 1.1801 and its antibacterial mechanism was elucidated. The minimum inhibitory concentration (MIC) of oridonin against A. hydrophila AS 1.1801 was 100 µg/mL. Oridonin at inhibitory concentrations could significantly increase the electrical conductivity in the supernatant and escalate nucleic acid leakage (p < 0.01). This effect was concomitant with observed distortions in bacterial cells, the formation of cytoplasmic cavities, cellular damage, and pronounced inhibition of protein and nucleic acid synthesis. Additionally, oridonin at inhibitory levels exhibited a noteworthy suppressive impact on A. hydrophila AS 1.1801 across biofilm formation, motility, hemolytic activity, lipase activity, and protease activity (p < 0.05), demonstrating a dose-dependent enhancement. qRT-PCR analysis showed that the gene expression of luxR, qseB and omp were significantly downregulated after oridonin treatment in A. hydrophila AS 1.1801 (p < 0.05). Our results indicated that oridonin possessed significant antibacterial and anti-virulence effects on A. hydrophila AS 1.1801.

Oridonin inhibits bladder cancer survival and immune escape by covalently targeting HK1.

BACKGROUND: Hexokinase I (HK1) is highly expressed in a variety of malignancies, regulates glycolytic pathway in cancer cells, and thus considered to be one of the promising molecular targets for cancer therapy. Nonetheless, the development of a specific inhibitor against HK1 remains elusive. PURPOSE: This study aims to elucidate the mechanism by which oridonin inhibits the proliferation and immune evasion of bladder cancer cells, specifically through the suppression of HK1. METHODS: To examine the mechanisms by which oridonin directly binds to cysteines of HK1 and inhibits bladder cancer growth, this study utilized a variety of methods. These included the Human Proteome Microarray, Streptavidin-agarose affinity assay, Biolayer Interferometry (BLI) ainding analysis, Mass Spectrometry, Cellular Thermal Shift Assay, Extracellular Acidification Rate measurement, and Xenotransplant mouse models. RESULTS: As indicated by our current findings, oridonin forms a covalent bond with Cys-813, located adjacently to glucose-binding domain of HK1. This suppresses the enzymatic activity of HK1, leading to an effective reduction of glycolysis, which triggers cell death via apoptosis in cells derived from human bladder cancer. Significantly, oridonin also inhibits lactate-induced PD-L1 expression in bladder cancer. Furthermore, pairing oridonin with a PD-L1 inhibitor amplifies the cytotoxicity of CD8+ T cells against bladder cancer. CONCLUSION: This research strongly suggests that oridonin serves as a covalent inhibitor of HK1. Moreover, it indicates that functional cysteine residue of HK1 could operate as viable targets for selective inhibition. Consequently, oridonin exhibits substantial potential for the evolution of anti-cancer agents targeting the potential therapeutic target HK1 via metabolism immunomodulation.