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.

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.

Oridonin suppresses the growth of glioblastoma cells via inhibiting Hippo/YAP axis.

Glioma is a brain tumor that originates from brain or spine glial cells. Despite alternative treatments, the overall survival rate remains low. Oridonin (ORI) is purified from the Chinese herb Rabdosia rubescens, which has exhibited positive effects on tumors. This study aimed to investigate the effect of ORI on U87MG glioblastoma cells and whether the Hippo/YAP-related signaling pathway was involved. Malignant glioblastoma U87MG cells and male athymic nude mice (BALB/cnu/nu) were used as the experimental models. The YAP inhibitor Verteporfin (VP) and the overexpression of YAP were used to investigate its potential relation with glioma. Here, we found that ORI inhibited cell proliferation and promoted cell apoptosis in a dose-dependent manner in U87MG cells. Moreover, ORI inhibited Bcl-2, YAP, and c-Myc protein expression but increased Bax, caspase-3, and p-YAP protein expression. Furthermore, the effect of ORI was also confirmed in a mouse model bearing glioma. ORI reversed the effect of overexpression of YAP. Collectively, oridonin suppressed glioblastoma oncogenesis via the Hippo/YAP signaling pathway and could be a potential therapeutic target in the treatment of glioblastoma.

Oridonin-induced ferroptosis and apoptosis: a dual approach to suppress the growth of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is one of the most common aggressive bone malignancy tumors in adolescents. With the application of new chemotherapy regimens, finding new and effective anti-OS drugs to coordinate program implementation is urgent for the patients of OS. Oridonin had been proved to mediate anti-tumor effect on OS cells, but its mechanism has not been fully elucidated. METHODS: The effects of oridonin on the viability, clonal formation and migration of 143B and U2OS cells were detected by CCK-8, colony formation assays and wound-healing test. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore the mechanism of oridonin on OS. Western blot (WB), real-time quantitative PCR (qRT-PCR) were used to detect the expression levels of apoptosis and ferroptosis-relative proteins and genes. Annexin V-FITC apoptosis detection kit and flow cytometry examination were used to detect the level of apoptosis. Iron assay kit was used to evaluate the relative Fe2+ content. The levels of mitochondrial membrane potential and lipid peroxidation production was determined by mitochondrial membrane potential detection kit and ROS assay kit. RESULTS: Oridonin could effectively inhibit the survival, clonal formation and metastasis of OS cells. The KEGG results indicated that oridonin is associated with the malignant phenotypic signaling pathways of proliferation, migration, and drug resistance in OS. Oridonin was capable of inhibiting expressions of BAX, cl-caspase3, SLC7A11, GPX4 and FTH1 proteins and mRNA, while promoting the expressions of Bcl-2 and ACSL4 in 143B and U2OS cells. Additionally, we found that oridonin could promote the accumulation of reactive oxygen species (ROS) and Fe2+ in OS cells, as well as reduce mitochondrial membrane potential, and these effects could be significantly reversed by the ferroptosis inhibitor ferrostatin-1 (Fer-1). CONCLUSION: Oridonin can trigger apoptosis and ferroptosis collaboratively in OS cells, making it a promising and effective agent for OS therapy.

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.

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.

Discovery of novel oridonin sulfamide derivatives as potent NLRP3 inhibitors by a visible-light photocatalysis-enabled peripheral editing.

The progress of organicsyntheticmethod can promote late-stage lead compound modification and novel active compound discovery. Molecular editing technology in the field of organic synthesis, including peripheral and skeletal editing, facilitates rapid access to molecular diversity of a lead compound. Peripheral editing of CH bond activation is gradually used in lead optimization to afford novel active scaffolds and chemical space exploitation. To develop oridonin derivatives with high anti-inflammatory potency, novel oridonin sulfamides had been designed and synthesized by a scaffoldhopping strategy based on a visible-light photocatalysis peripheral editing. All novel compounds revealed measurable inhibition of IL-1ß and low cytotoxicity in THP-1 cells. The docking study indicated that the best active compound ZM640 was accommodated in thebinding site of NLRP3 with two hydrogen bond interaction. These preliminary results confirm that α, ß-unsaturated carbonyl of oridonin is not essential for NLRP3 inhibitory effect. This new oridonin scaffold has its potential to be further developed as a promising class of NLRP3 inhibitors.

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.

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.

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 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.

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.

Angelica Sinensis Polysaccharide-Based Nanoparticles for Liver-Targeted Delivery of Oridonin.

The present work aimed to study the feasibility of Angelica sinensis polysaccharide (ASP) as an instinctive liver targeting drug delivery carrier for oridonin (ORI) in the treatment of hepatocellular carcinoma (HCC). ASP was reacted with deoxycholic acid (DOCA) via an esterification reaction to form an ASP-DOCA conjugate. ORI-loaded ASP-DOCA nanoparticles (ORI/ASP-DOCA NPs) were prepared by the thin-film water method, and their size was about 195 nm in aqueous solution. ORI/ASP-DOCA NPs had a drug loading capacity of up to 9.2%. The release of ORI in ORI/ASP-DOCA NPs was pH-dependent, resulting in rapid decomposition and accelerated drug release at acidic pH. ORI/ASP-DOCA NPs significantly enhanced the accumulation of ORI in liver tumors through ASGPR-mediated endocytosis. In vitro results showed that ORI/ASP-DOCA NPs increased cell uptake and apoptosis in HepG2 cells, and in vivo results showed that ORI/ASP-DOCA NPs caused effective tumor suppression in H22 tumor-bearing mice compared with free ORI. In short, ORI/ASP-DOCA NPs might be a simple, feasible, safe and effective ORI nano-drug delivery system that could be used for the targeted delivery and treatment of liver tumors.

Oridonin loaded peptide nanovesicles alleviate nonalcoholic fatty liver disease in mice.

This study was to construct a nanovesicle delivery system to improve the loading efficiency and stability of ORI for the treatment of nonalcoholic fatty liver disease (NAFLD). This nanovesicles (NVs) exerted a narrow size distribution (195.6 ± 11.49 nm) and high entrapment efficiency (84.46 ± 1.34%). In vitro cell studies demonstrated that the NVs treatment enhanced the cellular uptake of ORI and reduced lipid over-accumulation and total cholesterol levels in NAFLD cell model. At the same time, in vivo study proved that, compared with the normal group, the model group mice showed a decrease in body weight, a significant increase in liver index (6.71 ± 0.62, p < 0.01), and symptoms of liver lipid accumulation, lipid vesicles, and liver tissue fibrosis. Compared with the model group, after high-dose ORI NVs intervention, mice gained weight, decreased liver index (4.69 ± 0.55, p < 0.01), reduced hepatic lipid droplet vacuoles, reduced lipid accumulation (reduced oil red area, p < 0.001), and alleviated the degree of liver fibrosis (reduced blue collagen area, p < 0.001). In conclusion, ORI/HP-ß-CD/H9-HePC NVs showed specific liver accumulation and improved therapeutic effects, the nano drug loading system provides a promising strategy for the encapsulation of ORI to effectively alleviate the process of NAFLD.

Oridonin suppresses gastric cancer SGC-7901 cell proliferation by targeting the TNF-alpha/androgen receptor/TGF-beta signalling pathway axis.

Statistics provided by GLOBOCAN list gastric cancer as the sixth most common, with a mortality ranking of third highest for the year 2020. In China, a herb called Rabdosia rubescens (Hemsl.) H.Hara, has been used by local residents for the treatment of digestive tract cancer for hundreds of years. Oridonin, the main ingredient of the herb, has a curative effect for gastric cancer, but the mechanism has not been previously clarified. This study mainly aimed to investigate the role of TNF-alpha/Androgen receptor/TGF-beta signalling pathway axis in mediating the proliferation inhibition of oridonin on gastric cancer SGC-7901 cells. MTT assay, cell morphology observation assay and fluorescence assay were adopted to study the efficacy of oridonin on cell proliferation. The network pharmacology was used to predict the pathway axis regulated by oridonin. Western blot assay was adopted to verify the TNF-α/Androgen receptor/TGF-ß signalling pathway axis regulation on gastric cancer by oridonin. The results showed Oridonin could inhibit the proliferation of gastric cancer cells, change cell morphology and cause cell nuclear fragmentation. A total of 11signaling pathways were annotated by the network pharmacology, among them, Tumour necrosis factor alpha (TNF-α) signalling pathway, androgen receptor (AR) signalling pathway and transforming growth factor (TGF-ß) signalling pathway account for the largest proportion. Oridonin can regulate the protein expression of the three signalling pathways, which is consistent with the results predicted by network pharmacology. These findings indicated that oridonin can inhibit the proliferation of gastric cancer SGC-7901 cells by regulating the TNF-α /AR /TGF-ß signalling pathway axis.

Oridonin attenuates atherosclerosis by inhibiting foam macrophage formation and inflammation through FABP4/PPARγ signalling.

Both lipid accumulation and inflammatory response in lesion macrophages fuel the progression of atherosclerosis, leading to high mortality of cardiovascular disease. A therapeutic strategy concurrently targeting these two risk factors is promising, but still scarce. Oridonin, the bioactive medicinal compound, is known to protect against inflammatory response and lipid dysfunction. However, its effect on atherosclerosis and the underlying molecular mechanism remain elusive. Here, we showed that oridonin attenuated atherosclerosis in hyperlipidemic ApoE knockout mice. Meanwhile, we confirmed the protective effect of oridonin on the oxidized low-density lipoprotein (oxLDL)-induced foam macrophage formation, resulting from increased cholesterol efflux, as well as reduced inflammatory response. Mechanistically, the network pharmacology prediction and further experiments revealed that oridonin dramatically facilitated the expression of peroxisome proliferator-activated receptor gamma (PPARγ), thereby regulating liver X receptor-alpha (LXRα)-induced ATP-binding cassette transporter A1 (ABCA1) expression and nuclear factor NF-kappa-B (NF-κB) translocation. Antagonist of PPARγ reversed the cholesterol accumulation and inflammatory response mediated by oridonin. Besides, RNA sequencing analysis revealed that fatty acid binding protein 4 (FABP4) was altered responding to lipid modulation effect of oridonin. Overexpression of FABP4 inhibited PPARγ activation and blunted the benefit effect of oridonin on foam macrophages. Taken together, oridonin might have potential to protect against atherosclerosis by modulating the formation and inflammatory response in foam macrophages through FABP4/PPARγ signalling.

Oridonin Attenuates Thioacetamide-Induced Osteoclastogenesis Through MAPK/NF-κB Pathway and Thioacetamide-Inhibited Osteoblastogenesis Through BMP-2/RUNX2 Pathway.

Osteoporosis, an age-related metabolic bone disease, is mainly caused by an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. At present, there are many osteoporosis drugs that can promote bone formation or inhibit bone resorption. However, there were few therapeutic drugs that can simultaneously promote bone formation and inhibit bone resorption. Oridonin (ORI), a tetracyclic diterpenoid compound isolated from Rabdosia rubescens, has been proved to have anti-inflammatory, anti-tumor effects. However, little is known about the osteoprotective effect of oridonin. Thioacetamide (TAA) is a common organic compound with significant hepatotoxicity. Recent studies have found that there was a certain association between TAA and bone injury. In this work, we investigated the effect and mechanism of ORI on TAA-induced osteoclastogenesis and inhibition of osteoblast differentiation. The results showed that TAA could promote the osteoclastogenesis of RAW264.7 by promoting the MAPK/NF-κB pathway, and also promoted p65 nuclear translocation and activated intracellular ROS generation, and ORI can inhibit these effects to inhibit TAA-induced osteoclastogenesis. Moreover, ORI can also promote the osteogenic differentiation pathway and inhibit adipogenic differentiation of BMSCs to promote bone formation. In conclusion, our results revealed that ORI, as a potential therapeutic drug for osteoporosis, could protect against TAA-induced bone loss and TAA-inhibited bone formation.

Oridonin ameliorates caspase-9-mediated brain neuronal apoptosis in mouse with ischemic stroke by inhibiting RIPK3-mediated mitophagy.

Neuronal loss is a primary factor in determining the outcome of ischemic stroke. Oridonin (Ori), a natural diterpenoid compound extracted from the Chinese herb Rabdosia rubescens, has been shown to exert anti-inflammatory and neuroregulatory effects in various models of neurological diseases. In this study we investigated whether Ori exerted a protective effect against reperfusion injury-induced neuronal loss and the underlying mechanisms. Mice were subjected to transient middle cerebral artery occlusion (tMCAO), and were injected with Ori (5, 10, 20 mg/kg, i.p.) at the beginning of reperfusion. We showed that Ori treatment rescued neuronal loss in a dose-dependent manner by specifically inhibiting caspase-9-mediated neuronal apoptosis and exerted neuroprotective effects against reperfusion injury. Furthermore, we found that Ori treatment reversed neuronal mitochondrial damage and loss after reperfusion injury. In N2a cells and primary neurons, Ori (1, 3, 6 µM) exerted similar protective effects against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. We then conducted an RNA-sequencing assay of the ipsilateral brain tissue of tMCAO mice, and identified receptor-interacting protein kinase-3 (RIPK3) as the most significantly changed apoptosis-associated gene. In N2a cells after OGD/R and in the ipsilateral brain region, we found that RIPK3 mediated excessive neuronal mitophagy by activating AMPK mitophagy signaling, which was inhibited by Ori or 3-MA. Using in vitro and in vivo RIPK3 knockdown models, we demonstrated that the anti-apoptotic and neuroprotective effects of Ori were RIPK3-dependent. Collectively, our results show that Ori effectively inhibits RIPK3-induced excessive mitophagy and thereby rescues the neuronal loss in the early stage of ischemic stroke.

Preparation, characterization, and pharmacokinetics of oridonin-loaded liposomes.

The aim of this study was to prepare oridonin liposomes and evaluate the physicochemical characteristics and pharmacokinetics in rats. A three-level, three-factor Box-Behnken design was used to optimize the preparation of oridonin liposomes. A highly sensitive high-performance liquid chromatographic quantification method using ultraviolet detection was established and validated for the determination of oridonin in rat plasma. Twelve Sprague-Dawley rats were randomly assigned and injected with 15 mg/kg of oridonin or oridonin liposomes via the tail vein. Pharmacokinetic parameters were estimated using a compartmental modeling approach using PKsolver software. The optimum conditions were as follows: soybean phospholipids/cholesterol ratio, 3.9:1; soybean phospholipids/drug ratio, 8.5:1; and soybean phospholipid concentration, 1.1%. Under these conditions, the mean particle size, polydispersity index, zeta potential, and encapsulation efficiency of oridonin liposomes were 170.5 nm, 0.246, -30.3 mV, and 76.15%, respectively. The pharmacokinetic results showed that liposomes could significantly prolong the elimination half-life (from 2.88 ± 0.55 to 13.67 ± 3.52 h), increase the area under the concentration-time curve (from 1.65 ± 0.17 to 6.22 ± 0.83 µg h/ml), and decrease the clearance (from 6.62 ± 1.38 to 1.96 ± 0.24 L/kg h). The oridonin liposomes increased the elimination half-life and area under the concentration-time curve and provided a reference for the development of drugs with a short half-life.

Oridonin ameliorates acetaminophen-induced acute liver injury through ATF4/PGC-1α pathway.

Acetaminophen (APAP) overdose-induced acute liver injury (ALI) causes hepatocyte cell death, oxidative stress, and inflammation. Oridonin (Ori), a covalent NLRP3-inflammasome inhibitor, ameliorates APAP-induced ALI through an unclear molecular mechanism. This study found that Ori decreased hepatic cytochrome P450 2E1 level and increased glutathione content to prevent APAP metabolism, and then reduced the necrotic area, improved liver function, and inhibited APAP-induced proinflammatory cytokines and oxidative stress. Ori also decreased activating transcription factor 4 (ATF4) protein levels and increased peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) to reduce APAP-induced endoplasmic reticulum stress activation and mitochondrial dysfunction. Furthermore, western blot and luciferase assay found that ATF4 inhibited transcription in the PGC-1α promoter -507 to -495 region to reduce PGC-1α levels, while ATF4 knockdown neutralized the hepatoprotective effect of Ori. Molecular docking showed that Ori bound to ATF4's amino acid residue glutamate 302 through 6, 7, and 18 hydroxyl bands. Our findings demonstrated that Ori prevented metabolic activation of APAP and further inhibited the ATF4/PGC-1α pathway to alleviate APAP overdose-induced hepatic toxicity, which illuminated its potential therapeutic effects on ALI.