OBJECTIVE: To investigate the effects of oridonin on platelet function and related mechanisms. METHODS: Washed platelets from healthy adults and mice were incubated with different concentrations of oridonin (2.5, 5 and 10 μmol/L) in vitro . The surface expression level of P-selectin and the activation of integrin αIIbβ3 in platelets were detected by flow cytometry, and the aggregation ability of platelets under the stimulation by various agonists was detected by light transmission aggregometry. The expression of P-AKT (Ser473) was detected by protein immunoblotting. Arterial thrombosis model was established in mice with mesenteric injury induced by ferric chloride, and tail hemorrhage model was established by cutting off the tail of mice. The effect of intraperitoneal injection of oridonin (10 mg/kg) on thrombosis and haemostasis was tested. RESULTS: Oridonin inhibited platelet P-selectin expression and integrin αIIbβ3 activation. In the presence of different stimulants, oridonin inhibited platelet aggregation in a concentration-dependent manner. The phosphorylation level of AKT Ser473 was reduced in the groups treated with different concentrations of oridonin. Oridonin significantly prolonged the time of mesenteric artery thrombosis in mice, but did not affect the tail bleeding time. CONCLUSION Oridonin inhibits platelet activation, aggregation, and thrombosis by inhibiting AKT phosphorylation, and may be used as a potential antiplatelet drug.
Diterpenes, Kaurane/pharmacology* ; Animals ; Mice ; Blood Platelets/drug effects* ; Platelet Aggregation/drug effects* ; P-Selectin/metabolism* ; Thrombosis ; Platelet Glycoprotein GPIIb-IIIa Complex/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Humans ; Phosphorylation ; Platelet Activation/drug effects*

OBJECTIVE: To explore the key target molecules and potential mechanisms of oridonin against non-small cell lung cancer (NSCLC). METHODS: The target molecules of oridonin were retrieved from SEA, STITCH, SuperPred and TargetPred databases; target genes associated with the treatment of NSCLC were retrieved from GeneCards, DisGeNET and TTD databases. Then, the overlapping target molecules between the drug and the disease were identified. The protein-protein interaction (PPI) was constructed using the STRING database according to overlapping targets, and Cytoscape was used to screen for key targets. Molecular docking verification were performed using AutoDockTools and PyMOL software. Using the DAVID database, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted. The impact of oridonin on the proliferation and apoptosis of NSCLC cells was assessed using cell counting kit-8, cell proliferation EdU image kit, and Annexin V-FITC/PI apoptosis kit respectively. Moreover, real-time quantitative PCR and Western blot were used to verify the potential mechanisms. RESULTS: Fifty-six target molecules and 12 key target molecules of oridonin involved in NSCLC treatment were identified, including tumor protein 53 (TP53), Caspase-3, signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase kinase 8 (MAPK8), and mammalian target of rapamycin (mTOR). Molecular docking showed that oridonin and its key target molecules bind spontaneously. GO and KEGG enrichment analyses revealed cancer, apoptosis, phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), and other signaling pathways. In vitro experiments showed that oridonin inhibited the proliferation, induced apoptosis, downregulated the expression of Bcl-2 and Akt, and upregulated the expression of Caspase-3. CONCLUSION Oridonin can act on multiple targets and pathways to exert its inhibitory effects on NSCLC, and its mechanism may be related to upregulating the expression of Caspase-3 and downregulating the expressions of Akt and Bcl-2.
Diterpenes, Kaurane/chemistry* ; Carcinoma, Non-Small-Cell Lung/pathology* ; Humans ; Network Pharmacology ; Lung Neoplasms/pathology* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Cell Line, Tumor ; Signal Transduction/drug effects* ; Gene Expression Regulation, Neoplastic/drug effects* ; Reproducibility of Results ; Gene Ontology

OBJECTIVES: To evaluate the inhibitory effect of oridonin against proliferation of pancreatic cancer cells and the mechanism underlying the synergistic effect of low-intensity pulsed ultrasound (LIPUS). METHODS: PANC-1 cells treated with different concentrations of oridonin were examined for changes in cell proliferation using CCK-8 assay and in MDA, GSH and ATP levels using flow cytometry. The protein expressions of GPX4, Nrf2 and HO-1 in the treated cells were detected with Western blotting. The effect of Fer-1, a ferroptosis inhibitor, on proliferation of oridonin-treated cells were assessed, and the effects of oridonin combined with LIPUS on PIEZO1 protein expression was evalauted using Western blotting. A C57BL/6J mouse model bearing pancreatic cancer cell xenograft was established and treated with oridonin, LIPUS, or both, and the histological changes in the tumor tissues and tumor cell proliferation were examined with HE staining and immunohistochemistry for Ki67; the changes in GPX4 expression in the tumor tissues were detected using Western blotting and immunofluorescence staining. RESULTS: In PANC-1 cells, oridonin treatment significantly inhibited cell proliferation, increased intracellular Fe²⁺, ROS, and MDA levels, and decreased GSH and ATP levels. Oridonin also resulted in lowered GPX4 and increased HO-1 and Nrf2 protein expression levels in the cells. The combined treatment with LIPUS signficiantly enhanced the inhibitory effect of oridonin on PANC-1 cell viability in vitro and on xenograft growth in the mouse models, resulting also in more obvious reduction of the intensity of Ki67 staining and GPX4 protein expression and more pronounced increase of PIEZO1 protein expression in the tumor tissues in the mouse models. CONCLUSIONS LIPUS enhances the effect of oridonin to promote ferroptosis of pancreatic cancer cells by activating PIEZO1 through the Nrf2/HO-1/GPX4 pathway.
Ferroptosis/drug effects* ; Animals ; Pancreatic Neoplasms/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Humans ; Cell Line, Tumor ; Mice ; Heme Oxygenase-1/metabolism* ; Diterpenes, Kaurane/pharmacology* ; Cell Proliferation/drug effects* ; Mice, Inbred C57BL ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Ion Channels/metabolism* ; Ultrasonic Waves ; Signal Transduction

OBJECTIVE: To investigate the influence of oridonin on the killing activity of NK-92 MI cells targeting THP1 and the related mechanism. METHODS: The killing activity of NK-92 MI to THP1 before and after oridonin treatment was detected by LDH release assay; the expression of natural killer cell ligands activating receptor D (NKG2D, including MICA, MICB, ULBP1, ULBP2 and ULBP3) was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot respectively; the expression of cytokine TNF-α, TNF-β and IFN-γ in the co-culture supernatant of NK-92 MI cells and THP1 cells were measured by ELISA. RESULTS: The killing efficiency after oridonin treatment at different effector-target ratio (1:1, 5:1, 10:1) was all significantly up-regulated in comparison with that before oridonin treatment (P＜0.05). QRT-PCR and Western blot showed that the expressions of mRNA and protein levels of MICB, ULBP1, ULBP2 increased to varying degree (P＜0.05), but the expression levels of MICA and ULBP3 were not statistically significant between experimental group and control group (P＞0.05). ELISA results indicated that IFN-γ and TNF-β release were significantly increased after oridonin treatment (P＜0.05), however, the TNF-α release was not statistically different in comparison with control group (P＞0.05). CONCLUSION Oridonin can significantly improve killing efficiency of NK-92 MI on THP1, that might be related with up-regulation of MICB, ULBP1 and ULBP2 expression and promotion of IFN-γ and TNF-β release.
Cell Line, Tumor ; Diterpenes, Kaurane ; pharmacology ; GPI-Linked Proteins ; Histocompatibility Antigens Class I ; Humans

Nine new ent-kaurane diterpenoids, named scopariusols L-T (1-9), were isolated from the aerial parts of Isodon scoparius. Their structures were characterized mainly by analyzing the NMR and HR-ESI-MS data, and the absolute configuration of 1 was determined by single-crystal X-ray diffraction. Compound 1 was active against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480), and it also inhibited NO production in LPS-stimulated RAW264.7 cells, with an IC value of 0.6 μmol·L.
Animals ; Antineoplastic Agents, Phytogenic ; chemistry ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Crystallography, X-Ray ; Diterpenes, Kaurane ; chemistry ; isolation & purification ; pharmacology ; Drug Screening Assays, Antitumor ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; HL-60 Cells ; Humans ; Isodon ; chemistry ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; Mice ; Molecular Structure ; Nitric Oxide ; biosynthesis ; Nuclear Magnetic Resonance, Biomolecular ; Plant Components, Aerial ; chemistry ; RAW 264.7 Cells

OBJECTIVETo investigate the effect of oridonin on the human acute lymphocytic leukemia cell line Jurkat and its mechanism.

METHODSJurkat cells were cultured in vitro and treated with various concentrations (0, 1.25, 2.5, 5, and 10 μmol/L) of oridonin for different lengths of time (24, 48, and 72 hours). The proliferation of Jurkat cells was analyzed by MTT assay. The changes in nuclear morphology were evaluated by fluorescence microscopy at 12 hours after treatment with various concentrations of oridonin. The expression levels of Brg1, P53, and C-myc were determined by semi-quantitative Western blot in Jurkat cells treated with various concentrations of oridonin for 24 hours or 5 μmol/L oridonin for various lengths of time (0, 2, 6, 12, and 24 hours). The expression levels of P53 and C-myc and proliferation of Jurkat cells were evaluated after Brg1 expression was knocked down by Brg1-specific siRNA.

RESULTSCompared with the control group, the proliferation of oridonin-treated Jurkat cells was significantly inhibited in a concentration- and time-dependent manner (P<0.05). According to the florescence microscopic analysis, oridonin treatment led to nuclear pyknosis in Jurkat cells. Compared with the control group, Jurkat cells treated with 5 μmol/L oridonin had reduced expression of Brg1 and C-myc but elevated expression of P53. Brg1 knock-down led to a significant reduction in proliferation of Jurkat cells (P<0.05), up-regulated expression of P53, and down-regulated expression of C-myc.

CONCLUSIONSOridonin can inhibit the proliferation of Jurkat cells, probably via the Brg1 signaling pathway.

Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Proliferation ; drug effects ; DNA Helicases ; analysis ; physiology ; Diterpenes, Kaurane ; pharmacology ; Dose-Response Relationship, Drug ; Down-Regulation ; Humans ; Jurkat Cells ; Nuclear Proteins ; analysis ; physiology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; Proto-Oncogene Proteins c-myc ; analysis ; Signal Transduction ; physiology ; Transcription Factors ; analysis ; physiology ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVEThe aim of this study was to explore the molecular mechanism of apoptosis in esophageal cancer cells induced by Isodon rubescens.

METHODSThe DNA-damage effect of Jaridonin was detected by single cell gel electrophoresis (SCGE). The p53 protein was determined by Western blot. GSH assay kit was employed to determine the GSH content in human esophageal cancer EC-1 cells. Intracellular levels of hydrogen peroxide (H2O2) or superoxide (O(2).-) were determined using the redox-sensitive probes 2', 7'-dichlorodihydrofluorescein diacetate (DCF) or dihydroethidium (DHE), and the fluorescence signal was assayed by fluorescence microscopy and by flow cytometry.

RESULTSJaridonin induced DNA damage in EC-1 cells remarkably. The olive tail moments (OTM) of control and 20, 40 µmol/L Jaridonin were 3.2, 45.2 and 89.0, respectively. Compared with the control, the differences were significant (P < 0.01 for both). Jaridonin resulted in extensive p53 up-regulation in the EC-1 cells. More importantly, the p53 up-regulation occurred as early as 2 h after Jaridonin incubation, and in a time-dependent manner (P < 0.05). p53 siRNA transfection inhibited apoptosis in the EC-1 cells, and the Jaridonin-induced apoptosis rate was reduced from 38.5% to 8.8%. Intracellular level of H2O2 was increased by Jaridonin, whereas the level of O(2).- was barely changed. The GSH content in EC-1 cells was reduced from (10.3 ± 1.6) nmol/mg protein to (4.6 ± 2.1) nmol/mg protein after 20 µmol/L Jaridonin incubation for 8 h, and it was further reduced with the increase of Jaridonin concentration. Jaridonin induced DNA damage, H2O2 accumulation and apoptosis were significantly attenuated in the presence of GSH, but Jaridonin showed little effect on normal human liver L-02 cells.

CONCLUSIONSJaridonin selectively induces apoptosis in esophageal cancer EC-1 cells through H2O2-mediated DNA damage by depleting GSH.

Antineoplastic Agents ; pharmacology ; Apoptosis ; DNA Damage ; Diterpenes, Kaurane ; pharmacology ; Esophageal Neoplasms ; metabolism ; Humans ; Hydrogen Peroxide ; metabolism ; Up-Regulation

The present study was designed to determine the chemical constituents of EtOAc extracts of the aerial parts of Isodon wikstroemioides. Compounds 1-8 were isolated and purified by normal-phase silica gel and reversed-phase C18silica gel column chromatography and HPLC. Their structures were elucidated by extensive spectroscopic methods. Most of them were evaluated for their in vitro cytotoxicity against human cancer HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cells and their inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. Among the eight 11, 20-epoxy-ent-kauranoids isolated, compounds 1-6 (isowikstroemins H-M) were new diterpenoids. Compounds 1, 3, and 7 exhibited significant cytotoxicity with IC50 values ranging from (0.84 ± 0.02) to (4.09 ± 0.34) μmol · L(-1), while compounds 4 and 5 showed selective cytotoxicity. In addition, compounds 1, 3, 4, and 7 exhibited inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. These results provide a basis for future development of these compounds as anti-cancer and anti-inflammatory agents.
Anti-Inflammatory Agents ; isolation & purification ; Antineoplastic Agents, Phytogenic ; isolation & purification ; Cell Line, Tumor ; Diterpenes, Kaurane ; isolation & purification ; Humans ; Inhibitory Concentration 50 ; Isodon ; chemistry ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Neoplasms ; drug therapy ; Nitric Oxide ; antagonists & inhibitors ; biosynthesis ; Phytotherapy ; Plant Components, Aerial ; Plant Extracts ; isolation & purification

This study was purposed to investigate the antitumor effect of oridonin on human multiple myeloma cell line U266 and its possible mechanism. The CCK-8 test was used to determine the inhibitory effect of oridonin on proliferation of U266 cells. The morphological changes of U266 cells were observed under optical microscope. The apoptosis rate of U266 cells was detected by flow cytometry. The mRNA levels of FGFR3, BCL2, CCND1 and MYC genes were quantified by using real-time quantitative PCR method, and the protein levels of BCL2, MYC, CCND1, FGFR3 and P53 were detected by Western blot. The results showed that the oridonin obviously inhibited the growth of U266 cell in dose-and time-dependent manners. As for morphological changes, characteristic apoptotic cells presented in U266 cells treated with 10 µmol/L oridonin for 24 hours. The apoptotic rate of U266 cells increased in dose and time dependent manners; after treatment of U266 cells with oridonin the mRNA levels of FGFR3, BCL2, CCND1 and MYC as well as the their protein levels decreased. Occasionally, the oridonin up-regulated the protein levels of P53 in the same manner. It is concluded that the oridonin can exert its anti-tumor effect by inhibiting proliferation and inducing apoptosis of U266 cell in dose dependent and time dependent manners, that maybe give the clues about new program of target therapy for multiple myeloma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Diterpenes, Kaurane ; pharmacology ; Humans ; Multiple Myeloma ; pathology

Oridonin, a diterpenoid isolated from Rabdosia rubescens, has been proven to possess various pharmacological and physiological effects such as anti-inflammation, anti-bacterial, and anti-neoplastic, although in recent years, more attention has been paid to its anti-neoplastic effects. For example, oridonin can trigger cell cycle arrest, apoptosis, and autophagy in different neoplastic cell lines. This review summarizes the considerable knowledge about the action mechanisms of oridonin that has been studied in recent years. The present observations reveal the novel anti-neoplastic effects of oridonin, suggesting that it may be effective as a potent alternative or adjunct drug to conventional chemotherapy.
Animals ; Antineoplastic Agents ; adverse effects ; chemistry ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Diterpenes, Kaurane ; adverse effects ; chemistry ; pharmacology ; therapeutic use ; Humans ; Neoplasms ; drug therapy ; pathology ; Signal Transduction ; drug effects