Induction of apoptosis and G2/M cell cycle arrest by oridonin in human gastric cancer BGC-823 cells

Aim To investigate in vitro apoptosis-induction effects of oridonin on gastric tumor cells BGC-823 and its effects on cell cycle, mitochondrial membrane potential and intracellular Ca^2＋ to shed light on the mode of its anticancer action. Methods The MTT method was used to investigate the inhibitory effect of oridonin on BGC-823 cells. The apoptosis-induction effect was evaluated by confocal laser microscopy and flow cytometry. The change of mitochondrial membrane potential and the increase of intracellular Ca^2＋ were assessed by fluorescence probe rhodamine123 and Fluo 3-AM, respectively, with flow cytometry. The expression of apoptosis and cell cycle related proteins was studied using western blotting. Results Oridonin inhibited BGC-823 cells growth with IC50 of 22.21 p, mol.L^-1. It induced apoptosis in a dose-dependent manner. In addition, it decreased mitochondria membrane potential, increased intracellular Ca^2＋, and activated pro-caspase 3. BGC-823 cells were arrested in G2/M cell cycle phase with lower expression of cyclin A protein. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. Conclusion Oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca^2＋, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A.

Inhibitory Effect of Oridonin on the Proliferation of NB4 Cells and Its Mechanism

Objective: To investigate the anti-proliferation e?ect of oridonin on leukemic NB4 cells and its mechanism. Methods: NB4 cells in culture medium in vitro were given di?erent concentrations of oridonin. The inhibitory rate of the cells were measured by MTT assay, cell apoptotic rate was detected by ?ow cytometry(FCM), morphology of cell apoptosis was observed by hoechst 33258 ?uorescence staining , and the activity of telomerase was detected using TRAP-PCR-ELISA before and after apoptosis occurred. Results: Oridonin (over 8 μmol/L) could decrease the telomerase activity, inhibit the growth of NB4 cells and induce apoptosis signi?cantly in a time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed by hoechst 33258 ?uorescence staining especially after the cells treated by oridonin for 48–60 h. Conclusion: Oridonin could inhibit the proliferation and induce the apoptosis of NB4 cells in vitro. One of the mechanisms may be the decrease of the telomerase activity of NB4 cells.

oridonin部分通过TNFα信号转导途径诱导小鼠成纤维L929细胞凋亡

目的比较冬凌草甲素(oridonin)和TNFα诱导小鼠成纤维L929细胞凋亡的分子机制。方法MTT法、Hoechst33258荧光染色、DNA片断化分析和Western blot分析法。结果Oridonin和TNFα均能诱导L929细胞发生凋亡,其半数有效抑制浓度IC50分别为(24·8±1·2)μmol·L-1和(20·9±1·9)μg·L-1。Oridonin和TNFα均能引起L929细胞凋亡形态学变化,TNFα处理过的细胞在琼脂糖凝胶电泳上可见典型的凋亡DNA梯状条带,但是oridonin处理的细胞却不能,称之为非典型凋亡;caspase-3,-8抑制剂和caspase家族抑制剂能明显得促进25μmol·L-1oridonin和20μg·L-1TNFα诱导的L929细胞凋亡,caspase-9抑制剂只能促进oridonin诱导的L929细胞凋亡;ERK的抑制剂PD98059能明显的抑制oridonin和TNFα诱导的L929细胞凋亡,但是p38的抑制剂SB203580只能抑制TNFα诱导的L929细胞凋亡;在L929细胞中oridonin能促进内源性pro-TNFα的表达和其上游蛋白IκB的磷酸化。结论Oridonin通过激活转录因子NF-κB而促进内源性pro-TNFα的表达,并且部分通过细胞因子TNFα信号转到途径诱导L929细胞凋亡。

Effect of oridonin-mediated hallmark changes on inflammatory pathways in human pancreatic cancer(BxPC-3) cells

AIM: To investigate the effect of oridonin on nuclear transcription factors and to study the relationship between biological behavior and inflammatory factors in human pancreatic cancer (BxPC-3) cells.

Therapeutic effect of oridonin on mice with prostate cancer

Objective: To investigate the therapeutic effect and the related mechanism of oridonin on mice with prostate cancer. Methods: Sixty BALB/C male nude mice were selected. A model of RM-1 cell transplantation tumor of prostate cancer was built by the subcutaneous inoculation of RM-1 cells. After that, those 60 experimental mice were randomly divided into groups A, B and C. Each group had 20 mice. Mice in group A were treated with 0.2 m L of normal saline(0.9%) by intraperitoneal injection once a day; mice in group B received intraperitoneal injection of 1.875 mg/m L of oridonin once a day; and mice in group C received intraperitoneal injection of 7.5 mg/m L of oridonin once a day. Mice in the three groups were treated uninterruptedly for 5 weeks and were all killed. Then, tumors were excised and weighed to calculate their growth inhibitory rate, volume increment and anti-tumor rate. Thymus and spleen of mice in the three groups were collected to calculate the thymus and spleen index. Immunohistochemical staining was applied to observe the expression of caspase-3 in prostate cancer tissue of mice of the three groups. Results: The qualities and volume increment of tumors in groups B and C were significantly lower than those of group A(P < 0.05); the qualities and volume increment of tumors in groups C were evidently lower than those of group B(P < 0.05); the tumor volume increment and anti-tumor rate in group C were obviously higher than those of group B(P < 0.05); the thymus and spleen indexes of groups B and C were distinctly higher than those of group A(P < 0.05); comparison of the thymus and spleen indexes between group B and group C showed no statistical differences(P > 0.05). Immumohistochemical staining revealed that the caspase-3 protein in prostate cancer tissue of mice of group A expressed negatively with colourless or light-colored karyon; while the caspase-3 protein in prostate cancer tissue of mice of group B expressed positively with dark-colored karyon, centralized distribution and granular sensation; and the caspase-3 in prostate cancer tissue of mice of group C showed strong positive expression with big and darker colored karyon and dense distribution. Conclusions: Oridonin can inhibit the growth of RM-1 prostate cancer cells effectively and have great therapeutic effects on RM-1 cell transplantation tumor of prostate cancer.

ANTI-PROLIFERATION EFFECT OF ORIDONIN ONHL-60 CELLS AND ITS MECHANISM

ObjectiveTo investigate the anti-proliferation effect of oridonin on leukemic HL-60 cells and its mechanism. Methods HL-60 cells invitroin culture medium were given different concentrations of oridonin. The inhibitory rate of cells were measured by microculture tetrazolium (MTT) assay, cell apoptotic rate was detected by flow cytometry (FCM),morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was det-ected using telomere repeat amplification protocol (TRAP) PCR-ELISA before and after apoptosis occurred. Results Oridonin could decrease telomerase activity, inhibit growth of HL-60 cells, and cause apoptosis significantly. The suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by hoechst 33258 fluorescence staining especi-ally after cells were treated 48-60 hours by oridonin. Conclusions Oridonin has apparent anti-proliferation and apoptotic effects on HL-60 cells invitro, decreasing telomerase activity of HL-60 cells may be one of its most important mechanisms. These results will provide strong laboratory evidence of oridonin for clinical treatment of acute leukemia.

Static and dynamic studies of adsorption by four macroporous resins to enrich oridonin from Rabdosia rubescens

Oridonin,one of the active ingredients in Rabdosia rubescens(R.rubescens),has been reported to induce cell apoptosis and cell cycle arrest in many cancers.Conventional extraction methods tend to result in unsatisfied enrichment and poor quality of oridonin present in a given biomass.This paper aims to evaluate the performance and separation characteristics of four different macroporous resins to arrive at the most suitable methodology for the isolation and purification of highquality oridonin.Static absorption kinetics,thermodynamic and dynamic adsorption were evaluated.HP20 was selected for further study due to its high adsorption capacity of 32 mgg 1 and desorption ratio with 98.5%.The pseudosecondorder model was considered to be the most suitable for kinetic results,and Langmuir model was chosen to better describe the absorption thermodynamics.Under optimum conditions(flow rate of 4 ml min 1,bed depth with 6 cm and initial concentration of 2.15 mg·ml^1),the effective content of oridonin increased from 33.9%to 79.1%in the dry extract with a recovery of 81%and the purity of oridonin improved from 76%to 93%.The results confirm that HP20 provides an efficient method to purify most oridonin from R.rubescens.

The Inhibitory Effect of Oridonin on the Growth of Fifteen Human Cancer Cell Lines

OBJECTIVE To study the inhibitory effect of oridonin on the growth of cancer cells. METHODS Fifteen human cancer cell lines were subjected to various concentrations of oridonin in culture medium. The inhibitory rate of cell growth was measured by the MTT assay, and compared with a negative control and 5-Fu-positive control. RESULTS The 50% inhibiting concentration （IC50） and maximal inhibition （Imax） of oridonin shown by studying the growth of the cancer cell lines were as follows： leukemias （HL60 cells： 3.9 μg/ml and 73.8%, K562 cells： 4.3 μg/ml and 76.2%）; esophageal cancers（SHEEC cells： 15.4 μg/ml and 99.2%, Eca109 cells： 15.1 μg/ml and 84.6%, TEl cells： 4.0 μg/ml and 70.2%）; gastric cancers （BGC823 cells： 7.6 μg/ml and 98.7%, SGC7901 cells： 12.3 μg/ml and 85.7%）; colon cancers （HT29 cells： 13.6 μg/ml and 97.2%, HCT cells： 14.5 μg/ml and 96.5%）; liver cancers （Bel7402 cells： 15.2 μg/ml and 89.2%, HepG2 cells： 7.1 μg/ml and 88.3%）; pancreatic cancer （PC3 cells： 11.3 μg/ml and 68.4%）; lung cancer （A549 cells： 18.6 μg/ml and 98.0% ）; breast cancer （MCF7 cells： 18.4 μg/ml and 84.7%）; uterine cervix cancer （Hela cells： 13.7 μg/ml and 98.5%）. CONCLUSION Oridonin had a relatively wide anti-tumor spectrum, and a relatively strong inhibitory effect on the growth of the 15 human cancer cells. Inhibitory effects were concentration dependent.

Oridonin induces apoptosis in gastric cancer through Apaf-1,cytochrome c and caspase-3 signaling pathway

AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

Oridonin restores hepatic lipid homeostasis in an LXRa-ATGL/EPT1 axis-dependent manner

Hepatosteatosis is characterized by abnormal accumulation of triglycerides(TG),leading to prolonged and chronic inflammatory infiltration.To date,there is still a lack of effective and economical therapies for hepatosteatosis.Oridonin(ORI)is a major bioactive component extracted from the traditional Chinese medicinal herb Rabdosia rubescens.In this paper,we showed that ORI exerted significant protective effects against hepatic steatosis,inflammation and fibrosis,which was dependent on LXRa signaling.It is reported that LXRa regulated lipid homeostasis between triglyceride(TG)and phosphatidylethanolamine(PE)by promoting ATGL and EPT1 expression.Therefore,we implemented the lipidomic strategy and luciferase reporter assay to verify that ORI contributed to the homeostasis of lipids via the regulation of the ATGL gene associated with TG hydrolysis and the EPT1 gene related to PE synthesis in a LXRadependent manner,and the results showed the TG reduction and PE elevation.In detail,hepatic TG overload and lipotoxicity were reversed after ORI treatment by modulating the ATGL and EPT1 genes,respectively.Taken together,the data provide mechanistic insights to explain the bioactivity of ORI in attenuating TG accumulation and cytotoxicity and introduce exciting opportunities for developing novel natural activators of the LXRa-ATGL/EPT1 axis for pharmacologically treating hepatosteatosis and metabolic disorders.

Determination of Three-Dimensional Structures of Oridonin in Solution by NOESY Spectrum and Molecular Mechanics Calculation

The phase sensitive NOESY spectrum of oridonin was treated using Full Relaxation Matrix Analysis(FRMA) approach, and the cross relaxation rates of proton pairs were obtained by diagonalizing the NOE matrix of oridonin. The inter proton distances were calculated according to 1/r6 ij ∝σij. The three-dimensional structure of oridonin in solution was calculated by the combination of WUPH, WUPH-S method with molecular mechanics minimization on the basis of NMR experiment.

Altered gene expression reveals molecular mechanisms underlying oridonin-induced apoptosis of multiple myeloma LP-1 cells

Objective： To investigate the effect of oridonin on proliferation and invasion of human multiple myeloma LP-1 ceils and the underlying mechanism. Methods： LP-1 cells in culture medium in vitro were treated with oridonin at the different concentration Cell proliferation was measured by Microwave Theory and Techniques （MTT） assay and cell apoptotic rate was detected by flow cytometry. Morphology of cell apoptosis was observed by transmission electron microscope. Expressions of Bax, Bcl-2, Caspase-3, NFqcB as well as I-~B mRNA were detected by real-time PCR. Results： The MTT assays and flow cytometry revealed that oridonin could inhibit the growth of LP-1 cells and cause apoptosis significantly; the suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis were found under a transmission electron microscope after the cells were treated with oridonin at 25 ~rnol/L for 24 h. Along with the apoptotic process, Bcl-2, Caspase-3,NF-r,.B gene expressions were down-regulated （P〈0.05）. On the contrast, the Bax and I-~zB gene expressions were up-regulated （P〈0.05）. Conclusion： Oridonin could inhibit the proliferation of LP-1 cells via inducing apoptosis. We concluded that oridonin induces apoptosis in LP-1 cells via activation of caspase-3 as well as down-regulation of Bcl-2 and up-regulation of Bax expression. The results suggested that oridonin could induce apoptosis of LP-1 cells through mitochondria- and caspase3-dependent pathways. Meanwhile, the inhibition of NF-r,_B and the activation of I-~B indicate pro-apoptotic stimuli. In one word, oridonin might be an important potential anti-myeloma reagent.

Combination of oridonin and TRAIL induces apoptosis in uveal melanoma cells by upregulating DR5

AIM:To investigate the inhibitory effect of the combined use of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and oridonin on choroidal melanoma cell lines,and to explore its underlying mechanism.METHODS:MUM-2B and C918 cells were treated with different concentrations of TRAIL and oridonin,and MTT assay used to evaluate the inhibition rate of the two compounds on cells.Then,the cell cycle distribution and apoptosis were detected by flow cytometry,and changes in apoptosis-related proteins such as death receptor 5(DR5),a-caspase-3,and x-linked inhibitor of apoptosis protein(XIAP)were detected by Western blot.MUM-2B cells were transfected with si-DR5,which interfered with the expression of the DR5 gene.MTT and Western blot assay were used to detect cell activity and apoptosis-related proteins.RESULTS:When TRAIL and oridonin were simultaneously administered to the MUM-2B cells,the apoptosis rate was significantly higher than that by the two drugs individually.However,the effect of combined use of TRAIL and oridonin on C918 cells was not significantly different from that used alone.Cell cycle analysis showed that TRAIL and oridonin could induce G2/M arrest in MUM-2B cells.The Western blot results showed that the protein expression levels of the DR5,a-caspase-3,and BAX increased,while the expression levels of the anti-apoptosis-related proteins XIAP and BCL-2 were suppressed when TRAIL and oridonin simultaneously administered to MUM-2B cells.Interfering the expression of DR5 gene in MUM-2B cells could reverse the inhibitory effect of oridonin and TRAIL on the proliferation and apoptosis induction of MUM-2B cells.CONCLUSION:The inhibitory effects of oridonin and TRAIL on MUM-2B cells are significantly enhanced when they were administered as a combined treatment,which may ascribe to up-regulation of DR5.

Oridonin对急性肝衰竭小鼠肝细胞凋亡的影响及其机制研究

目的探讨冬凌草甲素(oridonin)对脂多糖/D-氨基半乳糖氨(LPS/D-Gal)联合诱导的急性肝衰竭(ALF)小鼠肝细胞凋亡的影响,并探讨其可能的机制。方法取25只小鼠,随机分成5组,每组5只。采用LPS/D-Gal腹腔注射建立小鼠ALF模型,设生理盐水对照组、LPS/D-Gal诱导模型组、LPS/D-Gal诱导和不同剂量oridonin干预组及oridonin处理组。采用末端转移酶介导的缺口末端标记法(TUNEL)检测肝细胞凋亡,采用real-time PCR法检测肝组织TNF-αm RNA水平,采用Western blot法检测凋亡相关蛋白的变化。结果模型组小鼠肝细胞凋亡率为(36.4±1.8)%,显著高于两个oridonin干预组的[(19.4±3.3)%和(11.4±0.3)%,P<0.01];模型组小鼠肝组织TNF-αm RNA水平显著高于正常对照组(P<0.01),而两个oridonin干预组肝组织TNF-αm RNA水平显著低于模型组(P<0.01);模型组小鼠线粒体凋亡通路相关的促凋亡蛋白c-Jun氨基末端激酶(JNK)、bax、细胞色素C、cleaved caspase9/3活化水平显著高于两个oridonin干预组(P<0.01),模型组小鼠抗凋亡蛋白bcl-xl水平显著低于两个oridonin干预组(P<0.01),模型组小鼠死亡受体凋亡通路相关的促凋亡蛋白caspase8活化水平与两个oridonin干预组并无明显差异(P>0.01)。结论 Oridonin可抑制LPS/D-Gal诱导的ALF小鼠肝细胞凋亡,其机制可能与下调促凋亡细胞因子TNF-α水平和抑制JNK介导的线粒体凋亡信号通路有关。

An iron-based metal-organic framework nanoplatform for enhanced ferroptosis and oridonin delivery as a comprehensive antitumor strategy

Ferroptosis is a recently discovered pathway for regulated cell death pathway.However,its efficacy is affected by limited iron content and intracellular ion homeostasis.Here,we designed a metalorganic framework(MOF)-based nanoplatform that incorporates calcium peroxide(CaO_(2))and oridonin(ORI).This platform can improve the tumor microenvironment and disrupt intracellular iron homeostasis,thereby enhancing ferroptosis therapy.Fused cell membranes(FM)were used to modify nanoparticles(ORI@CaO_(2)@Fe-TCPP,NPs)to produce FM@ORI@CaO_(2)@Fe-TCPP(FM@NPs).The encapsulated ORI inhibited the HSPB1/PCBP1/IREB2 and FSP1/COQ10 pathways simultaneously,working in tandem with Fe^(3+) to induce ferroptosis.Photodynamic therapy(PDT)guided by porphyrin(TCPP)significantly enhanced ferroptosis through excessive accumulation of reactive oxygen species(ROS).This selfamplifying strategy promoted robust ferroptosis,which could work synergistically with FM-mediated immunotherapy.In vivo experiments showed that FM@NPs inhibited 91.57%of melanoma cells within six days,a rate 5.6 times higher than chemotherapy alone.FM@NPs were biodegraded and directly eliminated in the urine or faeces without substantial toxicity.Thus,this study demonstrated that combining immunotherapy with efficient ferroptosis induction through nanotechnology is a feasible and promising strategy for melanoma treatment.

Recent Advances in the Molecular Basis of Anti-Neoplastic Mechanisms of Oridonin

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.

Effects and mechanism of oridonin on pulmonary hypertension induced by chronic hypoxia-hypercapnia in rats

Background Pulmonary arterial hypertension （PAH） is characterized by suppressing apoptosis and enhancing cell proliferation in the vascular wall. Inducing pulmonary artery smooth muscle cells （PASMC） apoptosis had been regarded as a therapeutic approach for PAH. Oridonin can cause apoptosis in many cell lines, while little has been done to evaluate its effect on PASMC. Methods Thirty male Sprague-Dawley rats were randomly assigned to three groups： normal control （NC）; hypoxia-hypercapnia （HH）; Hypoxia-hypercapnia ＋ oridonin （HHO）. Rats were exposed to hypoxia-hypercapnia for four weeks. Cultured human PASMC （HPASMC） were assigned to three groups： normoxia （NO）; hypoxia （HY）; hypoxia ＋ oridonin （HO）. The mean pulmonary artery pressure, mass ratio of right ventricle over left ventricle plus septum （RV/（LV＋S））, the ratio of thickness of the pulmonary arteriole wall to vascular external diameter （WT%） and the ratio of the vessel wall area to the total area （WA%） were measured. Morphologic changes of pulmonary arteries were observed under light and electron microscopes. The apoptotic characteristics in vitro and in vivo were detected. Results The mPAP, RW（LV＋S）, WT%, and WA% in the HH group were significantly greater than those in the NC （P 〈0.01） and HHO groups （P 〈0.01）; the activities of caspase-3 and caspase-9, and the expressions of Bax, cyt-C and apoptotic index （AI） in the group HH were less than those in the NC and HHO groups; and the expression of Bcl-2 in group HH was greater than that in the NC and HHO groups. HPASMC mitochondrial membrane potentials in group HO was lower than in group HY （P 〈0.01）, and cyt-C in the cytoplasm, AI, and caspase-9 in the HO group were greater than that in the HY group （P 〈0.01）, but the expression of Bcl-2 in the HO group was less than that in the HY group （P 〈0.05）. Conclusions The results suggest that oridonin can lower pulmonary artery pressure effectively, and inhibit pulmonary artery structural remodeling by inducing smooth cell apoptosis via a mitochondria-dependent pathway.

Inhalable oridonin-loaded poly(lactic-co-glycolic)acid large porous microparticles for in situ treatment of primary non-small cell lung cancer

Non-small cell lung cancer(NSCLC) accounts for about 85% of all lung cancers. Traditional chemotherapy for this disease leads to serious side effects. Here we prepared an inhalable oridonin-loaded poly(lactic-co-glycolic)acid(PLGA) large porous microparticle(LPMP) for in situ treatment of NSCLC with the emulsion/solvent evaporation/freeze-drying method. The LPMPs were smooth spheres with many internal pores. Despite a geometric diameter of 10 mm, the aerodynamic diameter of the spheres was only 2.72 mm, leading to highly efficient lung deposition. In vitro studies showed that most of oridonin was released after 1 h, whereas the alveolar macrophage uptake of LPMPs occurred after 8 h, so that most of oridonin would enter the surroundings without undergoing phagocytosis. Rat primary NSCLC models were built and administered with saline, oridonin powder, gemcitabine, and oridonin-loaded LPMPs via airway, respectively. The LPMPs showed strong anticancer effects. Oridonin showed strong angiogenesis inhibition and apoptosis. Relevant mechanisms are thought to include oridonin-induced mitochondrial dysfunction accompanied by low mitochondrial membrane potentials, downregulation of BCL-2 expressions, upregulation of expressions of BAX, caspase-3 and caspase-9. The oridonin-loaded PLGALPMPs showed high anti-NSCLC effects after pulmonary delivery. In conclusion, LPMPs are promising dry powder inhalations for in situ treatment of lung cancer.

Homoharringtonine synergy with oridonin in treatment of t(8;21) acute myeloid leukemia

Collaboration of c-KIT mutations with AML1-ETO (AE) has been demonstrated to induce t(8;21) acute myeloid leukemia (AML).Targeted therapies designed to eliminate AE and c-KIT oncoproteins may facilitate effective treatment of t(8;21) AML.Homoharringtonine (HHT) features activity against tumor cells harboring c-KIT mutations,whereas oridonin can induce t(8;21) AML cell apoptosis and AE cleavage.Therefore,studies should explore the efficacy of combination therapy with oridonin and HHT in t(8;21) AML.In this study,we investigated the synergistic effects and mechanism of oridonin combined with HHT in t(8;21) AML cell line and mouse model.The two drugs synergistically inhibited cell viability and induced significant mitochondrial membrane potential loss and apoptosis.Oridonin and HHT induced significant downregulation of c-KIT and its downstream signaling pathways and promoted AE cleavage.HHT increased intracellular oridonin concentration by modulating the expressions of MRP1 and MDR1,thus enhancing the effects of oridonin.The combination of oridonin and HHT prolonged t(8;21) leukemia mouse survival.In conclusion,oridonin and HHT exert synergistic effects against t(8;21) leukemia in vivo and in vitro,thereby indicating that their combination may be an effective therapy for t(8;21) leukemia.