ITRAQ-based proteomics analysis of human ectopic endometrial stromal cells treated by Maqian essential oil.

BACKGROUND: Endometriosis is a common and complex syndrome characterized by the presence of endometrial-like tissue outside the uterus. Chinese medicine has been recently found to show good efficacy in treating endometriosis. Our previous results revealed that Maqian fruit essential oil (MQEO) could inhibit the proliferation and induce apoptosis of ectopic endometrial stromal cells (EESCs), but the mechanisms remain unclear. In this study, we aim to explore the molecular mechanism of MQEO's specific effects in EESCs. METHODS: We conducted a quantitative proteomics analysis by iTRAQ on EESCs treated with MQEO or DMSO. Then deep analysis was performed based on differentially expressed proteins, including Gene Ontology enrichment analysis, pathway enrichment analysis and protein interaction analysis. Candidate protein targets were subsequently verified by western blotting. RESULTS: Among 6575 identified proteins, 435 proteins exhibited altered expression levels in MQEO-treated EESCs. Of these proteins, most were distributed in signal transduction as well as immune system and the most significantly altered pathway was complement and coagulation cascades. Moreover, two differentially expressed proteins (Heme oxygenase 1 and Acyl-CoA 6-desaturase) were verified and they can be potential biomarkers for endometriosis treatment. CONCLUSIONS: Our proteomic analysis revealed distinct protein expression patterns induced by MQEO treatment in EESCs, highlighting the potential of MQEO for endometriosis treatment and biomarker discovery.

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear. AIM OF THE STUDY: N6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC. MATERIAL AND METHODS: The alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed. RESULTS: Upon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin, two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL. CONCLUSIONS: These results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis.

Predictive value of left ventricular dyssynchrony for short-term outcomes in three-vessel disease patients undergoing coronary artery bypass grafting with preserved or mildly reduced left ventricular ejection fraction.

Background and objective: Coronary artery bypass grafting (CABG) is the reference standard intervention in coronary artery disease (CAD) patients with three-vessel disease (3VD). We aimed to evaluate the predictive value of left ventricular (LV) dyssynchrony for short-term adverse outcomes in patients with 3VD undergoing CABG with preserved or mildly reduced LV ejection fraction (LVEF). Materials and methods: This study involved ninety-five 3VD patients with preserved or mildly reduced LVEF undergoing scheduled on-pump CABG. The pre-operative diameters and volumes of LV and LVEF were obtained by two-dimensional echocardiography. LV dyssynchrony parameters were acquired by real-time three-dimensional echocardiography (RT-3DE) and analyzed by HeartModel quantification software. And the perfusion index of LV was obtained by contrast echocardiography. The clinical endpoints of short-term adverse outcomes comprised 30-day mortality and/or composite outcomes of postoperative complications. Univariate and multivariate logistic regression analyses were used to identify risk factors for the occurrence of post-CABG short-term adverse outcomes. Results: Short-term adverse outcomes occurred in 12 (12.6%) patients. These patients had higher LV dyssynchrony parameters obtained through RT-3DE. The standard deviation (SD) of the time to minimum systolic volume (Tmsv) corrected by heart rate over 16 segments (Tmsv16-SD%) [odds ratio (OR), 1.362; 95% confidence interval (CI) (1.090-1.702); P = 0.006], one of the LV dyssynchrony parameters, was independently associated with short-term adverse outcomes. Patients with poor synchronization tended to spend more time in the intensive care unit (ICU) and hospital after surgery. Conclusion: Pre-operative LV dyssynchrony parameter Tmsv16-SD% obtained through RT-3DE could be a useful additional predictor of postoperative short-term adverse outcomes in 3VD patients with preserved or mildly reduced LVEF undergoing CABG.

In Vitro Inhibitory Effects of Maqian Essential Oil against Ectopic Endometrial Stromal Cells and LPS-Induced Endometrial Epithelial Cells.

Previous studies revealed that MQEO (Maqian fruits essential oil), which is extracted from the fruit of Maqian (Zanthoxylum myriacanthum var. Pubescens), had a good anti-inflammatory effect, but the effect on endometriosis in vitro remains unknown. In the present study, the inhibitory effects of MQEO against the EESCs (ectopic endometrial stromal cells) were investigated. Cells were treated with a concentration gradient (from 0.025 % to 0.15 %) of MQEO for 24 h and cell viability was detected by CCK-8. In addition, apoptotic rates were investigated using flow cytometry. The effect of MQEO on cell migration was determined by wound-healing and transwell assay. The expression of apoptosis-associated and cell adhesion-related proteins was assessed by western blotting. The transcriptional levels of IL-1, IL-6 and TNF-α were determined by Real-time qPCR. RNA-seq was used to identify the DEGs (differentially expressed genes) in MQEO-pretreated EESCs. We found that the MQEO condition dosage-dependently reduced the cell viability of EESCs. Based on flow cytometry results, the number of apoptotic cells increased significantly with dosage. The wound-healing and transwell results showed that MQEO group exhibited a significantly decreased cell motility and migration ability in comparison with the normal group. Western blotting results showed that MQEO down-regulated the expression of Bcl-2, ICAM-1 (intercellular adhesion molecule 1) and CD44, but up-regulated the cleaved caspase-3 expression in EESCs. What's more, MQEO also inhibited the LPS-induced inflammation in human EECs (endometrial epithelial cells). RNA-seq revealed that 221 DEGs were up-regulated genes and 284 DEGs were down-regulated in MQEO-pretreated EESCs. Our data uncovered the beneficial effects of MQEO in endometriosis and provided new insights into the mechanism of the effect of MQEO on EESCs, suggesting MQEO could be a promising new therapeutic agent for endometriosis.

Protective effect of Du-Zhong-Wan against osteoporotic fracture by targeting the osteoblastogenesis and angiogenesis couple factor SLIT3.

ETHNOPHARMACOLOGICAL RELEVANCE: Du-Zhong-Wan (DZW) is a traditional Chinese medicine (TCM) composed of Eucommia ulmoides Oliv. and Dipsacus asper Wall. ex C.B. Clarke in the ratio 1:1. Based on the TCM theory, DZW nourishes the kidney to strengthen the bones. The literature research revealed that DZW possesses anti-fatigue, anti-depressant, and anti-osteoporotic properties. However, the action and mechanism of DZW on osteoporotic fracture remains slightly unclear. AIM OF THE STUDY: To evaluate the pharmacological effect of DZW on ovariectomized mice with an open femoral fracture and reveal the underlying mechanism. MATERIALS AND METHODS: We conducted ovariectomy for 5 weeks, followed by unilateral open transverse femoral fracture for another 3 weeks in C57BL/6 mice; during this process, DZW was administrated. The femur bone and vertebra tissues were collected and analyzed by micro-computed tomography, histomorphometry, mechanical strength testing, immunohistochemistry staining, and qRT-PCR analyses. In addition, alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to determine the extent of osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs). Western blotting was performed to examine the protein expression. RESULTS: DZW treatment significantly improved the bone histomorphometric parameters in mice undergoing ovariectomy when combined with the femoral fracture, including an increase in the bone volume, trabecular number, and bone formation rate and a decrease in the bone erosion area. Simultaneously, DZW treatment histologically promoted fractured callus formation. Mechanical strength testing revealed significantly higher stiffness and an ultimate load after treatment with DZW. The angiogenesis of H-type vessels was enhanced by DZW, as evidenced by increased levels of CD31 and endomucin (EMCN), the H-type vessel endothelium markers, at the fractured endosteum and metaphysis regions. Relative to the osteoporotic fracture mice, the DZW treatment group showed an increased proangiogenic factor SLIT3 level. The increased level of SLIT3 was also recorded during the process of DZW-stimulated osteoblastogenesis from BMSCs. CONCLUSIONS: For the first time, we demonstrated that DZW promoted osteoporotic fracture healing by enhancing osteoblastogenesis and angiogenesis of the H-type vessels. This enhanced combination of osteoblastogenesis and angiogenesis was possibly related to the production of proangiogenic factor SLIT3 induced by DZW.

Myocardial contrast echocardiography in the diagnosis of postoperative takotsubo myocardiopathy: case report and literature review.

BACKGROUND: Takotsubo cardiomyopathy (TCM) is a brief ventricular dysfunction that usually occurs after emotional or physical stress. Here, we report a patient who underwent cardiac surgery and then developed TCM during the postoperative period. CASE PRESENTATION: A 51-year-old woman was admitted to our hospital complaining of chest tightness, palpitations and dyspnoea after activity. An echocardiogram performed by our hospital showed rheumatic heart disease (severe mitral stenosis and regurgitation) with normal cardiac function and wall motion. After mitral valve replacement, this patient developed heart failure with low blood pressure and tachycardia. Urgent bedside echocardiography demonstrated akinesis in the middle and apical segments of the left ventricle and a depressed ejection fraction (EF) of 36%. Myocardial contrast echocardiography (MCE) showed similar enhancement intensity in the basal, middle and apical segments. Quantitative analysis showed approximately equivalent maximum intensity in these regions. The diagnosis was considered TCM instead of myocardial infarction. Then, an intra-aortic balloon pump was inserted to maintain effective circulation and reduce the postcardiac load. Given ventilation therapy, postoperative anticoagulation therapy and anti-infection treatment, the patient recovered quickly. In the follow-up examination, the patient remained asymptomatic and showed normalization of ventricular wall motion in the apical segment. CONCLUSION: This report presents a case of TCM in which MCE was used to demonstrate intact microvascular perfusion despite apical akinesis. This report might support the use of MCE as a substitute for invasive coronary angiography.

Wedelolactone inhibits osteoclastogenesis but enhances osteoblastogenesis through altering different semaphorins production.

Our previous study showed that wedelolactone, isolated from Ecliptae herba, enhanced osteoblastogenesis but inhibited osteoclastogenesis through Sema3A signaling pathway. This study aims to investigate the role of other semaphorins in wedelolactone-enhanced osteoblastogenesis and -inhibited osteoclastogenesis. Wedelolactone inhibited RANKL-induced Sema4D and Sema7A production, but had no effect on RANKL-reduced Sema6D expression in osteoclastic RAW264.7 cells. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone reversed osteogenic medium(OS)-reduced Sema7A expression and OS-enhanced Sema3E mRNA expression, but no effect on OS-reduced Sema3B mRNA expression. Addition of Sema4D antibody promoted wedelolactone-reduced TRAP activity and bone resorption pit formation. Wedelolactone combined with Sema4D antibody inhibited the formation of Sema4D-Plexin B1 complex. In co-culture of BMSC with RAW264.7 cells, Sema7A antibody, similar with Sema 3A antibody, reversed wedelolactone-enhanced ALP activity and mineralization level, but promoted wedelolactone-inhibited TRAP activity. However, Sema3E and Sema3B antibodies had no effect. Further, wedelolactone enhanced the binding of Sema7A with PlexinC1 and Beta1, but addition of Sema7A antibody partially blocked this binding. Our data demonstrated that wedelolactone inhibited Sema4D production and Sema4D-PlexinB1 complex formation in RAW264.7 cells, thereafter inhibiting osteoclastogenesis. At the same time, wedelolactone enhanced osteoblastogenesis through promoting Sema7A production and Sema7A-PlexinC1-Beta1 complex formation in BMSC.

Wedelolactone Enhances Osteoblastogenesis through ERK- and JNK-mediated BMP2 Expression and Smad/1/5/8 Phosphorylation.

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 µg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.

The inhibitory effect of Aconiti Sinomontani Radix extracts on the proliferation and migration of human synovial fibroblast cell line SW982.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconiti Sinomontani Radix is frequently used in the treatment of Bi syndrome in traditional Chinese medicine. Several reports indicate that Aconiti Sinomontani Radix has therapeutic effects for rheumatoid arthritis (RA). However, the cellular mode of action is still unclear. To investigate the effect of alkaloid extracts of Aconiti Sinomontani Radix on proliferation and migration of human synovial sarcoma SW982 cells as well as the molecular mechanism underlying. MATERIALS AND METHODS: SW982 cells were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Wound scratch assays were performed to assess the migrated rate of SW982 cells. Quantitative real-time PCR was used to measure the mRNA expression levels of Wnt5a, Runx2, MMP3, and Bmp2. Western blotting was used to measure the phosphorylated levels of JNK and NF-κB as well as the expression of MMP3. RESULTS: The alkaloid extract from Aconiti Sinomontani Radix (MQA) and MQB, which removed lappaconitine from MQA significantly inhibited the proliferation of SW982 in a dose-dependent manner. The proliferation inhibitory effect of MQB was more potent. Incubation with 10µg/ml MQB for 12, 24, and 36h inhibited the migration of SW982 cells by 83%, 58%, and 42%, respectively. Treatment with different concentrations of MQB for 24h inhibited mRNA expression of Wnt5a, Runx2, and MMP3, but Bmp2 mRNA expression was elevated by MQB. Further, MQB inhibited phosphorylation of JNK and NF-κB p65 as well as MMP3 expression by Western blotting analysis. CONCLUSION: The results showed that MQB inhibited proliferation and migration of SW982 cells possibly through suppressing Wnt5a-mediated JNK and NF-κB pathways. These results indicated that MQB might be an active extract of Aconiti Sinomontani Radix for targeting fibroblast-like synoviocytes (FLS) and be potential for RA therapy.

Wedelolactone enhances osteoblastogenesis by regulating Wnt/ß-catenin signaling pathway but suppresses osteoclastogenesis by NF-κB/c-fos/NFATc1 pathway.

Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3ß activity and enhanced the phosphorylation of GSK3ß, thereafter stimulated the nuclear translocation of ß-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3ß/ß-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis.

Inhibitory effect of Ecliptae herba extract and its component wedelolactone on pre-osteoclastic proliferation and differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ecliptae herba, also known as "Mo-Han-Lian", has long been used in China to nourish Kidney and thereafter strengthen bones. Accumulating evidence indicates that extracts of Ecliptae herba have antiosteoporotic effect. However, the effective compounds and cellular mode of action are still unclear. To investigate the effect of ethyl acetate extract of Ecliptae herba (EAE) and its component wedelolactone on proliferation and differentiation of preosteoclastic RAW264.7 cells as well as proliferation of bone marrow stromal cells (BMSC). MATERIALS AND METHODS: RAW264.7 and BMSC were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Tartrate-resistant acid phosphatase (TRAP) activity of RAW264.7 was measured by using p-nitrophenyl sodium phosphate (pNPP) assay after the cells were treated with 30ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) plus various concentrations of EAE, wedelolactone or alendronate. The formation of multinucleated TRAP-positive RAW264.7 cells was observed by using a TRAP-staining kit. RESULTS: Treatment of RAW264.7 cells with EAE at high doses (20µg/ml and 40µg/ml) or wedelolactone at 10µg/ml resulted in a decrease in proliferation of RAW264.7 cells. Low doses of EAE (5, 10µg/ml) and wedelolactone (2.5µg/ml) inhibited RANKL-induced TRAP activity by 20.3%, 37.9%, and 48.3%. The inhibitory effect of wedelolactone is more potent than that of alendronate, an anti-resorptive drug. Morphological changes revealed that 5µg/ml EAE and 2.5µg/ml wedelolactone reduced the number of multinucleated osteoclast-like cells. At the high doses, EAE (20µg/ml) and wedelolactone (10µg/ml) inhibited the growth of BMSC. CONCLUSIONS: EAE and its component wedelolactone inhibited osteoclast RAW264.7 proliferation and differentiation at the low doses, but at the high doses, showed cytotoxic effect on BMSC. These results indicated that EAE and wedelolatone might be potential alternative therapy for osteoporosis.

Taxol-induced alteration of intracellular amino-acid profile related to human cervical carcinoma HeLa cell death.

The anti-tumor action of Taxol was investigated in the changes of amino-acids involved in tumor cell survival. By tracing the intracellular amino-acid profiles of HeLa cells treated with non-conditioned and three conditioned media (Taxol, L-alanine, and Taxol + L-alanine), it was observed that an alteration of amino-acid metabolism participates in Taxol-induced death of HeLa cells. The contents of 18 out of 21 detected amino-acids are 5-95% and the ones of lysine and methionine are 158 and 117% of the corresponding contents in the control after treatment with Taxol for 24 h, respectively. Addition of L-alanine inhibited cell apoptosis upon Taxol treatment by partially blocking the increase of lysine and methionine and reversing decrease trend of alanine, glycine, and glutamic acid. These results suggest that interference of amino-acid metabolism might be an important mechanism of Taxol cytotoxicity.

[Smoking impairs endothelial function in ED patients].

OBJECTIVE: To determine whether smoking affects the endothelial function of young ED patients with no cardiovascular disease. METHODS: This study included 69 ED patients (21 smokers and 48 non-smokers) and 16 age-matched normal healthy controls. All underwent measurement of brachial artery flow-mediated vasodilation (FMD) and examinations of blood pressure, cholesterol, triglycerides and glucose. RESULTS: Brachial artery FMD was remarkably decreased in the ED patients, even more significantly in the smokers ([6.0 +/- 0.8]%) than in the non-smokers ([9.7 +/- 2.5]%) (P < 0.05), as compared with that in the normal healthy controls ([14.0 +/- 2. 5]%, P < 0.05). CONCLUSION: Endothelial function is impaired in ED patients, and is further damaged by smoking.

Functional analysis of cultured neural cells for evaluating cold/cool- and hot/warm-natured Chinese herbs.

Recently, modern scientific research has been required to understand pharmacological basis of traditional Chinese medicine (TCM) theory based on the ancient clinical experience, and to investigate the molecular mechanisms of action of Chinese herbs. Here, 20 Chinese herbs, classified into 4 properties (hot, warm, cold and cool) of TCM, were analyzed for their ability to exhibit antioxidant action, to enhance glucose uptake by murine microglia N9 cells, and to influence neurotransmitter norepinephrine (NE) release from rat pheochromocytoma PC12 cells. We found a generally protective effect of both hot/warm-natured and cold/cool-natured herbs against H(2)O(2)-induced N9 cell death, partially by elevating superoxide dismutase (SOD) activity. Glucose uptake was elevated after treatment with some hot/warm-natured herbs. In addition, most herbs with hot/warm nature tended to stimulate NE release, while such stimulatory effect was not observed in the herbs with cold/cool nature. Two cold/cool-natured herbs, Rhizoma coptidis and Radix scutellariae, even significantly suppressed the release. These results suggest that the distinct abilities of Chinese herbs to regulate neural cell functions appear to be correlated with their natures identified in traditional TCM theory, and may be a useful guide for their utility in neural degenerative diseases.

Fas/FasL signaling allows extracelluar-signal regulated kinase to regulate cytochrome c release in oridonin-induced apoptotic U937 cells.

Previously, we found that human histocytic lymphoma U937 cells possessed high susceptibility to oridonin-induced cell death, but the molecular mechanisms in response to oridonin remain unclear. In this study, U937 cells showed susceptible to apoptosis induced by 27 microM oridonin and an agonistic anti-Fas IgM mAb (CH-11) (500 ng/ml) as a Fas-sensitized positive control. Caspase 8 inhibitor z-IETD, but neither caspase 1 inhibitor Ac-YVAD nor caspase 10 inhibitor z-AEVD, effectively blocked oridonin-induced cell death as well as DNA fragmentation. Western blot analysis showed the up-regulated expression of Fas, FasL, and FADD, and down-regulated expression of procaspase 8, suggesting that Fas/FasL pathway was activated in oridonin-induced cell apoptosis. Further, stimulation of U937 cells with oridonin and CH11 resulted in significant ERK MAPK activation. However, inhibition of ERK by PD98059 reversed oridonin-induced cell death as well as the activation of caspase 8, indicating that ERK-mediated control occured upstream of caspase 8. Simultaneously, ERK activation accounted for the release of cytochrome c, but failed to influence decreased Bcl-2 expression induced by oridonin. Taken together, these results suggest that Fas/FasL signaling pathway-mediated ERK activation sensitized U937 cells to mitochondrial pathway-mediated apoptosis induced by oridonin.

Roles of Ras and extracellular signal-regulated kinase-dependent IkappaBalpha degradation in oridonin-enhanced phagocytosis of apoptotic cells by human macrophage-like U937 cells.

Rapid recognition and ingestion of apoptotic cells by phagocytes are important for the prevention of toxic intracellular contents release, thereby attenuate inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We have reported that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic U937 cells by macrophage-like U937 cells through TNFalpha and IL-1beta release. In this study, the molecular mechanisms involved in this phagocytic process are investigated. Inhibitors of Ras and Raf1 kinase significantly reduced oridonin-induced phagocytic stimulation as well as extracellular signal-regulated kinase (ERK) phosphorylation. Simultaneously, oridonin-enhanced engulfment was partially blocked by a nuclear factor (NF)-kappaB inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that oridonin induced IkappaBalpha degradation, which was prevented by Ras inhibitor manumycin A, ERK inhibitor PD98059, but not prevented by c-Jun N-terminal kinase (JNK) MAPK inhibitor SP600125, and up-regulated expression of IL-1beta precursor. These results demonstrate that Ras/Raf1/ERK signaling pathway-dependent IkappaBalpha degradation, resulting in NF-kappaB activation, participates in regulation of oridonin-enhanced phagocytosis, and one of its effector functions is to induce synthesis of IL-1beta, which partially contribute to phagocytic activity of oridonin.

Activation of phosphoinositide 3-kinase, protein kinase C, and extracellular signal-regulated kinase is required for oridonin-enhanced phagocytosis of apoptotic bodies in human macrophage-like U937 cells.

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity time- dependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB/Akt) and the increased expression of PLCgamma2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNFalpha and anti-IL-1beta antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of ERK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNFalpha or IL-1beta only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and ERK-dependent pathways.

Oridonin enhances phagocytosis of UV-irradiated apoptotic U937 cells.

We previously reported that oridonin, a major component isolated from the plant Rabdosia rubescens HEMSL, induced apoptosis in human melanoma A375-S2 and cervical cancer HeLa cells. In the present study, oridonin was first evaluated for its effect on phagocytosis of apoptotic cells by macrophages. Preincubation of human histocytic lymphoma U937 cell-derived macrophages with 2.7 microM oridonin significantly augmented phagocytosis of UV-irradiated (2.4 J/cm2, 4 min) U937 cells undergoing apoptosis in a dose- and time-dependent manner. However, less effect on synthetic fluoresbrite microspheres indicated that enhancement of apoptotic U937 cell uptake by oridonin was a selective effect. The oridonin-augmented phagocytosis was attenuated by anti-human TNFalpha and IL-1beta antisera, suggesting that TNFalpha and IL-1beta participate in the phagocytosis by oridonin-treated U937 cell-derived macrophages. In addition, the similar effect of phagocytosis was observed in oridonin-treated human monocyte-derived macrophages at 4 d maturation. Taken together, oridonin facilitates the phagocytic activity against apoptotic cells through TNFalpha and IL-1beta release, which may be contribute to its antitumor activities.