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.

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.

Prevalence of the methylenetetrahydrofolate reductase 677C>T polymorphism in the pregnant women of Yunnan Province, China.

Mutations in the methylenetetrahydrofolate reductase (MTHFR) gene can result in a reduced ability to utilize folic acid. The MTHFR 677C>T polymorphism in particular has been linked to both birth defects and pregnancy-associated diseases. This study aimed to evaluate the prevalence of the MTHFR 677C>T mutation among pregnant women in Yunnan Province so as to collect baseline data that may be utilized to guide folic acid supplementation efforts and to support related disease prevention programs. We retrospectively reviewed 3387 pregnant women from Yunnan Province. The MTHFR 677C>T polymorphism was identified using polymerase chain reaction (PCR) and DNA sequencing. In total, 1350 (39.9%) subjects were homozygous for the C allele (CC), 1540 (45.4%) subjects were heterozygous (CT), and 497 (14.7%) subjects were homozygous for the T allele (TT). The MTHFR 677C>T polymorphism was found to be present within the studied population, with ∼60% of these patients being either heterozygous or homozygous for the mutant allele and with an overall T allele frequency of 0.37. The frequency of the T allele was significantly higher among pregnant women with complications relative to women with healthy pregnancies, particularly among women <30 years old. As such, the maternal MTHFR 677C>T polymorphism may be a genetic risk factor associated with pregnancy complications and may help identify pregnant women at a high risk of such complications.

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.

Rapid Screening of Active Components with an Osteoclastic Inhibitory Effect in Herba epimedii Using Quantitative Pattern-Activity Relationships Based on Joint-Action Models.

Screening of bioactive components is important for modernization and quality control of herbal medicines, while the traditional bioassay-guided phytochemical approach is time-consuming and laborious. The presented study proposes a strategy for rapid screening of active components from herbal medicines. As a case study, the quantitative pattern-activity relationship (QPAR) between compounds and the osteoclastic inhibitory effect of Herba epimedii, a widely used herbal medicine in China, were investigated based on joint models. For model construction, standard mixtures data showed that the joint-action models are better than the partial least-squares (PLS) model. Then, the Good2bad value, which could reflect components' importance based on Monte Carlo sampling, was coupled with the joint-action models for screening of active components. A compound (baohuoside I) and a component composed of compounds with retention times in the 6.9-7.9 min range were selected by our method. Their inhibition rates were higher than icariin, the key bioactive compound in Herba epimedii, which could inhibit osteoclast differentiation and bone resorption in a previous study. Meanwhile, the half-maximal effective concentration, namely, EC50 value of the selected component was 7.54 µg/mL, much smaller than that of baohuoside I-77 µg/mL-which indicated that there is synergistic action between compounds in the selected component. The results clearly show our proposed method is simple and effective in screening the most-bioactive components and compounds, as well as drug-lead components, from herbal medicines.

[Degradation Process of Exogenous Naphthenic Acids and Their Effects on Microbial Community Structure in Soil].

Naphthenic acids (NAs) are a natural component of petroleum, which account for about 2% of severe ecological toxicity in addition to polycyclic aromatic hydrocarbons. With the growth in demand for energy, a large number of NAs have leaked into soil environments through oil industry processes, which have caused enormous potential threats to human health and ecosystems. However, there are few studies about the degradation process of exogenous NAs and their effects on microbial community structures in soil. This research explores the degradation process of NAs and their dynamics in microbial communities in soil by adding a high concentration of 180 mg·kg-1 of NAs to natural, clean soil with the aid of liquid chromatography and high-throughput sequencing technologies. This study found that:① Natural clean soil has a strong capability to degrade high concentration of NAs with about 50% of the NAs degraded within 5 days, which stabilized at 80% after 30 days of the experiment; ② Pollution with NAs obviously alters the microbial community structure as the number of specific OTU increased and were mainly distributed in phylum of unidentified Proteobacteria and Bacteroidetes; ③ Under high concentrations of NAs, the content of Bacteroidetes and Acidobacteria phylum and the γ-Proteobacteria of Proteobacteria phylum all increased swiftly and were speculated to be a potential agents for NA degradation, with the relative abundance ratio of Bacteroidetes and Acidobacteria increasing from 4.2% and 2% to 20.3% and 5.5%, respectively, while a 24.8% decrease was found in Actinobacteria phylum; ④ This study revealed the degradation process of exogenous NAs and their effects on microbial community structure in soil, which provided scientific support for the ecological restoration of petroleum pollution and further study in this area.

A cell-based model of bone remodeling for identifying activity of icarrin in the treatment of osteoporosis.

The activity of icarrin (a flavonoid from Herba epimedii) was investigated in the regulation of bone remodeling, a process coupled by osteoblast-mediated bone forming and osteoclast-mediated bone resorption. By directly co-culturing mouse bone marrow stromal cells and mouse preosteoclastic RAW264.7, and transwell co-culturing rat ovarian follicular granulosa cells (FGC), a 30 % increase in alkaline phosphatase (ALP) activity and 25 % increase in estradiol level occurred. Compared with the antiresorptive drug, alendronate, and an anabolic drug, PTH1-34, icarrin possessed all of the positive effects on the co-culture by increasing ALP activity, estradiol production and decreasing tartrate-resistant acid phosphatase activity. A similar action of icarrin occurred on co-culture of mesenchymal stem cells, mouse peripheral blood mononuclear cells, and FGC. Overall, by using a co-cultured cell-based in vitro screening assay, icarrin is suggested as a new class of dual-action therapeutic agent for osteoporosis.

Synergistic inhibitory effect of Icariside II with Icaritin from Herba Epimedii on pre-osteoclastic RAW264.7 cell growth.

Increasing evidence shows the therapeutic superiority of herbal extracts in comparison to isolated single constituents. One of the reasons may be attributed to the synergy effect of compound combinations. Flavonoids from Herba Epimedii have been shown to have therapeutic effect against bone loss. Our previous study showed that Icariside II inhibited pre-osteoclast RAW264.7 growth. The aim of this study was to investigate whether the activity of Icariside II is synergized by other components of Herba Epimedii. The inhibitory activity of Icariside II was significantly enhanced in the presence of the extract of Herba Epimedii (EHE) at the ratio of 1:1, 1:5 and 1:10. Icaritin, another flavonoid constituent, was shown here to inhibit RAW264.7 growth in a dose-dependent manner. Further, we found that Icariside II, together with Icaritin, synergistically inhibited RAW264.7 growth. The synergistic effect is significant when the ratio of Icariside II and Icaritin was 10:1, 5:1, 1:1, 1:2, and 1:5, respectively. In conclusion, Icaritin were an active component. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as 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.

[Screening of bioactive components in epimedium for osteoporosis treatment by model population analysis].

Epimedium was obtained from different habitats, and their bioactive components for inhibiting RAW264.7 were screened by MTT assay. Results indicate that epimedium from different habitats displayed significant different activities. By means of model population analysis (MPA), a latent bioactive component, baohuoside-I was got. Activity of baohuoside-I wasvalidated and prior to icariin. MPA can be used for bioactive components screening.

Rhizoma coptidis and berberine-induced activation of murine microglia N9 cells.

AIMS OF THE STUDY: To investigate the effect of water extract of Rhizoma coptidis (WEC) and berberine on the activation of murine microglia N9 cells and corresponding mechanism related to mitochondria. MATERIALS AND METHODS: Phagocytic activity of murine microglia N9 cells was measured by neutral red staining method after the cells were treated with various concentrations of WEC and alkaloids for 24h. Flow cytometric analysis was performed to determine the level of intracellular ROS, Ca(2+), and mitochondrial transmembrane potential (Delta psi) after 87 microg/ml of WEC and 12.4 microg/ml of berberine treatment. Global changes of gene expression in WEC- and berberine-treated N9 cells were measured using cDNA microarray. RESULTS: WEC and berberine, but not palmatine and jatrorrhizine, enhanced phagocytic activity of murine N9 cells in a dose-dependent manner. Both of WEC and berberine stimulated free radical generation, enhanced mitochondrial Delta psi and induced gene expression of Ndufab1, Cox6a2 and Atp5a1. However, a more significant phagocytic effect was observed for WEC. WEC, but not berberine, increased intracellular Ca(2+) concentration. The gene expression of Atp5c1 was selectively up-regulated by WEC, while three genes of Uqcrq, Cox8b, and Atp5g2 were induced by berberine. CONCLUSIONS: WEC and berberine activated murine microglia N9 cells by the regulation of mitochondrial function and mitochondria-related signal molecules. The action of WEC is stronger than that of berberine, indicating that the effect of WEC is ascribed partially, but not totally, to berberine.

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.

[Oridonin induced U937 cell apoptosis through ERK pathway].

OBJECTIVE: To study the mechanisms of oridonin-induced U937 cell apoptosis, and to examine the role of ERK MAPK. METHOD: MTT, Hoechst 33258 staining, DNA agarose gel electrophoresis and Western blot analysis were used. RESULT: Oridonin inhibited U937 cell growth in a time- and dose-dependent manner. Apoptotic bodies were found with Hoechst 33258 staining after treatment with 27 micromol x L(-1) oridonin. Simultaneously, ERK phosphorylation was significant. ERK inhibitor PD98059 partially blocked the growth-inhibitory effect as well as DNA fragmentation. The expression of antiapoptotic mitochondrial protein Bcl-XL decreased time-dependently, and that of proapoptotic protein Bax increased. However, PD98059 reversed the effect of oridonin on Bcl-XL and Bax. CONCLUSION: Oridonin induces U937 cell apoptosis through activation of ERK and alteration of the ratio of Bax/Bcl-XL.