Metabolic landscape and pathogenic insights: a comprehensive analysis of high ovarian response in infertile women undergoing in vitro fertilization.

BACKGROUND: In the realm of assisted reproduction, a subset of infertile patients demonstrates high ovarian response following controlled ovarian stimulation (COS), with approximately 29.7% facing the risk of Ovarian Hyperstimulation Syndrome (OHSS). Management of OHSS risk often necessitates embryo transfer cancellation, leading to delayed prospects of successful pregnancy and significant psychological distress. Regrettably, these patients have received limited research attention, particularly regarding their metabolic profile. In this study, we aim to utilize gas chromatography-mass spectrometry (GC-MS) to reveal these patients' unique serum metabolic profiles and provide insights into the disease's pathogenesis. METHODS: We categorized 145 infertile women into two main groups: the CON infertility group from tubal infertility patients and the Polycystic Ovary Syndrome (PCOS) infertility group. Within these groups, we further subdivided them into four categories: patients with normal ovarian response (CON-NOR group), patients with high ovarian response and at risk for OHSS (CON-HOR group) within the CON group, as well as patients with normal ovarian response (PCOS-NOR group) and patients with high ovarian response and at risk for OHSS (PCOS-HOR group) within the PCOS group. Serum metabolic profiles were analyzed using GC-MS. The risk criteria for OHSS were: the number of developing follicles > 20, peak Estradiol (E2) > 4000pg/mL, and Anti-Müllerian Hormone (AMH) levels > 4.5ng/mL. RESULTS: The serum metabolomics analysis revealed four different metabolites within the CON group and 14 within the PCOS group. Remarkably, 10-pentadecenoic acid emerged as a discernible risk metabolite for the CON-HOR, also found to be a differential metabolite between CON-NOR and PCOS groups. cysteine and 5-methoxytryptamine were also identified as risk metabolites for the PCOS-HOR. Furthermore, KEGG analysis unveiled significant enrichment of the aminoacyl-tRNA biosynthesis pathway among the metabolites differing between PCOS-NOR and PCOS-HOR. CONCLUSION: Our study highlights significant metabolite differences between patients with normal ovarian response and those with high ovarian response and at risk for OHSS within both the tubal infertility control group and PCOS infertility group. Importantly, we observe metabolic similarities between patients with PCOS and those with a high ovarian response but without PCOS, suggesting potential parallels in their underlying causes.

Transcriptome analysis unveils the mechanisms of lipid metabolism response to grayanotoxin I stress in Spodoptera litura.

Background: Spodoptera litura (tobacco caterpillar, S. litura) is a pest of great economic importance due to being a polyphagous and world-distributed agricultural pest. However, agricultural practices involving chemical pesticides have caused resistance, resurgence, and residue problems, highlighting the need for new, environmentally friendly methods to control the spread of S. litura. Aim: This study aimed to investigate the gut poisoning of grayanotoxin I, an active compound found in Pieris japonica, on S. litura, and to explore the underlying mechanisms of these effects. Methods: S. litura was cultivated in a laboratory setting, and their survival rate, growth and development, and pupation time were recorded after grayanotoxin I treatment. RNA-Seq was utilized to screen for differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to determine the functions of these DEGs. ELISA was employed to analyze the levels of lipase, 3-hydroxyacyl-CoA dehydrogenase (HOAD), and acetyl-CoA carboxylase (ACC). Hematoxylin and Eosin (H & E) staining was used to detect the development of the fat body. Results: Grayanotoxin I treatment significantly suppressed the survival rate, growth and development, and pupation of S. litura. RNA-Seq analysis revealed 285 DEGs after grayanotoxin I exposure, with over 16 genes related to lipid metabolism. These 285 DEGs were enriched in the categories of cuticle development, larvae longevity, fat digestion and absorption. Grayanotoxin I treatment also inhibited the levels of FFA, lipase, and HOAD in the hemolymph of S. litura. Conclusion: The results of this study demonstrated that grayanotoxin I inhibited the growth and development of S. litura. The mechanisms might, at least partly, be related to the interference of lipid synthesis, lipolysis, and fat body development. These findings provide valuable insights into a new, environmentally-friendly plant-derived insecticide, grayanotoxin I, to control the spread of S. litura.

Effects of Yulin Tong Bu formula on modulating gut microbiota and fecal metabolite interactions in mice with polycystic ovary syndrome.

Background: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder characterized by hyperandrogenism, ovarian dysfunction and polycystic ovarian morphology. Gut microbiota dysbiosis and metabolite are associated with PCOS clinical parameters. Yulin Tong Bu formula (YLTB), a traditional Chinese medicine formula, has been recently indicated to be capable of ameliorating polycystic ovary symptoms and correcting abnormal glucose metabolism. However, the therapeutic mechanism of YLTB on PCOS has not been fully elucidated. Methods: A pseudo sterile mouse model was established during this four-day acclimatization phase by giving the animals an antibiotic cocktail to remove the gut microbiota. Here, the therapeutic effects of YLTB on PCOS were investigated using dehydroepiandrosterone plus high-fat diet-induced PCOS mice model. Female prepuberal mice were randomly divided into three groups; namely, the control group, PCOS group and YLTB (38.68 g·kg-1·day-1) group. To test whether this effect is associated with the gut microbiota, we performed 16S rRNA sequencing studies to analyze the fecal microbiota of mice. The relationships among metabolites, gut microbiota, and PCOS phenotypes were further explored by using Spearman correlation analysis. Then, the effect of metabolite ferulic acid was then validated in PCOS mice. Results: Our results showed that YLTB treatment ameliorated PCOS features (ovarian dysfunction, delayed glucose clearance, decreased insulin sensitivity, deregulation of glucolipid metabolism and hormones, etc.) and significantly attenuated PCOS gut microbiota dysbiosis. Spearman correlation analysis showed that metabolites such as ferulic acid and folic acid are negatively correlated with PCOS clinical parameters. The effect of ferulic acid was similar to that of YLTB. In addition, the bacterial species such as Bacteroides dorei and Bacteroides fragilis were found to be positively related to PCOS clinical parameters, using the association study analysis. Conclusion: These results suggest that YLTB treatment systematically regulates the interaction between the gut microbiota and the associated metabolites to ameliorate PCOS, providing a solid theoretical basis for further validation of YLTB effect on human PCOS trials.

Role of TRPM2 in brain tumours and potential as a drug target.

Ion channels are ubiquitously expressed in almost all living cells, and are the third-largest category of drug targets, following enzymes and receptors. The transient receptor potential melastatin (TRPM) subfamily of ion channels are important to cell function and survival. Studies have shown upregulation of the TRPM family of ion channels in various brain tumours. Gliomas are the most prevalent form of primary malignant brain tumours with no effective treatment; thus, drug development is eagerly needed. TRPM2 is an essential ion channel for cell function and has important roles in oxidative stress and inflammation. In response to oxidative stress, ADP-ribose (ADPR) is produced, and in turn activates TRPM2 by binding to the NUDT9-H domain on the C-terminal. TRPM2 has been implicated in various cancers and is significantly upregulated in brain tumours. This article reviews the current understanding of TRPM2 in the context of brain tumours and overviews the effects of potential drug therapies targeting TRPM2 including hydrogen peroxide (H2O2), curcumin, docetaxel and selenium, paclitaxel and resveratrol, and botulinum toxin. It is long withstanding knowledge that gliomas are difficult to treat effectively, therefore investigating TRPM2 as a potential therapeutic target for brain tumours may be of considerable interest in the fields of ion channels and pharmacology.

Genetic Polymorphisms of Long Non-coding RNA Linc00312 Are Associated With Susceptibility and Predict Poor Survival of Nasopharyngeal Carcinoma.

BACKGROUND: Linc00312 is dysregulated in nasopharyngeal carcinoma (NPC) and participates in the initiation and progression of NPC. Our previous studies suggested that linc00312 was able to enhance the sensitivity of NPC cells to irradiation and NPC patients with higher expression of linc00312 was associated with better short-term curative effect and overall survival. The single nucleotide polymorphisms (SNPs) of lncRNAs may influence the disease course and outcome by affecting the expression, secondary structure or function of lncRNAs. However, the role of SNPs in linc00312 on the occurrence and survival of NPC remains unknown. METHODS: We recruited 684 NPC patients and 823 healthy controls to evaluate the association between linc00312 SNPs and NPC susceptibility by using multivariate logistic regression analysis. Kaplan-Meier analysis and Cox proportional hazards regression were applied to assess the effect of linc00312 SNPs on the survival of NPC patients. The relative expression of linc00312 in NPC tissues was determined by real-time PCR. The interaction between linc00312 and mir-411-3p was explored by luciferase reporter assay. In silico prediction of the changes on linc00312 folding structure was conducted by RNAfold WebServer. RESULT: We demonstrated that rs12497104 (G > A) GA genotype carriers had a higher risk than others for suffering from NPC (GA vs GG, OR = 1.437, P = 0.003). Besides, patients with rs12497104 AA genotype showed a poorer overall survival in contrast to GG genotype (AA vs GG, HR = 2.117, P = 0.011). In addition, the heterozygous carriers of rs15734 (G > A) and rs164966 (A > G) were correlated with decreased risk of NPC (GA vs GG, OR = 0.778, P = 0.031; GA vs AA, OR = 0.781, P = 0.033, respectively). We found that the three SNPs might influence the expression of linc00312 in a genotype specific feature. The local centroid secondary structure as well as the minimum free energy of linc00312 were changed following the candidate SNPs alterations. Besides, we revealed that the G to A alteration at rs12497104 disrupted the binding between mir-411-3p and linc00312. CONCLUSION: Our results indicated genetic polymorphisms of linc00312 might serve as potential biomarkers for NPC carcinogenesis and prognosis.

Identification of key genes in ruptured atherosclerotic plaques by weighted gene correlation network analysis.

The rupture of atherosclerotic plaques is essential for cardiovascular and cerebrovascular events. Identification of the key genes related to plaque rupture is an important approach to predict the status of plaque and to prevent the clinical events. In the present study, we downloaded two expression profiles related to the rupture of atherosclerotic plaques (GSE41571 and GSE120521) from GEO database. 11 samples in GSE41571 were used to identify the differentially expressed genes (DEGs) and to construct the weighted gene correlation network analysis (WGCNA) by R software. The gene oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment tool in DAVID website, and the Protein-protein interactions in STRING website were used to predict the functions and mechanisms of genes. Furthermore, we mapped the hub genes extracted from WGCNA to DEGs, and constructed a sub-network using Cytoscape 3.7.2. The key genes were identified by the molecular complex detection (MCODE) in Cytoscape. Further validation was conducted using dataset GSE120521 and human carotid endarterectomy (CEA) plaques. Results: In our study, 868 DEGs were identified in GSE41571. Six modules with 236 hub genes were identified through WGCNA analysis. Among these six modules, blue and brown modules were of the highest correlations with ruptured plaques (with a correlation of 0.82 and -0.9 respectively). 72 hub genes were identified from blue and brown modules. These 72 genes were the most likely ones being related to cell adhesion, extracellular matrix organization, cell growth, cell migration, leukocyte migration, PI3K-Akt signaling, focal adhesion, and ECM-receptor interaction. Among the 72 hub genes, 45 were mapped to the DEGs (logFC > 1.0, p-value < 0.05). The sub-network of these 45 hub genes and MCODE analysis indicated 3 clusters (13 genes) as key genes. They were LOXL1, FBLN5, FMOD, ELN, EFEMP1 in cluster 1, RILP, HLA-DRA, HLA-DMB, HLA-DMA in cluster 2, and SFRP4, FZD6, DKK3 in cluster 3. Further expression detection indicated EFEMP1, BGN, ELN, FMOD, DKK3, FBLN5, FZD6, HLA-DRA, HLA-DMB, HLA-DMA, and RILP might have potential diagnostic value.

TRPM2-AS inhibits the growth, migration, and invasion of gliomas through JNK, c-Jun, and RGS4.

Gliomas are a group of brain cancers with high mortality and morbidity. Understanding the molecular mechanisms is important for the prevention or treatment of gliomas. The present study was to investigate the effects and mechanisms of long noncoding RNA TRPM2-AS in gliomas proliferation, migration, and invasion. We first compared the levels of TRPM2-AS in 111 patients with glioma to that of the normal control group by a quantitative polymerase chain reaction. The results indicated a significant increase of TRPM2-AS in patients with glioma (2.43 folds of control, p = .0135). MTT methods, wound healing assays, transwell analysis, and clone formation analysis indicated the overexpression of TRPM2-AS promoted the proliferation, migration, and invasion of U251 and U87 cells, while downregulation of TRPM2-AS inhibited the cell proliferation, migration, and invasion significantly (p < .05). To further uncover the mechanisms, bioinformatics analysis was conducted on the expression profiles, GSE40687 and GSE4290, from the Gene Expression Omnibus database. One hundred fifty-six genes were differentially expressed in both datasets (FC > 2.0; p = .05). Among these differentially expressed genes, the level of RGS4 messenger RNA was drastically regulated by TRPM2-AS. Further western-blot analysis indicated the increase of RGS4 protein expression and decrease of p-JNK/JNK and p-c-Jun/c-Jun ratio after TRPM2-AS overexpression. On the other hand, inhibition of TRPM2-AS by small interfering RNA suppressed the expression of RGS4 and promoted the ratios of p-JNK/JNK and p-c-Jun/c-Jun. The present work indicated the mechanisms of the participation of TRPM2-AS in the progression of gliomas might, at least partly, be related to JNK, c-Jun, and RGS4. Our work provided new insights into the underlying mechanisms of glioma cellular functions.

Pharmacological approaches promoting stem cell-based therapy following ischemic stroke insults.

Stroke can lead to long-term neurological deficits. Adult neurogenesis, the continuous generation of newborn neurons in distinct regions of the brain throughout life, has been considered as one of the appoaches to restore the neurological function following ischemic stroke. However, ischemia-induced spontaneous neurogenesis is not suffcient, thus cell-based therapy, including infusing exogenous stem cells or stimulating endogenous stem cells to help repair of injured brain, has been studied in numerous animal experiments and some pilot clinical trials. While the effects of cell-based therapy on neurological function during recovery remains unproven in randomized controlled trials, pharmacological agents have been administrated to assist the cell-based therapy. In this review, we summarized the limitations of ischemia-induced neurogenesis and stem-cell transplantation, as well as the potential proneuroregenerative effects of drugs that may enhance efficacy of cell-based therapies. Specifically, we discussed drugs that enhance proliferation, migration, differentiation, survival and function connectivity of newborn neurons, which may restore neurobehavioral function and improve outcomes in stroke patients.

Long Noncoding RNA LINC00657 Acting as a miR-590-3p Sponge to Facilitate Low Concentration Oxidized Low-Density Lipoprotein-Induced Angiogenesis.

Angiogenesis in atherosclerotic plaque promotes plaque growth, causes plaque hemorrhage, and violates plaque stability. LINC00657 is a long noncoding RNA highly conserved and abundantly expressed in vascular endothelial cells. The present study was designed to investigate the effects and mechanisms of LINC00675 on low concentrations of oxidized low-density lipoprotein (oxLDL)-induced angiogenesis. Cell proliferation, transwell, wound healing, and tube formation assays were conducted to detect the effects of low concentrations of oxLDL on angiogenesis; the results discovered that oxLDL promoted cell proliferation, migration, and tube formation. oxLDL also upregulated LINC00657 expression. Inhibition of LINC00657 by siRNA significantly suppressed oxLDL-induced endothelial cell proliferation, migration, and tube formation. Bioinformatic assay indicated six binding sites in the LINC00657 sequence to miR-590-3p. The upregulation of LINC00657 was related to the downregulation of miR-590-3p in oxLDL-treated endothelial cells; while downregulation of LINC00657 resulted in upregulation of miR-590-3p. The antiangiogenesis effects of si-LINC00657 were partly abrogated by miR-590-3p inhibitor. Further dual-luciferase assay found miR-590-3p inhibited the expression of hypoxia-inducible factor 1α (HIF-1α) by binding to the position of 689-696 in HIF-1α 3'-untranslated region directly. MiR-590-3p also inhibited the oxLDL-induced upregulation of HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9). These results suggested that in oxLDL-treated endothelial cells, LINC00657 acted as a miR-590-3p sponge to attenuate the suppression of miR-590-3p on HIF-1α, and to promote angiogenesis through VEGF, MMP-2, and MMP-9. The present study provided new insight into the roles of LINC00657 and miR-590-3p in preventing oxLDL-induced angiogenesis and may provide a novel strategy for atherosclerosis treatment.

Effect of Oridonin on Cytochrome P450 Expression and Activities in HepaRG Cell.

Oridonin, the major terpene found in Rabdosia rubescens, is widely used as a dietary supplement or therapeutic drug. However, the effects of oridonin on major CYP450s are still unclear. As oridonin can enhance the effect of other clinical drugs, in this study, we investigated the influence of oridonin on CYP450s mRNA expression and its impact on activities in human HepaRG cell to evaluate the safety by studying its potential drug interaction. HepaRG cells were cultured with series concentrations of oridonin (1, 5, 10, and 20 µmol/L), and the major CYP450s mRNA and protein expression, as well as enzyme activities were analyzed by real-time polymerase chain reaction, Western blot analysis and UPLC-MS/MS-based metabolite assay. In general, ordonin has induced effects on the major member of CYP450s mRNA and protein expression, as well as on the enzyme activity in human HepaRG cells, especially on CYP3A4 and CYP2C9. To our knowledge, this is the first systematic research about the inductive effects of oridonin on the major member of CYP450s in human cell line. These results may provide at least partly of the basis for potential drug-drug interactions and oridonin should be used with caution to avoid potential risk.

Long non-coding RNAs in ischemic stroke.

Stroke is one of the leading causes of mortality and disability worldwide. Uncovering the cellular and molecular pathophysiological processes in stroke have been a top priority. Long non-coding (lnc) RNAs play critical roles in different kinds of diseases. In recent years, a bulk of aberrantly expressed lncRNAs have been screened out in ischemic stroke patients or ischemia insulted animals using new technologies such as RNA-seq, deep sequencing, and microarrays. Nine specific lncRNAs, antisense non-coding RNA in the INK4 locus (ANRIL), metastasis-associate lung adenocarcinoma transcript 1 (MALAT1), N1LR, maternally expressed gene 3 (MEG3), H19, CaMK2D-associated transcript 1 (C2dat1), Fos downstream transcript (FosDT), small nucleolar RNA host gene 14 (SNHG14), and taurine-upregulated gene 1 (TUG1), were found increased in cerebral ischemic animals and/or oxygen-glucose deprived (OGD) cells. These lncRNAs were suggested to promote cell apoptosis, angiogenesis, inflammation, and cell death. Our Gene Ontology (GO) enrichment analysis predicted that MEG3, H19, and MALAT1 might also be related to functions such as neurogenesis, angiogenesis, and inflammation through mechanisms of gene regulation (DNA transcription, RNA folding, methylation, and gene imprinting). This knowledge may provide a better understanding of the functions and mechanisms of lncRNAs in ischemic stroke. Further elucidating the functions and mechanisms of these lncRNAs in biological systems under normal and pathological conditions may lead to opportunities for identifying biomarkers and novel therapeutic targets of ischemic stroke.

Transient receptor potential melastatin 2 channels (TRPM2) mediate neonatal hypoxic-ischemic brain injury in mice.

Transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, is reported to mediate brain damage following ischemic insults in adult mice. However, the role of TRPM2 channels in neonatal hypoxic-ischemic brain injury remains unknown. We hypothesize that TRPM2+/- and TRPM2-/- neonatal mice have reduced hypoxic-ischemic brain injury. To study the effect of TRPM2 on neonatal brain damage, we used 2,3,5-triphenyltetrazolium chloride (TTC) staining to assess the infarct volume and whole brain imaging to assess morphological changes in the brain. In addition, we also evaluated neurobehavioral outcomes for sensorimotor function 7days following hypoxic-ischemic brain injury. We report that the infarct volumes were significantly smaller and behavioral outcomes were improved in both TRPM2+/- and TRPM2-/- mice compared to that of wildtype mice. Next, we found that TRPM2-null mice showed reduced dephosphorylation of GSK-3ß following hypoxic ischemic injury unlike sham mice. TRPM2+/- and TRPM2-/- mice also had reduced activation of astrocytes and microglia in ipsilateral hemispheres, compared to wildtype mice. These findings suggest that TRPM2 channels play an essential role in mediating hypoxic-ischemic brain injury in neonatal mice. Genetically eliminating TRPM2 channels can provide neuroprotection against hypoxic-ischemic brain injury and this effect is elicited in part through regulation of GSK-3ß.

NF-κB-Regulated miR-99a Modulates Endothelial Cell Inflammation.

Objective. The present study was performed to investigate the effects and mechanisms of miR-99a on LPS-induced endothelial cell inflammation, as well as the regulation of NF-κB on miR-99a production. Methods and Results. ELISA showed that LPS treatment significantly promoted the secretion of inflammatory factors (TNF-α, IL-6, IL-1ß, and MCP-1). LPS treatment also inhibited miR-99a production and promoted mTOR expression and NF-κB nuclear translocation. Overexpression of miR-99a suppressed the LPS-induced TNF-α, IL-6, IL-1ß, and MCP-1 overproduction, mTOR upregulation, and NF-κB nuclear translocation. The PROMO software analysis indicated NF-κB binding site in the -1643 to -1652 region of miR-99a promoter. Dual luciferase reporter analysis, electrophoretic mobility shift assays (EMSA), and chromosome immunoprecipitation (ChIP) assays demonstrated that NF-κB promoted the transcription of miR-99a by binding to the -1643 to -1652 region of miR-99a promoter. Further studies on HUVECs verified the regulatory effects of NF-κB on miR-99a production. Conclusion. MiR-99a inhibited the LPS-induced HUVECs inflammation via inhibition of the mTOR/NF-κB signal. NF-κB promoted miR-99a production by binding to the -1643 to -1652 region of miR-99a promoter. Considering the importance of endothelial inflammation on cardiovascular diseases, such as atherosclerosis, our results may provide a new insight into the pathogenesis and therapy of atherosclerosis.

Effects of miR­590 on oxLDL­induced endothelial cell apoptosis: Roles of p53 and NF­κB.

Oxidized low­density lipoprotein (oxLDL)­induced endothelial cell apoptosis is considered to be important in atherogenesis. MicroRNA (miR)­590 has been reported to inhibit oxLDL­induced endothelial cell apoptosis. However, the mechanism underlying the inhibition of oxLDL­induced endothelial cell apoptosis by miR­590 remains to be elucidated. In the present study, the expression levels of miR­590 were quantified using reverse transcription­quantitative polymerase chain reaction analysis. Cell apoptosis was investigated using Hoechst staining and flow cytometry, and cell viability was measured using an MTS method. The protein expression levels of p53, B cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), caspase­3, lectin­like low­density lipoprotein receptor 1 (LOX­1), p38 mitogen­activated protein kinase (MAPK) and nuclear factor (NF)­κB were quantified using western blot analyses. The results of the present study showed that oxLDL treatment inhibited the expression levels of miR­590 in a time­dependent and concentration­dependent manner. The overexpression of miR­590 inhibited oxLDL­induced endothelial cell apoptosis, expression of p53 and Bax, reduction of Bcl­2 and activation of caspase­3. miR­590 also inhibited the oxLDL­induced upregulation of the expression of LOX­1, overproduction of reactive oxygen species (ROS), phosphoryation of p38MAPK and translocation of NF­κB. These findings demonstrated the anti­apoptotic effects of miR­590 in oxLDL­treated endothelial cells, with the mechanisms underlying the effects of miR­590 involved, in part, in the LOX­1­ROS­p38MAPK­NF­κB signaling cascade and the p53­Bcl­2/Bax­caspase­3 signaling pathway. The present study may provide novel insights into the protective properties of miR­590 in preventing atherosclerosis.

Unstable simple volatiles and gas chromatography-tandem mass spectrometry analysis of essential oil from the roots bark of Oplopanax horridus extracted by supercritical fluid extraction.

Volatile oil from the root bark of Oplopanax horridus is regarded to be responsible for the clinical uses of the title plant as a respiratory stimulant and expectorant. Therefore, a supercritical fluid extraction method was first employed to extract the volatile oil from the roots bark of O. horridus, which was subsequently analyzed by GC/MS. Forty-eight volatile compounds were identified by GC/MS analysis, including (S,E)-nerolidol (52.5%), τ-cadinol (21.6%) and S-falcarinol (3.6%). Accordingly, the volatile oil (100 g) was subjected to chromatographic separation and purification. As a result, the three compounds, (E)-nerolidol (2 g), τ-cadinol (62 mg) and S-falcarinol (21 mg), were isolated and purified from the volatile oil, the structures of which were unambiguously elucidated by detailed spectroscopic analysis including 1D- and 2D-NMR techniques.

Induction of human CYP3A4 by huperzine A, ligustrazine and oridonin through pregnane X receptor-mediated pathways.

The pregnane X receptor (PXR) is a key regulator of CYP3A4, which is involved in catalyzing the metabolic conversion of a number of endogenous substrates. In this study, we screened 22 compounds isolated from traditional Chinese herbal medicines using luciferase reporter gene assays for inspecting their capabilities in inducing PXR-mediated transactivation of CYP3A4 expression. In addition, the mRNA and protein expressions of CYP3A4 and PXR as well as the enzymatic activites of CYP3A4 were analyzed by real-time PCR, Western blot analysis and UPLC-MS/MS-based metabolite assay in LS174T cells. Huperzine A, ligustrazine and oridonin were identified to be the inducers of CYP3A4. These compounds induced the CYP3A4 reporter luciferase activity, and up-regulated CYP3A4 mRNA and protein levels significantly. Besides, huperzine A, ligustrazine and oridonin significantly up-regulated enzymatic activities of CYP3A4. However, the three compounds showed no effects on PXR mRNA and protein expression. To our knowledge, it is the first identification of these three compounds as PXR activators to induce CYP3A4. These results indicate that huperzine A, ligustrazine and oridonin induced CYP3A4 expression and activation via PXR dependent pathways, and might contribute to drug-drug interactions.

Puerarin protects endothelial cells from oxidized low density lipoprotein induced injuries via the suppression of LOX-1 and induction of eNOS.

Oxidized low density lipoprotein (oxLDL) induced injury of endothelial cells is considered to be the first step in the pathogenesis of atherosclerosis. This study aimed to investigate some of the effects and mechanisms of puerarin on oxLDL-induced endothelial injuries. We measured cell viability, and the release of lactate dehydrogenase (LDH), nitric oxide (NO), and interleukin-8 (IL-8) to evaluate the protective effects of puerarin. Intracellular reactive oxygen species (ROS) were detected using 2',7'-dichlorofluorescein diacetate (DCFH-DA). The expression of lectin-like low-density lipoprotein receptor-1 (LOX-1), endothelial nitric oxide synthase (eNOS), cyclooxygenase 2 (COX-2), p38MAPK, and protein kinase B (PKB) phosphorylation, nuclear factor-κB (NF-κB) nuclear translocation, and inhibitor of κB (IκB) degradation were detected using quantitative real-time PCR or Western blot. The results showed that oxLDL significantly decreased cell viability, increased LDH and IL-8 release, inhibited NO production, and induced COX-2 expression. Pretreatment with puerarin led to a strong inhibition of these effects. OxLDL stimulated the expression of LOX-1, the overproduction of ROS, the phosphorylation of p38MAPK, the dephosphorylation of PKB, activation of NF-κB, and the degradation of IκB. These oxLDL-induced effects were suppressed after puerarin pretreatment. These results suggest that puerarin inhibits oxLDL-induced endothelial cell injuries, at least in part, via inhibition of the LOX-1-mediated p38MAPK-NF-κB inflammatory and the PKB-eNOS signaling pathways.

Paeonol suppresses oxidized low-density lipoprotein induced endothelial cell apoptosis via activation of LOX-1/p38MAPK/NF-κB pathway.

Paeonol is an active compound isolated from traditional Chinese medicine, and has been shown to have anti-atherosclerosis, anti-inflammatory, antioxidant effects. The present investigation was undertaken to determine the suppression effects of paeonol on oxidized low-density lipoprotein (ox-LDL) induced endothelial cell line HUVEC apoptosis and to uncover some of the underlying mechanisms of these effects. Cell viability and lactate dehydrogenase (LDH) were measured to evaluate the cell injuries. Apoptosis was evaluated by Hoechst 33342 staining and flow cytometry. Intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Real-time PCR was used to confirm the expression of LOX-1 mRNA. Western blotting was used to evaluate the protein expression of LOX-1 and Bcl-2, as well as caspase-3 cleavage, p38-mitogen-activated protein kinase (p38MAPK) phosphorylation. NF-κB nuclear translocation was detected by Western blotting and immunofluorescence. Caspase-3 activity was measured using a colorimetric protease assay kit. The results showed that ox-LDL significantly decreased cell viability and increased the LDH release, as well as the apoptotic rate (P<0.01). Pre-treatment of paeonol resulted in remarkable increase of cell viability, decrease of LDH release and cell apoptosis in a concentration-dependent manner. Besides, ox-LDL caused the up-regulation of LOX-1, the down-regulation of Bcl-2, the phosphorylation of p38MAPK, the translocation of NF-κB and the activation of caspase-3. Paeonol pre-treatment reversed these effects introduced by ox-LDL. Moreover, paeonol also showed its inhibition effects on ox-LDL induced ROS overproduction. These results indicate the preventive effects of paeonol on ox-LDL induced endothelial cell apoptosis. The effects might, at least partly, be obtained via inhibition of LOX-1-ROS- p38MAPK-NF-κB signaling pathway.