The m6A reader HNRNPC promotes glioma progression by enhancing the stability of IRAK1 mRNA through the MAPK pathway.

Glioma is the most common and aggressive type of primary malignant brain tumor. The N6-methyladenosine (m6A) modification widely exists in eukaryotic cells and plays an important role in the occurrence and development of human tumors. However, the function and mechanism of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an RNA-binding protein and m6A reader in gliomas remains to be comprehensively and extensively explored. Herein, we found that HNRNPC mRNA and protein overexpression were associated with a poor prognosis for patients with gliomas, based on the data from TCGA, the CGGA, and the TMAs. Biologically, HNRNPC knockdown markedly repressed malignant phenotypes of glioma in vitro and in vivo, whereas ectopic HNRNPC expression had the opposite effect. Integrative RNA sequencing and MeRIP sequencing analyses identified interleukin-1 receptor-associated kinase 1 (IRAK1) as a downstream target of HNRNPC. The glioma public datasets and tissue microarrays (TMAs) data indicated that IRAK1 overexpression was associated with poor prognosis, and IRAK1 knockdown significantly repressed malignant biological behavior in vitro. Mechanistically, HNRNPC maintains the mRNA stability of IRAK1 in an m6A-dependent manner, resulting in activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which was necessary for the malignant behavior of glioma. Our findings demonstrate the HNRNPC-IRAK1-MAPK axis as a crucial carcinogenic factor for glioma and the novel underlying mechanism of IRAK1 upregulation, which provides a rationale for therapeutically targeting epitranscriptomic modulators in glioma.

The neuroprotective effect of near infrared light therapy in aged mice with postoperative neurocognitive disorder by upregulating IRF7.

BACKGROUND: Postoperative neurocognitive disorder (PND) is a common central nervous system complication after undergoing surgery and anesthesia especially in elderly patients, while the therapeutic options are very limited. This study was carried out to investigate the beneficial effects of transcranial near infrared light (NIRL) which was employed to the treatment of PND and propose the involved mechanisms. METHODS: The PND mice were established through left carotid artery exposure under isoflurane anesthesia and received transcranial NIRL treatment. Behavioral testing was performed to evaluate the cognitive function of PND mice after transcranial NIRL therapy. Changes in the transcriptomic profiles of prefrontal cortex (PFC) and hippocampus (HP) were identified by next generation sequencing (NGS), and the molecular mechanisms involved were examined by both in vivo mouse model and in vitro cell culture studies. RESULTS: We found that transcranial NIRL therapy effectively ameliorated learning and memory deficit induced by anesthesia and surgery in aged mice. Specifically, we identified down-regulation of interferon regulatory factor 7 (IRF7) in the brains of PND mice that was mechanistically associated with increased pro-inflammatory M1 phenotype of microglia and elevated neuroinflammatory. NIRL treatment produced protective effects through the upregulation of IRF7 expression and reversing microglial phenotypes from pro-inflammatory to neuroprotective, resulting in reduced brain damage and improved cognitive function in PND mice. CONCLUSION: Our results indicate that transcranial NIRL is an effective and safe therapy for PND via alleviating neuroinflammation, and IRF7 plays a key transcription factor in regulating the M1-to-M2 switch of microglia.

[Mechanism of Marsdenia tenacissima against ovarian cancer based on network pharmacology and experimental verification].

The present study aimed to explore the main active components and underlying mechanisms of Marsdenia tenacissima in the treatment of ovarian cancer(OC) through network pharmacology, molecular docking, and in vitro cell experiments. The active components of M. tenacissima were obtained from the literature search, and their potential targets were obtained from SwissTargetPrediction. The OC-related targets were retrieved from Therapeutic Target Database(TTD), Online Mendelian Inheritance in Man(OMIM), GeneCards, and PharmGKB. The common targets of the drug and the disease were screened out by Venn diagram. Cytoscape was used to construct an "active component-target-disease" network, and the core components were screened out according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened out according to the node degree. GO and KEGG enrichment analyses of potential therapeutic targets were carried out with DAVID database. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock. Finally, the anti-OC activity of M. tenacissima extract was verified based on SKOV3 cells in vitro. The PI3K/AKT signaling pathway was selected for in vitro experimental verification according to the results of GO function and KEGG pathway analyses. Network pharmacology results showed that 39 active components, such as kaempferol, 11α-O-benzoyl-12ß-O-acetyltenacigenin B, and drevogenin Q, were screened out, involving 25 core targets such as AKT1, VEGFA, and EGFR, and the PI3K-AKT signaling pathway was the main pathway of target protein enrichment. The results of molecular docking also showed that the top ten core components showed good binding affinity to the top ten core targets. The results of in vitro experiments showed that M. tenacissima extract could significantly inhibit the proliferation of OC cells, induce apoptosis of OC cells through the mitochondrial pathway, and down-regulate the expression of proteins related to the PI3K/AKT signaling pathway. This study shows that M. tenacissima has the characteristics of multi-component, multi-target, and multi-pathway synergistic effect in the treatment of OC, which provides a theoretical basis for in-depth research on the material basis, mechanism, and clinical application.

Tongguanteng injection reverses paclitaxel resistance via upregulation of TAB1 expression in ovarian cancer in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongguanteng injection (TGT), the water extract from the stem of the Traditional Chinese hebal medicine of Marsdenia tenacissima (Roxb.) Wight et Arn. has been used as anticancer remedy for decades. TGT was not only used in the treatment of many malignant cancers extensively, but also an adjuvant anticancer drug with chemotherapeutics clinically. AIM OF THE STUDY: To evaluate the effects of TGT on reversing paclitaxel (PTX) resistance and investigate the potential mechanism related to TAB1 in ovarian cancer (OC) in vitro and in vivo. MATERIALS AND METHODS: The synergistic effect and reversal ratio were determined by CCK8 assay and median-effect principle after the combination of TGT and PTX in OC A2780 and its PTX-resistant (A2780/T) cells. The biological functions in cell apoptosis, migration and invasion of A2780/T cells treated by PTX 4 µM with TGT 20, 40, 80 mg⋅mL-1 for 24 h were evaluated by colony formation, flow cytometry, wound healing and transwell assays. Proteomics technique and bioinformatic analysis were used to indentify the change of TAB1 expression in A2780/T cells induced by TGT. The association between TAB1 expression and human OC was analyzed by gene expression databases. In A2780/T cells, western blotting and colony formation assays were used to investigate the relationship between TAB1 expression and PTX resistance after TAB1 overexpression by TAB1 plasmids. The mechanism of TGT and PTX regulating TAB1 and its related proteins were explored by western blotting and flow cytometry assays after TAB1 knock-down using siTAB1. Moreover, TUNEL staining, immunohistochemistry (IHC) and histopathology were used to observe the antitumor effects, TAB1 and p-p38 expression and the tissues impairments in nude mice xenograft model established by A2780/T cells after the co-treatment with TGT and PTX by in vivo. RESULTS: TGT combined with PTX showed the synergistic effect (CI<1), which could reverse the IC50 values of PTX in OC A2780 and A2780/T cells about 23.50 and 6.44 times, respectively. Besides, TGT combined with PTX could significantly inhibit the migration, invasion and promote apoptosis of A2780/T cells. We identified that TGT could induce TAB1 expression in A2780/T cells by proteomics analysis. TAB1 downregulation was significantly associated with tumorigenesis and poor prognosis in OC patients and PTX resistance in A2780/T cells. Furthermore, TGT could activate TAB1/TAK1/p38 MAPK signaling pathway targeting TAB1 and regulate the expression of Bax, Bcl-2 proteins to improve the sensitivity of A2780/T cells to PTX. TGT combined with PTX also showed a greater inhibition in tumor growth than PTX monotherapy in vivo. These promising results show the efficacy of TGT in reversing PTX resistance and provide a potential strategy that targeting TAB1/TAK1/p38 MAPK signaling pathway may improve the chemotherapy sensitivity in OC. CONCLUSIONS: Our results revealed that Tongguanteng injection could reverse paclitaxel resistance and the potential mechanism might be associated with the activation of TAB1/TAK1/p38 MAPK signaling pathway in OC in vitro and in vivo. TAB1 might be a pivotal target for reversing PTX resistance. This study will provide a theoretical basis for the combination of Tongguanteng injection and paclitaxel in clinic.

lncRNA ZNF674-AS1 inhibits the migration, invasion and epithelial-mesenchymal transition of thyroid cancer cells by modulating the miR-181a/SOCS4 axis.

Thyroid cancer (TC) is a very common endocrine cancer worldwide. Further understanding and revealing the molecular mechanism underlying thyroid cancer are indispensable for the development of effective diagnosis and treatments. Long non-coding RNAs (lncRNAs), a series of non-coding RNAs with a length of >200 nts, have been regarded as crucial regulators of many cancers playing a tumor suppressive or oncogenic role, depending on circumstances. lncRNA ZNF674-AS1 was reported to be abnormally expressed in TC, but the exact mechanism remains unclear. This study aims to probe the mechanism and roles of ZNF674-AS1 in TC. The expression patterns of RNAs and proteins were determined via qRT-PCR and western blotting, respectively. Cell proliferation, migration and invasion were detected using MTT and Transwell assays. ZNF674-AS1 and SOCS4 expression were remarkably reduced while miR-181a was upregulated in TC tissues and cells. Enforced expression of ZNF674-AS1 inhibited proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and reduced tumour growth in vivo. Mechanistic assays verified that ZNF674-AS1 directly interacted with miR-181a to increase SOCS4 expression. In addition, miR-181a overexpression aggravated proliferation, metastasis and EMT by inhibiting SOCS4. Interestingly, inhibition of miR-181a diminished the promoting effects of ZNF674-AS1 silencing on the malignant behaviours of TC cells. These data illustrated that ZNF674-AS1 alleviated TC progression by modulating the miR-181a/SOCS4 axis (graphical abstract), further suggesting that ZNF674-AS1 might be used as a therapheutic target in TC treatment.

Biomarkers for Predicting the Occurrence and Progression of Atrial Fibrillation: Soluble Suppression of Tumorigenicity 2 Protein and Tissue Inhibitor of Matrix Metalloproteinase-1.

Background: Soluble suppression of tumorigenicity 2 protein (sST2) and tissue inhibitor of matrix metalloproteinase (TIMP)-1 are involved in multiple pathogenic pathways, including cardiac remodeling, which is the main pathology of atrial fibrillation (AF). This study aims to investigate the previously unexplored relationship between the serum levels of sST2, TIMP-1, and AF. Methods: This was a prospective cross-sectional study conducted at the Capital Medical University Affiliated Beijing Anzhen Hospital between June 2019 and July 2020, with a total of 359 participants. The clinical characteristics and laboratory results of the patients were compared, and multivariable ordinal logistic regression was used to evaluate the relationship between serum sST2, TIMP-1, and AF. Results: The participants included 110 patients with sinus rhythm (SR), 113 with paroxysmal AF (the paroxysmal AF group), and 136 with persistent AF (the persistent AF group). It was found that the sST2 levels gradually increased in these three groups, from 9.1 (6.7-12.4 pg/ml) in the SR group to 14.0 (10.4-20.8 pg/ml) in the paroxysmal AF group and to 19.0 (13.1-27.8) pg/ml) in the persistent AF group (p < 0.001). The multivariable ordinal logistic regression model for sST2 and TIMP-1 demonstrated that sST2 had an area under the receiver operating characteristic (ROC) curve (AUC) of 0.797 (95% confidence interval (CI) 0.749-0.846, p < 0.001) and TIMP-1 had an AUC of 0.795 (95% CI 0.750-0.841, p=0.000). The multivariable ordinal logistic regression model for sST2 and TIMP-1 showed good discrimination between SR and AF, with an AUC of 0.846, and the addition of clinical factors, such as brain natriuretic peptide (BNP), left atrial diameter, age, and gender, to the biomarker model improved the detection of SR and AF (AUC 0.901). Conclusions: In this cohort study, sST2 and TIMP-1 were associated with AF progression, independent of clinical characteristics and biomarkers. Soluble ST2 and TIMP-1 combined with age, elevated N-terminal-pro hormone BNP(NT-BNP), and an enlarged left atrium were able to demonstrate the progression of AF reliably.

Construction of a competitive endogenous RNA network and analysis of potential regulatory axis targets in glioblastoma.

BACKGROUND: Glioblastoma is the most common primary malignant brain tumor. Because of the limited understanding of its pathogenesis, the prognosis of glioblastoma remains poor. This study was conducted to explore potential competing endogenous RNA (ceRNA) network chains and biomarkers in glioblastoma by performing integrated bioinformatics analysis. METHODS: Transcriptome expression data from The Cancer Genome Atlas database and Gene Expression Omnibus were analyzed to identify differentially expressed genes between glioblastoma and normal tissues. Biological pathways potentially associated with the differentially expressed genes were explored by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, and a protein-protein interaction network was established using the STRING database and Cytoscape. Survival analysis using Gene Expression Profiling Interactive Analysis was based on the Kaplan-Meier curve method. A ceRNA network chain was established using the intersection method to align data from four databases (miRTarBase, miRcode, TargetScan, and lncBace2.0), and expression differences and correlations were verified by quantitative reverse-transcription polymerase chain reaction analysis and by determining the Pearson correlation coefficient. Additionally, an MTS assay and the wound-healing and transwell assays were performed to evaluate the effects of complement C1s (C1S) on the viability and migration and invasion abilities of glioblastoma cells, respectively. RESULTS: We detected 2842 differentially expressed (DE) mRNAs, 2577 DE long non-coding RNAs (lncRNAs), and 309 DE microRNAs (miRNAs) that were dysregulated in glioblastoma. The final ceRNA network consisted of six specific lncRNAs, four miRNAs, and four mRNAs. Among them, four DE mRNAs and one DE lncRNA were correlated with overall survival (p < 0.05). C1S was significantly correlated with overall survival (p= 0.015). In functional assays, knockdown of C1S inhibited the proliferation and invasion of glioblastoma cell lines. CONCLUSIONS: We established four ceRNA networks that may influence the occurrence and development of glioblastoma. Among them, the MIR155HG/has-miR-129-5p/C1S axis is a potential marker and therapeutic target for glioblastoma. Knockdown of C1S inhibited the proliferation, migration, and invasion of glioblastoma cells. These findings clarify the role of the ceRNA regulatory network in glioblastoma and provide a foundation for further research.

Skip participates in formation and lipid metabolism of beige adipose and might mediate the effects of SIRT1 activator BTM-0512 on beige remodeling.

Dissipating energy by activating thermogenic adipose to combating obesity attracts many interests. Ski-interacting protein (Skip) has been known to play an important role in cell proliferation and differentiation, but whether it participates in energy metabolism is not known. Our previous study revealed that BTM-0512 could induce beige adipose formation, accompanying with up-regulation of Skip, but the role of Skip in metabolism was unknown. In this study, we mainly investigated whether Skip was involved in beige remodeling of subcutaneous white preadipocytes as well as in lipid metabolism of differentiated beige adipocytes. The results showed that in high fat diet-induced obesity mice, the protein levels of Skip in subcutaneous and visceral white adipose as well as in brown adipose were all down-regulated, especially in subcutaneous white adipose. Then we cultured subcutaneous adipose derived-stem cells (ADSCs) and found knock-down of Skip (siSkip) inhibited the expressions of thermogenic adipose specific genes including PRDM16 and UCP1 in both undifferentiated ADSCs and differentiated beige adipocytes, which could abolish the effects of BTM-0512 on beige remodeling. We further observed that siSkip affected multiple rate-limiting enzymes in lipid metabolism. The expressions of ACC, GPAT-1, HSL and ATGL were down-regulated, while CPT1α expression was up-regulated by siSkip. The expression of AMPK was also decreased by siSkip. In conclusion, our study demonstrated that Skip might play an important role in the beige remodeling of white adipocytes as well as lipid metabolism of beige adipose.

M6A RNA Methylation Regulator HNRNPC Contributes to Tumorigenesis and Predicts Prognosis in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most malignant glioma with a high death rate. N6-methyladenosine (m6A) RNA methylation plays an increasingly important role in tumors. The current study aimed to determine the function of the regulators of m6A RNA methylation in GBM. We evaluated the difference, interaction, and correlation of these regulators with TCGA database. HNRNPC, WTAP, YTHDF2 and, YTHDF1 were significantly upregulated in GBM. To explore the expression characteristics of regulators in GBM, we defined two subgroups through consensus cluster. HNRNPC, WTAP, and YTHDF2 were significantly upregulated in the cluster2 which had a good overall survival (OS). To investigate the prognostic value of regulators, we used lasso cox regression algorithm to screen an independent prognostic risk characteristic based on the expression of HNRNPC, ZC3H13, and YTHDF2. The prognostic feature between the low and high-risk groups was significantly different (P < 0.05), which could predict significance of prognosis (area under the curve (AUC) = 0.819). Moreover, we used western blot, RT-PCR, and immunohistochemical staining to verify the expression of HNRNPC was associated with malignancy and development of gliomas. Similarly, the high expression of HNRNPC had a good prognosis. In conclusion, HNRNPC is a vital participant in the malignant progression of GBM and might be valuable for prognosis.

Xiaoaiping injection enhances paclitaxel efficacy in ovarian cancer via pregnane X receptor and its downstream molecules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoaiping injection, a traditional Chinese medical injection extracted from root of Marsdenia tenacissima (Roxb.) Moon, has been exclusively used on curing malignant tumor in China and as adjuvant therapeutic agent for chemotherapeutics, including paclitaxel. AIM OF THE STUDY: The goal of this study was to investigate the synergistic inhibitory efficacy of Xiaoaiping injection and paclitaxel on ovarian cancer. The mechanism may be associated with nuclear receptor pregnane X receptor (PXR) regulating its downstream molecules. MATERIALS AND METHODS: In vitro, MTT assay, flow cytometry and Hoechst dyeing were used to evaluate the SK-OV-3 cell proliferation, apoptosis and cell cycle respectively. The mRNA and protein expression of PXR and its downstream CYP450 enzymes, transporters and Bcl-2 families were measured by qRT-PCR and Western blot. Rhodamine 123 efflux experiment was conducted to detect the P-gp efflux ability. PXR plasmid and PXR siRNA were transiently transfected into SK-OV-3 cells respectively to establish PXR-overexpressed or PXR-interfered cells. In vivo, xenograft tumor mice model was established by SK-OV-3 cells to estimate the antitumor effect of Xiaoaiping injection combined with paclitaxel. The expressions of PXR and its downstream molecules in tumor tissues were determined to further clarify the potential mechanism. RESULTS: Xiaoaiping injection significantly enhanced the anti-proliferation, pro-apoptosis effect of paclitaxel on SK-OV-3 cells. The synergetic effect was displayed by Xiaoaiping injection inhibiting paclitaxel-induced PXR and CAR expression, which subsequently inhibited CYP450 enzymes CYP2C8 and CYP3A4, transporter P-gp and anti-apoptotic proteins Bcl-2 and Bcl-xl in SK-OV-3 cells. In PXR-overexpressed cells, Xiaoaiping injection down-regulated the expression of PXR and its downstream molecules. The result of xenograft tumor model showed that Xiaoaiping injection combined with paclitaxel enhanced anti-tumor effect on ovarian cancer in vivo. CONCLUSIONS: Xiaoaiping injection enhances anti-tumor effect of paclitaxel by inhibiting cell proliferation, inducing apoptosis process. The mechanism may be associated with Xiaoaiping injection inhibiting PXR and its downstream metabolic enzymes CYP2C8, CYP3A4, transporter P-gp and anti-apoptosis protein Bcl-2.

Potential association between Fusobacterium nucleatum enrichment on oral mucosal surface and oral lichen planus.

OBJECTIVE: We determined the bacterial community structure of the buccal mucosa in patients with oral lichen planus (OLP) and evaluated the potential association of Fusobacterium nucleatum with OLP. SUBJECTS AND METHODS: We collected buccal mucosal swab samples of patients with OLP (n = 20) and healthy controls (n = 10) and performed 16S rRNA gene sequencing and real-time PCR to determine potentially different bacteria. Damaged and adjacent non-damaged mucosal swab samples of 25 OLP patients were used to detect the amount of F. nucleatum by real-time PCR. RESULTS: Compared with healthy controls, enrichment of Fusobacterium and Granulicatella was more abundant in patients with OLP (p = .0146 and 0.0034). The abundance of Fusobacterium and F. nucleatum was significantly enriched on buccal mucosa of patients with OLP compared with healthy controls (p = .0043 and 0.0235). Compared with adjacent non-damaged buccal mucosa of OLP patients, the amount of F. nucleatum in the damaged mucosa was significantly increased (p = .001). We examined third-level KEGG pathways for bacteria on mucosal surface and found that genes controlling sporulation and ether lipid metabolism were enriched in patients with OLP. CONCLUSIONS: A high amount of F. nucleatum may be associated with OLP. Further studies are required to investigate the precise association of F. nucleatum with OLP.

Effect of Xiao-Ai-Ping injection on paclitaxel pharmacokinetics in rats by LC-MS/MS method.

Xiao-Ai-Ping injection (XAP) has been shown to be clinically effective in treatment of gastric carcinoma, liver cancer and lung cancer, when it was combined with anticancer drug paclitaxel (PTX). To analyze the effect of XAP on the pharmacokinetics of PTX, a liquid chromatography-tandem mass spectroscopy (LCMS/MS) assay method was developed and validated to quantify PTX simultaneously and its main metabolite 3'-p-hydroxypaclitaxel (C3'-OHP) in rat plasma. PTX and C3'-OHP were quantified using positive MRM mode. The analysis method was validated for specificity, recovery, carry-over, accuracy, precision, sample stability and dilution integrity under various storage conditions. The pharmacokinetic parameters were determined in rats after tail intravenous administration of 6 mg/mL PTX in the absence (control group) or presence of intraperitoneal administration of 10 mL/kg、20 mL/kg XAP (study groups). Compared to control group, the area under the plasma concentration-time curve (AUC) of PTX and C3'-OHP in study groups increased significantly following consecutive administration with XAP for 10 days. In conclusion, pretreatment with XAP enhanced the exposure of PTX and C3'-OHP. There would be herb-drug interaction happening between XAP and PTX in rats.

Alpha-glucosidase inhibitor 1-Deoxynojirimycin promotes beige remodeling of 3T3-L1 preadipocytes via activating AMPK.

Obesity is a serious health challenge in the world, and searching effective drugs to cure obesity is of great importance. 1-Deoxynojirimycin (DNJ) is extracted from mulberry leaves and acts as an α-glucosidase inhibitor to lower blood glucose. Recent studies demonstrated that it also has anti-obesity effect, but the mechanisms remain unknown. In our present study, we mainly examined the effects of DNJ on beige remodeling of 3T3-L1 preadipocytes. We observed that DNJ didn't affect the mRNA levels of fatty acid binding protein 4 (aP2), peroxisome proliferator-activated receptor γ (PPARγ), preadipocyte factor-1 (Pref-1) as well as the mitochondrial uncoupling protein 1 (UCP1), PR domain containing protein 16 (PRDM16), transmembrane protein 26 (TMEM26) in undifferentiated preadipocytes. But after inducing 3T3-L1 preadipocytes to differentiation with white or beige adipogenic medium, DNJ significantly reduced aP2, PPARγ and Pref-1 expressions, while up-regulated the expressions of UCP1, PRDM16 and TMEM26, accompanying with decreased lipid deposition. The ratio of p-AMPK/AMPK was up-regulated by DNJ (10 µM) treatment for 10 days, and the effects of DNJ on p-AMPK/AMPK, UCP1 and PRDM16 could be blocked by AMPK inhibitor Compound C. These results demonstrated that hypoglycemic agent DNJ could suppress the adipogenesis during the differentiation of white preadipocytes, and promote the switch of white preadipocytes to beige adipocytes via activating AMPK, which provided new mechanisms for explaining the benefits of DNJ on obesity-related disorders.

Effects of Danhong injection, a traditional Chinese medicine, on nine cytochrome P450 isoforms in vitro.

Danhong injection (DHI) is made from Salvia miltiorrhiza Bunge. and Carthamus tinctorius L. extract and is widely used in the clinical treatment of cardiovascular and cerebrovascular diseases. This study aimed to evaluate the effect of DHI on cytochrome P450 (CYP450) enzymes in vitro to predict drug-drug interactions based on CYP450 as combination therapy. To assess the inhibitory effect of DHI on CYP450, we detected the IC50 value of DHI on CYP450 in vitro by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Simultaneously, the induction effect of DHI on CYP450s was also evaluated. The relative induction ratios of DHI on CYP1A2, CYP2B6 and CYP3A4 activity were calculated by LC-MS/MS. The expression level of CYP3A4 mRNA was determined by reverse transcription PCR (RT-PCR). The LC-MS/MS data showed DHI intensively inhibit CYP2A6 activity and the intensity of inhibition was followed by CYP2C8, CYP3A4, CYP2C19, CYP2B6, CYP2D6, CYP1A2, CYP2E1 and CYP2C9 in vitro. The results of RT-PCR showed that there is a certain induction of DHI on CYP3A4 mRNA in human primary hepatocytes in vitro. The study suggested that drug-drug interactions might occur in clinical co-administration of drugs owing to the CYP2A6 inhibition and CYP3A4 induction.

Clinicopathologic and prognostic significance of VEGF, JAK2 and STAT3 in patients with nasopharyngeal carcinoma.

BACKGROUND: The aim of the study was to investigate the effect associated with the protein expression of VEGF, JAK2 and STAT3 on the clinicopathologic characteristics and prognosis in the development and progression of nasopharyngeal carcinoma (NPC). METHODS: Fifty NPC patients in addition to 20 patients with chronic nasopharyngitis (CNP) were recruited for the purposes of the study. Western blotting and immunohistochemistry methods were employed to evaluate the protein expressions of JAK2, STAT3 and VEGF in the NPC and CNP tissues, with their respective correlations with the clinicopathologic characteristics of NPC patients subsequently analyzed. Spearman's rank correlation coefficient and Kaplan-Meier method were conducted to evaluate the respective correlations of JAK2, STAT3 and VEGF with NPC as well as the survival rates of patients with NPC. Cox regression analyses was performed in determine the prognostic NPC factors. RESULTS: Compared with the CNP tissues, the NPC tissues exhibited elevated levels of JAK2, STAT3 and VEGF which were subsequently determined to share a positive correlation with T stages, lymph node metastasis (LNM), N stages and clinical stages, while a negative correlation with survival rates were observed in the NPC patients. Positive correlations between the expressions of JAK2, STAT3 and VEGF were detected among the NPC tissues. NPC patients survival time with negative expressions of JAK2, STAT3 and VEGF were observed to be longer than that of NPC patients with positive expressions of JAK2, STAT3 and VEGF. T stage, LNM, N stage, clinical stage. The expressions of JAK2, STAT3 and VEGF were discovered to be independent risk factors associated with the prognosis of patients with NPC. CONCLUSION: The results obtained from the present study support the notion that higher expressions of JAK2, STAT3 and VEGF may be correlated with the clinicopathologic characteristics and prognosis of patients suffering from NPC.

MicroRNA-185 inhibits cell proliferation while promoting apoptosis and autophagy through negative regulation of TGF-ß1/mTOR axis and HOXC6 in nasopharyngeal carcinoma.

OBJECTIVE: Accumulating studies have revealed that microRNAs (miRs) play a critical role in the development and progression of nasopharyngeal carcinoma (NPC), which is a disease with a remarkable racial and geographical distribution. In our study, through the alteration in the expression of microRNA-185 (miR-185) in NPC cells by microarray-based gene expression profiling, we subsequently evaluated its ability to influence NPC cells and associated mechanism. METHODS: The expressions of miR-185 and HOXC6 in NPC and paracancerous tissues collected from patients with NPC were detected. The CNE-2 cells with the lowest miR-185 among the five NPC cell lines (CNE-1, CNE-2, HNE-1, HNE-2, and 5-8F) were selected and transfected with a series of mimic or inhibitor of miR-185, or shRNA-against HOXC6. The Kaplan-Meier method was used to analyze the survival of patients. Besides, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to determine the levels of related genes/proteins. By means of cell counting kit-8 (CCK-8) assay, transwell assay, flow cytometry, and AO staining, the influences miR-185 has on the processes associated with NPC, including cell proliferation, invasion, apoptosis and autophagy were evaluated. RESULTS: NPC was observed to decrease miR-185 but increase HOXC6. Dual luciferase reporter gene assay demonstrated that HOXC6 is a target gene of miR-185. Increased mRNA and protein levels of Bax, caspase-3, LC3 and Beclin1 and reduced levels of HOXC6, TGF-ß1, mTOR, Cyclin D1, PCNA, Bcl-2 were found by overexpression of miR-185. High expression of miR-185 and low expression of HOXC6 had longer survival time of NPC patients. Overexpressed miR-185 enhanced cell apoptosis and autophagy, and reduced cell proliferation and invasion, while miR-185 inhibitor was observed to have induced effects on the CNE-2 cells. CONCLUSION: Overall, the data show that miR-185 could negatively target HOXC6 to suppress cell proliferation, promotes apoptosis and autophagy through inhibiting TGF-ß1/mTOR axis in NPC. Thus, miR-185 is useful strategy for the treatment of NPC.

Effects of diosmetin on nine cytochrome P450 isoforms, UGTs and three drug transporters in vitro.

Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), a natural flavonoid from traditional Chinese herbs, has been used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic and anti-inflammatory activity. However, flavonoids could affect the metabolic enzymes and cause drug-drug interactions (DDI), reducing the efficacy of co-administered drugs and potentially resulting in serious adverse reactions. To evaluate its potential to interact with co-administered drugs, the IC50 value of phase I cytochrome P450 enzymes (CYPs), phase II UDP-glucuronyltransferases (UGTs) and hepatic uptake transporters (organic cation transporters (OCTs), organic anion transporter polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptides (NTCPs)) were examined in vitro by LC-MS/MS. Diosmetin showed strong inhibition of CYP1A2 in a concentration-dependent manner. The intensity of the inhibitory effect was followed by CYP2C8, CYP2C9, CYP2C19 and CYP2E1. For CYP2A6, CYP2B6, CYP2D6 and CYP3A4, diosmetin was found to have no significant inhibitory effects, and the induction effect on CYPs was not significant. For UGTs, diosmetin had a minimal inhibitory effect. In addition, the inhibitory effects of diosmetin on OATP and OCT1 were weak, and it had little effect on NTCP. This finding indicated that drug-drug interactions induced by diosmetin may occur through co-administration of drugs metabolized by CYP1A2.

Cardioprotective effects of timosaponin B II from Anemarrhenae asphodeloides Bge on isoproterenol-induced myocardial infarction in rats.

The aim of the present study was to investigate the cardioprotective effects of Timosaponin B II (TB), a main bioactive constituent from Anemarrhenae asphodeloides Bge, on an isoproterenol (ISO)-induced myocardial infarction model in rats and explore its underlying mechanisms. Rats were treated with TB (50 mg/kg, 100 mg/kg) or diltiazem hydrochloride (DH, 5 mg/kg) by gastric gavage for five days. At the 4th and 5th days, myocardial injury was induced by ISO injection (85 mg/kg) at an interval of 24 h for 2 consecutive days. After the induction, rats were anaesthetized with pentobarbital sodium (30 mg/kg) to record the electrocardiogram. Our research showed that ISO administration resulted in significant elevations in the ST-segment, the levels of cardiac injury biomarkers creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), and concentrations of serum proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Pretreatment with TB significantly reversed these alterations induced by ISO challenge. The cardioprotective effects of TB were further proved by the histopathological examination. Exploration of the underlying mechanisms of its actions revealed that TB pretreatment restored the ISO-induced decrease of super oxide dismutase (SOD) and the increase of malondialdehyde (MDA). Meanwhile, we found that the enhancement of antioxidant defense system might be associated with the increased heme oxygenase isoform 1 (HO-1) induction and activated nuclear respiratory factor 2 (Nrf-2) translocation. Furthermore, the present research also demonstrated that nuclear translocation of Nrf-2 and subsequent HO-1 expression might be associated with nuclear factor kappa B (NF-κB) pathway activation. Taken together, our finding demonstrated that TB might have a potential benefit in preventing ischemic heart diseases like myocardial infarction.

Thymol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in mice.

Thymol, a bioactive monoterpene isolated from Thymus vulgaris, has displayed inspiring neuroprotective properties. The present study was designed to evaluate the antidepressant-like effects of thymol on a chronic unpredictable mild stress (CUMS) model of depression in mice and explore the underlying mechanisms. It was observed that thymol treatment (15 mg/kg and 30 mg/kg) significantly reversed the decrease of sucrose consumption, the loss of body weight, the reduction of immobile time in the tail suspension tests (TST) and forced swimming tests (FST) induced by CUMS paradigm. The levels of norepinephrine (NE) and serotonin (5-HT) in the hippocampus decreased in the CUMS-treated mice. Chronic treatments with thymol significantly restored the CUMS-induced alterations of monoamine neurotransmitters in the hippocampus. Our results further demonstrated that thymol administration negatively regulated the induction of proinflammatory cytokines including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α in CUMS mice. Furthermore, thymol inhibited the activation of nod-like receptor protein 3 (NLRP3) inflammasome and its adaptor, and subsequently decreased the expression of caspase-1. In sum, our findings suggested that thymol played a potential antidepressant role in CUMS mice model through up-regulating the levels of central neurotransmitters and inhibiting the expressions of proinflammatory cytokines, which might provide potential for thymol in the light of opening up new therapeutic avenues for depression.