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Due to the rising abuse of ketamine usage in recent years, ketamine-induced urinary tract syndrome has received increasing attention. The present study aimed to investigate the molecular mechanism underlying ketamine-associated cystitis in a mouse model. Female C57BL/6 mice were randomly divided into two groups: One group was treated with ketamine (100 mg/kg/day of ketamine for 20 weeks), whereas, the control group was treated with saline solution. In each group, micturition frequency and urine volume were examined to assess urinary voiding functions. Mouse bladders were extracted and samples were examined for pathological and morphological alterations using hematoxylin and eosin staining, Masson's trichrome staining and scanning electron microscopy. A cDNA microarray was conducted to investigate the differentially expressed genes following treatment with ketamine. The results suggested that bladder hyperactivity increased in the mice treated with ketamine. Furthermore, treatment with ketamine resulted in a smooth apical epithelial surface, subepithelial vascular congestion and lymphoplasmacytic aggregation. Microarray analysis identified a number of genes involved in extracellular matrix accumulation, which is associated with connective tissue fibrosis progression, and in calcium signaling regulation, that was associated with urinary bladder smooth muscle contraction. Collectively, the present results suggested that these differentially expressed genes may serve critical roles in ketamine-induced alterations of micturition patterns and urothelial pathogenesis. Furthermore, the present findings may provide a theoretical basis for the development of effective therapies to treat ketamine-induced urinary tract syndrome. [ABSTRACT FROM AUTHOR]

The aberrant proliferation of nucleus pulposus (NP) cells has been reported to be implicated in the pathogenesis of intervertebral disc degeneration (IDD). Previous studies have demonstrated that microRNAs (miRNAs), which are a group of small noncoding RNAs, are critical regulators of cell proliferation in various pathologies. However, the role of miRNA‑96 (miR‑96) in the proliferation of NP cells remains to be determined. In the present study, reverse transcription‑quantitative polymerase chain reaction was used to investigate the expression of miR‑96 in NP tissues from patients with IDD and healthy tissues from patients with traumatic lumbar fracture as the control. A dual‑luciferase reporter assay was used to investigate whether AT‑rich interaction domain 2 (ARID2) may be a direct target gene for miR‑96. Furthermore, isolated NP cells from patients with IDD were transfected with miR‑96 mimics and ARID2‑targeting small interfering RNAs; cell proliferation, and the protein expression of Akt, phosphorylated Akt and ARID2 were examined, whereas the effects of an Akt inhibitor on NP cell proliferation were also evaluated. The present results demonstrated that miR‑96 expression was significantly upregulated in IDD samples, and the level of miR‑96 expression was positively associated with disc degeneration grade, which was evaluated by a modified Pfirrmann grading system. In addition, the current study identified ARID2 as a direct gene target of miR‑96. Furthermore, it was demonstrated that ARID2 mRNA expression was inversely correlated with the expression of miR‑96 in NP tissues. In addition, miR‑96 overexpression promoted NP cell proliferation and induced Akt phosphorylation, which led to increased cyclin D1 translation. Notably, overexpression of ARID2 or treatment with an Akt inhibitor decreased the effect of miR‑96 on NP cell proliferation. In conclusion, the results of the present study indicate that miR‑96 may promote the proliferation of human degenerated NP cells by targeting ARID2 via activation of the Akt pathway, and potentially serves as a therapeutic target for IDD. [ABSTRACT FROM AUTHOR]

A strategy to suppress the expression of the DNA repair enzyme O6‑methylguanine‑DNA methyltransferase (MGMT) by inhibition of Wnt/β‑catenin signaling may be useful as a novel treatment for pituitary adenoma. Previous studies have reported that Tanshinone IIA (TSA), a major quinone compound isolated from Salvia miltiorrhiza, had antitumor effects. However, whether TSA has antitumor effects against pituitary adenoma and whether the mechanisms are associated with the Wnt/β‑catenin/MGMT pathway remains to be clarified. In the present study, TSA treatment caused apoptosis in AtT‑20 cells in a concentration‑dependent manner, as demonstrated by cell viability reduction, phophatidylserine externalization detected by Annexin V staining and mitochondrial membrane potential disruption detected by JC‑1 staining, which were associated with activation of caspase‑3 and DNA fragmentation detected by TUNEL in AtT‑20 cells. T‑cell factor (TCF)‑lymphoid‑enhancing factor (LEF) reporter activity was determined by dual luciferase reporter assay and the interaction between β‑catenin and TCF‑4 were detected using a co‑immunoprecipitation kit. The results indicated TSA treatment increased β‑catenin phosphorylation, inhibited β‑catenin nuclear translocation, reduced β‑catenin/TCF‑4 complex formation and TCF‑LEF luciferase reporter activity, and subsequently reduced the expression of cyclin D1 and MGMT. Notably, overexpression of MGMT in β‑catenin knock down AtT‑20 cells abrogated the TSA‑mediated effects in AtT‑20 cells. In conclusion, TSA induced apoptosis via inhibition of Wnt/β‑catenin‑dependent MGMT expression, which may provide novel insights into the understanding of the mechanism of the antitumor effects of Salvia miltiorrhiza. [ABSTRACT FROM AUTHOR]

Retinoic acid inducible gene I (RIG‑I) is upregulated during all‑trans retinoic acid (ATRA)‑induced terminal granulocytic differentiation of NB4 acute promyelocytic leukemia (APL) cells. However, the function and mechanism of RIG‑I in NB4 cells remains to be fully elucidated. In the present study, lentivirus‑mediated RIG‑I‑knockdown was used to investigate the proliferation, cell cycle and apoptotic processes of ATRA‑induced NB4 cells in vitro using an MTT assay and flow cytometry, respectively. The roles of RIG‑I and the AKT‑FOXO3A signaling pathway were investigated using western blot analysis. The results showed that the ATRA‑induced expression of RIG‑I was specifically and effectively knocked down at the mRNA and protein levels by lentivirus mediated RIG‑I short hairpin RNA. In addition, silencing of RIG‑I reduced the ATRA‑induced inhibition of NB4 cell proliferation, cell cycle arrest and apoptosis. Further investigations indicated that with ATRA‑induced expression of RIG‑I, levels of phosphorylated (p)AKT‑Thr308 and pForkhead Box (FOX) O3A‑Thr32 were decreased, the expression levels of cell cycle arrest protein p27 and the apoptotic protein, tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL), directly transcribed by FOXO3A were increased. By contrast, following the knockdown of ATRA‑induced expression of RIG‑I, the levels of pAKT‑Thr308 and pFOXO3A‑Thr32 were increased, and the protein expression levels of p27 and TRAIL were decreased. Taken together, these results showed that the knockdown of RIG‑I reduced the inhibition of cell proliferation, cell cycle arrest and apoptosis in the ATRA‑induced NB4 cells via the AKT‑FOXO3A signaling pathway. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effect of and mechanism underlying interleukin (IL)-17 on human retinal vascular endothelial cell (HREC) capillary tube formation in vitro. The expression of IL-17 receptor (IL-17R) in human HRECs was quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses. The roles of IL-17 in HREC migration and capillary tube formation were detected using a wound scratching assay and three-dimensional Matrigel assay, respectively, in vitro. HREC proliferation was examined using a cell counting kit-8 assay with administration of serial doses of IL-17. The effects of IL-17 on the expression of vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule (ICAM)-1, IL-6 and IL-8 in HRECs were evaluated using RT-PCR and western blot analyses. The results revealed that the HRECs expressed IL-17R, and the number of intact capillary tubes formed by HRECs in the presence of IL-17 was markedly higher, compared with that in the blank control group. The wound scratching assay showed that the numbers of migrated HRECs stimulated with IL-17 at concentrations of 100 or 500 ng/ml were significantly higher, compared with the number in the control group. The RT-PCR and western blot analyses showed that IL-17 significantly promoted the expression of VEGF, ICAM-1, IL-6 and IL-8 by the HRECs. The proliferation of HRECs in the presence of IL-17 was also significantly increased. Therefore, IL-17 increased HREC capillary tube formation through promoting HREC migration, proliferation, and expression levels of VEGF, ICAM-1, IL-6 and IL-8. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) have important roles in the parthenogenesis of malignancies. While it has been suggested that deregulation of miR-101 is involved in bladder cancer, the underlying mechanisms have remained largely elusive. The present study aimed to investigate the roles of miR-101 in the regulation of bladder cancer cell proliferation and invasion. Reverse-transcription quantitative polymerase chain reaction analysis revealed that the expression of miR-101 was significantly reduced in the HT-1376, BIU87, T24 and 5637 several human bladder cancer cell lines compared to that in the SV-HUC-1 normal bladder epithelial cell line. Furthermore, a Targetscan search and a luciferase assay were used to identify c-FOS as a novel target of miR-101, and western blot analysis indicated that the protein expression of c-FOS was shown to be negatively regulated by miR-101 in bladder cancer T24 cells; however, c-FOS mRNA expression was not affected. In addition, plasmid-mediated overexpression of miR-101 and small hairpin RNA-mediated inhibition of c-FOS significantly inhibited the proliferation and invasive capacity of T24 cells, as indicated by an MTT and a Transwell assay, respectively. However, plasmid-mediated overexpression of c-FOS reversed the inhibitory effects of miR-101 overexpression on T24-cell proliferation and invasion. In conclusion, the present study demonstrated that miR-101 inhibits the proliferation and invasion of bladder cancer cells, at least partly via targeting c-FOS, suggesting that miR-101/c-FOS signaling may represent a potential therapeutic target for bladder cancer. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the expression of sirtuin (Sirt)1 in tendon stem cells (TSCs) and to elucidate its association with osteogenic and adipogenic differentiation of TSCs. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to detect Sirt1 mRNA and protein levels in TSCs, respectively. TSCs were positive for Sirt1 expression, which was elevated by Sirt1 activator SRT1720 in a time- and concentration- dependent manner, and decreased by Sirt1 inhibitor EX527. TSCs were treated with SRT1720 and EX527 for various time periods and resulting changes in osteogenic and adipogenic protein markers were analyzed using alizarin red and oil red O staining. According to RT-qPCR and western blot analyses, the associated factors β-catenin, Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 were elevated following increases of Sirt1 levels, while CCAAT/enhancer binding protein (CEBP)α and peroxisome proliferator-activated receptor (PPAR)γ were decreased. These results suggested that osteogenic differentiation capacity was enhanced, while adipogenic differentiation capacity declined. Further mechanistic study revealed that phosphoinositide-3 kinase (PI3K) and AKT were decreased following activation of Sirt1. In conclusion, the present study suggested that Sirt1 promotes the osteogenic differentiation of TSCs through upregulating β-catenin and Runx2 and inhibits the adipogenic differentiation of TSCs through the PI3K/AKT pathway with downregulation of CEBPα and PPARγ. [ABSTRACT FROM AUTHOR]

[Sar9, Met(O2)11] termed Substance P (SP), is an effective and selective agonist for the neurokinin-1 (NK-1) receptors, which are synthetic peptides, similar in structure to SP. SP is an important neurotransmitter or neuromodulator mediated by neurokinin receptors, namely the SP receptor in the central nervous system. The excitatory effects induced by SP may be selectively inhibited by a neurokinin-1 receptor antagonist, such as SR140333B. It has been proposed that Parkinson's disease (PD) is primarily caused by the loss of trophic peptidergic neurotransmitter, possibly SP, which may lead to the degeneration of neurons. In previous studies, 1-methyl-4-phenylpyridinium (MPP+) has been frequently utilized to establish animal or cell models of PD. In the present study, to further investigate the effects of SP in PD, MPP+ was employed to investigate the promising anti-apoptotic effects of SP, and examine the underlying mechanisms of the pathology in the MES23.5 dopaminergic cell line. The results indicated that MPP+-triggered apoptosis was prevented by treatment with SP. SP treatment also decreased the MPP+-triggered Ca2+ influx, caspase-3 re-activity, reactive oxygen species production and mitochondrial membrane potential decrease. Treatment with MPP+ also induced phosphorylation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. In addition, treatment with SP inhibited the MPP+-triggered neurotoxicity in MES23.5 cells. However, no changes were observed in SR140333B+SP+MPP+-treated MES23.5 cell lines. In conclusion, SP could protect the cells from MPP+-induced cytotoxicity by inhibiting the apoptosis via NK-1 receptors. [ABSTRACT FROM AUTHOR]

Inflammation and immunity are important in the pathogenesis of cerebral ischemia. Toll-like receptor 4 (TLR4) is involved in the inflammatory responses of injured brain tissues. Emerging studies have focused on the effect of isoflurane (ISO) pretreatment on cerebral ischemia, however, the association between ISO pretreatment and TLR4 during cerebral ischemia remains to be elucidated. In the present study, the protective role of ISO pretreatment in rats with focal cerebral ischemia reperfusion was investigated and the molecular mechanism was discussed. Using a middle cerebral artery occlusion (MCAO) model, triphenyltetrazolium chloride staining was utilized to measure the infarct volume and brain edema and immunofluorescence staining was used to detect the MCAO-induced TLR4 expression and localization. Western blot analyses were conducted to quantify the protein expression levels of TLR4, myeloid differentiation primary response 88 (MyD88) and nuclear factor (NF)- κB in ischemic brain tissue at different time points. The results demonstrated that, following ISO pretreatment, the neurological deficits, brain edema and cerebral infarct size caused by ischemia/reperfusion were attenuated. The astrocyte and microglial activation in the brain tissue was decreased. In addition, the expression levels of TLR4, MyD88 and NF-κB were decreased. The present study indicated that ISO pretreatment may protect the brain from ischemic damage by downregulating the expression levels of TLR4, MyD88 and NF-κB. [ABSTRACT FROM AUTHOR]

The poor therapeutic effect of traditional antiangiogenic therapy on glioblastoma multiforme (GBM) may be attributed to vasculogenic mimicry (VM), which was previously reported to be promoted by cancer stem-like cells (SLCs). All-trans retinoic acid (ATRA), a potent reagent which drives differentiation, was reported to be able to eradicate cancer SLCs in certain malignancies. The aim of the present study was to investigate the effects of ATRA on the VM formation ability of U87 glioblastoma SLCs. The expression of cancer SLC markers CD133 and nestin was detected using immunocytochemistry in order to identify U87 SLCs. In addition, the differentiation of these SLCs was observed through detecting the expression of glial fibrillary acidic protein (GFAP), β-tubulin III and galactosylceramidase (Galc) using immunofluorescent staining. The results showed that the expression levels of GFAP, β-tubulin III and Galc were upregulated following treatment with ATRA in a dose-dependent manner. Furthermore, ATRA significantly reduced the proliferation, invasiveness, tube formation and vascular endothelial growth factor (VEGF) secretion of U87 SLCs. In conclusion, the VM formation ability of SLCs was found to be negatively correlated with differentiation. These results therefore suggested that ATRA may serve as a promising novel agent for the treatment of GBM due to its role in reducing VM formation. [ABSTRACT FROM AUTHOR]

Mesenchymal stem/stromal cells (MSCs) have a wide application in cell-based therapies and tissue engineering. In the present study, the differentiation, survivin (SVV)-modified effects and molecular basis of human umbilical cord-derived MSCs (HUMSCs) and dental pulp-derived stem cells (DPSCs) were investigated. The HUMSCs were found to differentiate into adipocytes more readily than the DPSCs and the HUMSCs and DPSCs were each able to differentiate into osteoblasts and chondroblasts. Following modification of the MSCs by SVV, the secretion of SVV in the modified HUMSCs was significantly higher compared with that in the modified DPSCs. In vivo, survival of the SVV-modified DPSCs was observed at 4 and 14 days after intrastriatal transplantation, as was the expression of SVV and differentiation into astrocytes. The gene expression profiles of the control and modified HUMSCs and DPSCs were compared using RNA sequencing and an association was observed between gene expression and variability in cell line function. These findings provide novel information regarding the differences between HUMSCs and DPSCs and insight into optimal cell sources for therapeutic applications. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate and compare the anti-inflammatory activities of curcumin and its three metabolites, tetrahydrocurcumin, hexahydrocurcumin and octahydrocurcumin in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results demonstrated that overproduction of nitric oxide (NO) was potently inhibited following treatment with curcumin and its three metabolites. In addition, curcumin and tetrahydrocurcumin significantly inhibited the release of prominent cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); however, hexahydrocurcumin and octahydrocurcumin did not significantly alter cytokine release. Furthermore, the present study investigated the effect of curcumin and its metabolites on the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and activated-nuclear factor kappa B (NF-κB); the results showed that curcumin and its three metabolites significantly inhibited LPS-mediated upregulation of iNOS and COX-2 as well as NF-κB activation. However, curcumin exerted a more potent effect on LPS-stimulated RAW 264.7 cells compared to that of its three metabolites, of which tetrahydrocurcuim was found to be the most pharmacologically active. In conclusion, the results of the present study demonstrated that curcumin and its major metabolites inhibited the LPS-induced inflammatory response via the mechanism of inhibiting NF-κB translocation to the nucleus. [ABSTRACT FROM AUTHOR]

Omentin is a novel adipokine, which is expressed in and released from omental adipose tissue. In the present study, the effect of omentin on neural stem cells (NSCs) was investigated. NSCs are a subtype of stem cell in the nervous system, which are able to self-renew and generate neurons and glia for repairing neural lesions. Mouse NSCs were isolated and cultured in vitro. Treatment with recombinant omentin for 3 and 5 days significantly increased the size of NSC neurospheres (P<0.01) and enhanced NSC cell viability in normal conditions. In addition, omentin protected against the decrease in cell viability induced by the pro-inflammatory cytokine tumor necrosis factor-α. In the NSCs, incubation of omentin for 2, 4, 6, 8 and 16 h enhanced the phosphorylation of Akt at the Thr308 site and of AS160 at the Ser318 site, peaking 6 h after treatment. Additionally, treatment with LY294002 (10 μM), a specific inhibitor of phosphatidylinositol 3-kinase/Akt signaling, eliminated the omentin-induced increase in neurosphere size and cell viability. Overall, the present study provided the first evidence, to the best of our knowledge, that omentin promotes the growth and survival of NSCs in vitro through activation of the Akt signaling pathway. These results may contribute to the understanding of the role of omentin in the nervous system. [ABSTRACT FROM AUTHOR]

Picrasma quassioides (P. quassioides) has been widely used as a traditional Chinese medicine for clearing fever and detoxification. Previous phytochemical studies have identified a novel dihydrobenzofuran type neolignan, picrasmalignan A, isolated from the stems of P. quassioides. In the present study, the in vitro anti inflammatory effects and molecular mechanisms of picrasmalignan A have been investigated in cultured RAW 264.7 cells, a mouse macrophage like cell line. A Griess assay was used to demonstrate the inhibitory effect of picrasmalignan A on the overproduction of nitric oxide (NO). An enzyme linked immunosorbent assay was used to determine the levels of pro inflammatory cytokines, including tumor necrosis factorα (TNFα) and interleukin 6 (IL 6). The inhibitory effect on the enzymatic activity of cyclooxygenase 2 (COX 2) and inducible nitric oxide synthase (iNOS) was observed by colorimetric and fluorimetric assays, respectively. Western blotting was conducted to detect the expression of iNOS and COX 2. Results showed that picrasmalignan A suppressed lipopolysaccharide stimulated NO production and pro inflammatory cytokine secretion, including TNFα and IL 6, in a dose dependent manner. It also significantly inhibited the expression and enzymatic activity of iNOS and COX 2. These results may demonstrate the anti inflammatory mechanism of picrasmalignan A. [ABSTRACT FROM AUTHOR]