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Children with autism spectrum disorders (ASD) may not develop symbolic play skills, so such skills need to be taught specifically. We report an experiment regarding a procedure targeting “object-substitution” symbolic play skills. The “object-substitution” symbolic play behavior occurred when the child labeled a common object with the name of a substitute and used the object to perform a play action (e.g., As she put a bowl on her head, she called it a hat). A multiple probe across behaviors design was employed with five children (four boys and one girl, aged 3 to 6 years) with ASD. All children had verbal communication and demonstrated functional play and generalized imitation, but no symbolic play skills prior to the study. The instruction consisted of intraverbal training, picture prompts, and modeling of play actions. All children demonstrated object-substitution symbolic play skills after the instruction. The occurrences of response generalization were also discussed.

Atherosclerosis (AS) is a progressive disease of multifactorial origin, which occurs in response to endothelial injury. Increased homocysteine (Hcy) is considered a major cause of endothelial dysfunction, oxidative stress and DNA methylation; however, the mechanisms remain to be fully elucidated. The aim of the present study was to investigate whether Hcy causes injury to endothelial cells (ECs) by the effect of lectin‑like oxidized‑low density lipoprotein receptor‑1 (LOX‑1) DNA methylation through toll‑like receptor 4(TLR4)/nuclear factor (NF)‑κB/DNA methyltransferase (DNMT)1. The ECs were treated with different concentrations of Hcy, and it was found that Hcy promoted the expression of TLR4, leading to EC injury. The effect of oxidative stress was analyzed by measuring superoxide dismutase, malondialdehyde and hydrogen peroxide in the ECs. In addition, the association between NF‑κB and DNMT1 was examined by treatment of the ECs with pyrrolidine dithiocarbamate (PDTC). The results suggested that Hcy induced LOX‑1 DNA hypomethyaltion to promote the expression levels of LOX‑1. Taken together, Hcy injured the ECs through the effect of methylation and trans‑sulfuration metabolism of LOX‑1 through TLR4/NF‑κB/DNMT1. Following injury to the ECs, lipids, particularly ox‑LDL, accumulated in the sub‑endothelial layer to promote the formation of AS.

Gentiopicroside (Gent) is promising as an important protective secoiridoid compound against pain. The present study was designed to investigate whether administration of Gent would alleviate the expression of nociceptive behaviors and whether it would cause the relevant electrophysiological changes in a chronic constriction injury (CCI) model of neuropathic pain in mice. Gent was administered from the seventh day after surgery for 8 consecutive days. Behavioral parameters and sciatic functional index were assessed immediately before surgery and on days 7, 8, 10, 12, and 14 post-CCI, and electrophysiological activities of sciatic nerve were recorded immediately after the behavioral test on the last day. The present study has shown that administration of Gent (at a dose of 50 and 100 mg/kg) increased behavioral parameters from day 8 compared with the CCI-NS group. Electrophysiological data indicated that CCI caused a significant reduction in nerve conduction velocities in the sciatic nerves and the amplitudes of compound action potential, while Gent at a dose of 50 or 100 mg/kg caused a significant recovery of electrophysiological changes induced by CCI. Our data indicated that Gent has antinociceptive effects on neuropathic pain induced by CCI.

Aloperine (ALO), one of the alkaloids isolated from Sophora alopecuroides L., is traditionally used for various diseases including neuronal disorders. This study investigated the protective effects of ALO on neonatal rat primary-cultured hippocampal neurons injured by oxygen–glucose deprivation and reperfusion (OGD/RP). Treatment with ALO (25, 50, and 100 mg/l) attenuated neuronal damage (p

Oxysophocarpine is an alkaloid extracted from Sophora alopecuroides. We investigated the analgesic effect of oxysophocarpine on carrageenan-induced inflammatory pain in mice, in order to explore its possible mechanisms. Mouse ear swelling tests and carrageenan-induced paw edema tests were used to investigate the effects of oxysophocarpine on inflammatory pain in mice. Morphological changes on inflamed paw sections were measured by hematoxylin-eosin staining. The mRNA and protein expression of extracellular signal-regulated kinase, phosphorylation of extracellular signal-regulated kinase 1/2, cyclooxygenase-2, tumor necrosis factor α, interleukin-1 beta, interleukin-6 and prostaglandin E2 were investigated by real-time quantitative polymerase chain reaction, immunohistochemistry, western-blot and enzyme-linked immunosorbent assay. In our results, oxysophocarpine shows a significant anti-inflammatory effect in the mouse ear swelling test. Oxysophocarpine also significantly reduced the paw edema volume and improved mechanical allodynia threshold value on carrageenan-induced inflammatory pain, as well as relieved paw tissues inflammatory damage and reduced the numbers of neutrophils in mice. Oxysophocarpine significantly suppressed over-expression of cyclooxygenase-2, tumor necrosis factor α, interleukin-1 beta, interleukin-6 and prostaglandin E2, and inhibited the over-phosphorylation of extracellular signal-regulated kinase 1/2. Based on these findings we propose that oxysophocarpine attenuates inflammatory pain by suppressing the levels of phosphorylation of extracellular signal-regulated kinase 1/2, cyclooxygenase-2, prostaglandin E2, tumor necrosis factor α, interleukin-1 beta and interleukin-6.

Oxysophoridine (OSR) is a major active alkaloid extracted from Sophoraalopecuroides L. The aim of the present study was to investigate the induction of the apoptotic effects of OSR on colorectal cancer cells in vivo and in vitro. The results of the MTT and colony formation assays demonstrated that the proliferation of HCT116 cells was inhibited by OSR in vitro. The characteristics of cellular apoptosis in OSR-treated HCT116 cells were analyzed by Hoechst 33258 staining. It was also observed that the expression of caspase-3, B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) and cytochrome c increased significantly upon OSR treatment. However, the expression of Bcl-2 and poly ADP-ribose polymerase-1 (PARP-1) was downregulated in OSR-treated cells compared with untreated cells. The in vivo experiments identified that OSR significantly inhibited the growth of the transplanted mouse CT26 tumor tissue, upregulated the expression of caspase-3, Bax and cytochrome c and downregulated the expression of Bcl-2 and PARP-1, as detected by reverse transcription-quantitative polymerase chain reaction and western blotting. It may be concluded that OSR significantly induced apoptotic effects on colorectal cancer cells in vivo and in vitro, and that its mechanism may be associated with the Bcl-2/Bax/caspase-3 signaling pathway.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in atherosclerosis (AS), and homocysteine (Hcy) is an independent risk factor for AS. However, the underlying mechanisms are still lagging. Studies have used the combination of methylation of promoters of multiple genes to diagnose tumors, thus the aim of the current study was to investigate the role of methylation status of several genes in VSMCs treated with Hcy. CpG islands were identified in the promoters of platelet‑derived growth factor (PDGF), p53, phosphatase and tensin homologue on chromosome 10 (PTEN) and mitofusin 2 (MFN2). Hypomethylation was observed to occur in the promoter region of PDGF, hypermethylation in p53, PTEN and MFN2, and hypomethylation in two global methylation indicators, aluminium (Alu) and long interspersed nucleotide element‑1 (Line‑1). This was accompanied by an increase in the expression of PDGF, and reductions of p53, PTEN and MFN2, both in mRNA and protein levels. An elevation of S‑adenosylmethionine (SAM) and a reduction of S‑adenosylhomocysteine (SAH) and the SAM/SAH ratio were also identified. In conclusion, Hcy impacted methylation the of AS‑associated genes and global methylation status that mediate the cell proliferation, which may be a character of VSMCs treated with Hcy. The data provided evidence for mechanisms of VSMCs proliferation in AS induced by Hcy and may provide a new perspective for AS induced by Hcy.

Lanthanum, a rare earth element, has been used to decrease serum phosphorus level in patients with chronic renal disease and hyperphosphatemia. We aimed to observe the effect and mechanism of two doses of lanthanum acetate (375 and 750 mg/kg/day) on vascular calcification induced by vitamin D3 plus nicotine treatment in rats for 4 weeks. As compared with control rats, rats with calcification showed widespread calcified nodules and irregular elastic fibers in calcified aorta on von Kossa calcium staining and increased aortic calcium and phosphorus contents, alkaline phosphatase (ALP) activity and bone-related protein expressions for osteopontin (OPN) and type III sodium dependent phosphate cotransporter Pit-1 (Pit-1). After treatment with either dose of lanthanum acetate, the calcified nodules and degree of irregular elastic fibers decreased in aortas. Lanthanum acetate at 750 mg/kg/day was more effective than 375 mg/kg/day in lessening vascular calcification by significantly reducing plasma phosphorus level, calcium × phosphorus product and ALP activity, by 30.3%, 28.6%, and 68.6%, respectively; reducing aortic phosphorus and calcium contents and ALP activity, by 48%, 53.1%, and 63.5% (all P < 0.01), respectively; reducing aortic mRNA level of OPN and Pit-1, by 55.8% ( P < 0.01) and 38.8% ( P < 0.05) and protein level of OPN and Pit-1, by 37.2% and 27.2% (both P < 0.01), respectively; and increasing carboxylated matrix Gla-protein (MGP) protein expression by 33.7% ( P < 0.05), as compared with rats treated with vitamin D3 and nicotine alone. Lanthanum acetate could effectively inhibit the pathogenesis of vascular calcification.

The purpose of this article is to research on whether MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma (BUC). In this study, the expression of MACC1 gene was knocked down by RNA interference (RNAi) in the T24 cell (human BUC cell). The transcription level of MACC1 was detected by RT-PCR. Activities of MACC1, caspase-3, caspase-8, Bax and Met (mesenchymal-epithelial transition factor) protein were measured by Western blot. The cell proliferation and apoptosis were detected by MTT and flow cytometry. The cell’s invasion ability was performed on Matrigel transwell assay. We also detect MMP2 (metalloproteinase-2) proteins by ELISA. The results showed that the level of MACC1 mRNA and protein was significantly reduced after RNAi. MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference. Apoptosis studies also showed that MACC1 gene interference in T24 loses its anti-apoptotic effects. The expression of apoptosis proteins (Caspase-3, Caspase-8 and Bax) increased significantly due to the MACC1 RNAi. The level of Met protein was down-regulated obviously due to RNAi. Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to MACC1 RNAi. Further studies showed that the secretion of MMP-2 was reduced by RNAi. It can conclude that the ability of proliferation and invasion in T24 cells can be inhibited by RNAi-targeting MACC1. As a result, MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma.

Objective To investigate whether aloperine (ALO) has antinociceptive effects on neuropathic pain induced by chronic constriction injury, whether ALO reduces ROS against neuropathic pain, and what are the mechanisms involved in ALO attenuated neuropathic pain. Methods Mechanical and cold allodynia, thermal and mechanical hyperalgesia and spinal thermal hyperalgesia were estimated by behavior methods such as Von Frey filaments, cold-plate, radiant heat, paw pressure and tail immersion on one day before surgery and days 7, 8, 10, 12 and 14 after surgery, respectively. In addition, T-AOC, GSH-PX, T-AOC and MDA in the spinal cord (L4/5) were measured to evaluate anti-oxidation activity of ALO on neuropathic pain. Expressions of NF-κB and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in the spinal cord (L4/5) were analyzed by using Western blot. Results Administration of ALO (80 mg/kg and 40 mg/kg, i.p.) significantly increased paw withdrawal threshold, paw pressure, paw withdrawal latencies, tail-curling latencies, T-AOC, GSH-PX and T-SOD concentration, reduced the numbers of paw lifts and MDA concentration compared to CCI group. ALO attenuated CCI induced up-regulation of expressions of NF-κB, TNF-α, IL-6, IL-1β at the dose of 80 mg/kg (i.p.). Pregabalin produced similar effects serving as positive control at the dose of 10 mg/kg (i.p.). Conclusion ALO has antinociceptive effects on neuropathic pain induced by CCI. The antinociceptive effects of ALO against neuropathic pain is related to reduction of ROS, via suppression of NF-κB pathway.

Oxysophocarpine (OSC), a quinolizidine alkaloid extracted from leguminous plants of the genus Robinia, is traditionally used for various diseases including neuronal disorders.This study investigated the protective effects of OSC on neonatal rat primary-cultured hippocampal neurons were injured by oxygen-glucose deprivation and reperfusion (OGD/RP).Cultured hippocampal neurons were exposed to OGD for 2 h followed by a 24 h RP. OSC (1, 2, and 5 μmol/L) and nimodipine (Nim) (12 μmol/L) were added to the culture after OGD but before RP. The cultures of the control group were not exposed to OGD/RP. MTT and LDH assay were used to evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca(2+)]i and mitochondrial membrane potential (MMP) were determined to evaluate the degree of neuronal damage. Morphologic changes of neurons following OGD/RP were observed with a microscope. The expression of caspase-3 and caspase-12 mRNA was examined by real-time quantitative PCR.The IC50 of OSC was found to be 100 μmol/L. Treatment with OSC (1, 2, and 5 μmol/L) attenuated neuronal damage (p 0.001), with evidence of increased cell viability (p 0.001) and decreased cell morphologic impairment. Furthermore, OSC increased MMP (p 0.001), but it inhibited [Ca(2+)]i (p 0.001) elevation in a dose-dependent manner at OGD/RP. OSC (5 μmol/L) also decreased the expression of caspase-3 (p 0.05) and caspase-12 (p 0.05).The results suggested that OSC has significant neuroprotective effects that can be attributed to inhibiting endoplasmic reticulum (ER) stress-induced apoptosis.

Aims of the Study To investigate the neuroprotective effect of Lycium barbarum polysaccharide (LBP) on focal cerebral ischemic injury in mice and to explore its possible mechanism. Materials and Methods Male ICR mice were used to make the model of middle cerebral artery occlusion (MCAO) after intragastric administration with LBP (10, 20 and 40 mg/kg) and Nimodipine (0.4 mg/kg) for seven successive days. After 24 h of reperfusion, neurological scores were estimated and infarct volumes were measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Morphological changes in ischemic brains were performed for hematoxylin-eosin (HE) staining. The number of apoptotic neurons was detected by TUNEL staining. The Bax, Bcl-2 protein expression and CytC, Caspase-3, -9 and cleaved PARP-1 activation were investigated by immunofluorescence and western-blot analysis. Results LBP (10, 20 and 40 mg/kg) treatment groups significantly reduced infract volume and neurological deficit scores. LBP also relieved neuronal morphological damage and attenuated the neuronal apoptosis. LBP at the dose of 40 mg/kg significantly suppressed overexpression of Bax, CytC, Caspase-3, -9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. Conclusions Based on these findings we propose that LBP protects against focal cerebral ischemic injury by attenuating the mitochondrial apoptosis pathway.

Our previous studies have demonstrated that oxysophoridine (OSR) has protective effects on cerebral neurons damage in vitro induced by oxygen and glucose deprivation. In this study, we further investigated whether OSR could reduce ischemic cerebral injury in vivo and its possible mechanism. Male Institute of cancer research mice were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days, then subjected to brain ischemia induced by the model of middle cerebral artery occlusion. After reperfusion, neurological scores and infarct volume were estimated. Morphological examination of tissues was performed. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Oxidative stress levels were assessed by measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. The expression of various apoptotic markers as Caspase-3, Bax and Bcl-2 were investigated by immunohistochemistry and Western-blot analysis. OSR pretreatment groups significantly reduced infract volume and neurological deficit scores. OSR decreased the percentage of apoptotic neurons, relieved neuronal morphological damage. Moreover, OSR markedly decreased MDA content, and increased SOD, GSH-Px activities. Administration of OSR (250 mg/kg) significantly suppressed overexpression of Caspase-3 and Bax, and increased Bcl-2 expression. These findings indicate that OSR has a protective effect on focal cerebral ischemic injury through antioxidant and anti-apoptotic mechanisms.

Escherichia coli is a common cause of community‑ and hospital‑acquired urinary tract infections, and class 1 integrons are the prior elements of gene transference in the capture and distribution of gene cassettes among clinical gram-negative bacillus. In the present study, the resistance of Escherichia coli to antimicrobial agents was investigated. A total of 97 isolates were found to be susceptible to 16 antimicrobial agents and were detected in the production of extended β‑lactamases (ESBLs), distribution of CTX‑M‑type β‑lactamases, presence and characterization of class 1 integrons and a variable region of integron‑positive isolates. Escherichia coli isolates possessing CTX‑M (31; 32%) were detected in 19 isolates (61.5%). The presence of ESBLs was associated with resistance to penicillins, third-generation cephalosporins, ciprofloxacin, aminoglycosides and monocyclic β‑lactam antibiotics. Escherichia coli isolates (69; 71.1%) possessed class 1 integrons associated with resistance to ciprofloxacin and numerous third-generation cephalosporins, penicillins, tobramycin and trimethoprim‑sulfamethoxazole. The four gene cassette arrangements were as follows: dfrA17‑aadA5, aadA1, aacC4‑cmlA1 and dfr2d, and 8 carried two disparate class 1 integrons. Five isolates presented class 1 integrons containing no gene cassettes. The distribution of ESBLs and class 1 integrons in Escherichia coli were prevalent with drug resistance in Chengdu. In addition, the resistance range of Escherichia coli isolates that harboured ESBLs and carried class 1 integrons were similar. The current study demonstrated the presence of class 1 integrons and ESBLs, which jointly mediate the resistance of Escherichia coli isolates to a number of antibacterial agents.

Both tyrosine kinase and topoisomerase II (TopII) are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK) detection kit using a horseradish peroxidase (HRP)-conjugated phosphotyrosine (pY20) antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P

Oxymatrine-carbenoxolone sodium inclusion compound (OCSIC) has been reported as a novel painkiller, but its effectiveness and mechanism remain unknown. This study investigated the analgesic effects of OCSIC and the possible alterations in the expression of gamma-aminobutyric acid type A alpha1 (GABA(A)alpha1) receptors in the central nervous system caused by the compound. The antinociceptive action of the OCSIC was assessed in thermal and chemical behavioral models of nociception. The hot-plate test, tail immersion test, acetic acid-induced abdominal constriction and formalin-induced pain were used in ICR mice. OCSIC was administered intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.). In all cases, OCSIC produced significant dose-dependent antinociceptive effects. In addition, OCSIC (150 mg/kg) increased the expression of positive staining cells of GABA(A)alpha1 receptors. These results reveal that the antinociceptive effects of OCSIC may be involved in the central nervous system and the peripheral nervous system. The involvement of GABA(A) receptors in the antinociceptive effect of OCSIC is now under investigation.

To study the analgesic effects and sites of oxymatrine-carbenoxolone sodium complex (OCSC).Adopting formalin test, warm water tail-flick test and intracerebroventricularly (icv) injection to observe the analgesic effects of OCSC in mice.Intraperitoneally injecting (ip) OCSC (75, 150 mg x kg(-1)) remarkedly inhibited the pain of mice in the formalin test and prolonged latent phases of tail-shrinking of mice, icy OCSC (1.875, 3.75, 7.5 mg x kg(-1)) significantly prolonged latent phases of tail-shrinking of mice, it had dose-dependent effect with concentration.The result indicated that OCSC has obvious analgesic effects and its mechanism may be involved in central nervous system (CNS).

To study the action of pseudoephedrine salicylate on the central nervous system of mice.Following the administration of pseudoephedrine salicylate at various dose levels and schedules, the alterations in general behavior and autonomic activities of the mice were recorded using photoelectric counter Type GJ-1, the changes in hypnotic action indices (sleep latency, sleep-lasting time) for the mice placed on pentobarbital in advance were observed, and the muscular clonic spasm of forefoot and neck muscles as well as the deaths from convulsions induced by previously given pentylenetrazol, nicotin and picrotoxin were observed and recorded respectively.Pseudoephedrine salicylate (12.5, 25.0, 50.0, 100.0 mg/kg) administrated ip significantly inhibited the autonomic activities and the hypnotic action induced by previously given pentobarbital. Pseudoephedrine salicylate (100.0 mg/kg) given ip was noted to have synergistic effect on convulsions of the mice previously given pentylenetrazol and picrotoxin at subthreshold doses respectively, but no synergistic effect of pseudoephedrine salicylate (100 mg/kg) and nicotin (at subthreshold dose 0.018 mg/kg) was observed.Pseudoephedrine salicylate has excited effect on the central nervous system of mice.

Background: Our previous studies demonstrated that oxysophoridine (OSR) had neuroprotective effects on mice through antioxidant and anti-apoptotic mechanisms. In this study, we investigated whether OSR could influence the release of amino acids in ischemic mice brains. Materials and Methods: Male ICR mice were scheduled to undergo 2 h middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Before MCAO, mice in corresponding groups were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days. After reperfusion, neurological scores were estimated, infarct volume and the brain water content were assessed. The levels of glutamate (Glu), aspartate (Asp), γ-aminobutyric acid (GABA) and Glycine (Gly) were measured by amino acid analyzer. Results: OSR significantly decreased neurological scores, reduced infarct volume and the brain water content. After treatment with OSR of 250 mg/kg, the contents of Glu, Asp, GABA and Gly in mice brains could maintain at a normal level compared with MCAO group mice. The Glu/GABA ratio was significantly decreased in OSR group mice. Conclusion: These findings indicate that OSR has a protective effect on cerebral ischemic injury and helps to maintain the amino acids homeostasis after reperfusion for a long time.