Omega-3 Rich Oils Attenuate ADHD-Like Behaviour Induced by Dietary Monosodium Glutamate in Rats.

<b>Background and Objective:</b> Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity and cognitive dysfunction. The present study was designed to examine the possible modulatory effect of Fish, Walnuts or Fenugreek Oils against Attention Deficit Hyperactivity Disorder (ADHD)-like Behavior induced by Monosodium Glutamate (MSG) in Rats. <b>Materials and Methods:</b> Fifty weaning rats were divided into five groups, (each group contain 10 rats) as follows: Group 1: Normal control rats were fed on a balanced diet. Groups from 2-5 rats were fed on a balanced diet+MSG (0.4 g kg¹ diet), Group 2 served as a positive control group whereas group 3, 4 and 5 treated with Fish, Walnuts and Fenugreek oil, respectively, (200 mg kg¹ b.wt.) by intra-gastric tube. Biochemical and behavioural parameters were tested as well as microscopic examination of brain tissue was done. <b>Results:</b> MSG ingestion caused marked disruption in locomotors activity, memory function and brain tissue structure along with significant abnormalities in some bio-markers and reduction in the gene expression level of Bcl-2 in brain tissue. However, treatment with the tested oils showed remarkable effect by reversing the condition. <b>Conclusion:</b> Dietary supplementation with walnut; fenugreek or fish oils at the tested dose could modulate the condition of ADHD in rats.

Palmitate reduces starvation-induced ER stress by inhibiting ER-phagy in hypothalamic cells.

Palmitate is a saturated fatty acid that is well known to induce endoplasmic reticulum (ER) stress and autophagy. A high-fat diet increases the palmitate level in the hypothalamus, the main region of the brain regulating energy metabolism. Interestingly, hypothalamic palmitate level is also increased under starvation, urging the study to distinguish the effects of elevated hypothalamic palmitate level under different nutrient conditions. Herein, we show that ER-phagy (ER-targeted selective autophagy) is required for progress of ER stress and that palmitate decreases ER stress by inhibiting ER-phagy in hypothalamic cells under starvation. Palmitate inhibited starvation-induced ER-phagy by increasing the level of B-cell lymphoma 2 (Bcl-2) protein, which inhibits autophagy initiation. These findings suggest that, unlike the induction of ER stress under nutrient-rich conditions, palmitate protects hypothalamic cells from starvation-induced stress by inhibiting ER-phagy.

Herb pair of Ephedrae Herba-Armeniacae Semen Amarum alleviates airway injury in asthmatic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedrae Herba (EH, Ephedra sinica Stapf.) and Armeniacae Semen Amarum (ASA, Prunus armeniaca L. var. ansu Maxim.) have been used to treat asthma, cold, fever, and cough in China for thousands of years. AIM OF THE STUDY: In this study, we aimed to investigate the optimal ratio of EH and ASA compatibility (EAC) to reduce airway injury in asthmatic rats and its possible mechanism. METHODS: Rats were sensitized with a mixture of acetylcholine chloride and histamine bisphosphate 1 h before sensitization by intragastric administration of EAC or dexamethasone or saline for 7 days. Subsequently, the ultrastructure of rat airway epithelial tissue changes, apoptosis of the airway epithelial cells, and the expression of mRNA and protein of EGRF and Bcl-2 were detected. RESULTS: Transmission electron microscope: EAC (groups C and E) had the most prominent effect on repairing airway epithelial cells' ultrastructural changes in asthmatic rats. TUNEL: dexamethasone and EAC (groups B、C、E and F) inhibited the apoptosis of airway epithelial cells in asthmatic rats (P < 0.05). In situ hybridization: EAC (group E) inhibited the overexpression of EGFR and Bcl-2 mRNA (P < 0.05).Western Blotting: EAC (groups A、B、C、E and F) inhibited the upregulation of airway epithelial EGFR and Bcl-2 protein expression (P < 0.01). CONCLUSIONS: Our findings indicate that EAC can inhibit abnormal changes in airway epithelial structure and apoptosis of airway epithelial cells, thereby alleviating airway injury. In this study, the best combination of EH and ASA to alleviate airway epithelial injury in asthmatic rats was group E (EH: ASA = 8: 4.5).

Naringin Targets NFKB1 to Alleviate Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury in PC12 Cells Via Modulating HIF-1α/AKT/mTOR-Signaling Pathway.

This study was designed to investigate the effect of naringin in oxygen-glucose deprivation/reoxygenation (OGD/R) model and its mechanism. The target gene of naringin and the enriched pathways of the gene were searched and identified using bioinformatics analysis. Then OGD/R model was built using PC12 cells, after which the cells were treated with different concentrations of naringin. Subsequently, cell proliferation and apoptosis were evaluated by cell counting kit-8 (CCK-8) and flow cytometry assays, respectively. Meanwhile, the expression of NFKB1 in PC12 cells underwent OGD/R-induced injury was detected by qRT-PCR, while apoptosis-related and pathway-related proteins were checked by Western blot. DCF-DA kit was utilized to measure the level of ROS. Our results revealed that NFKB1, which was upregulated in MACO rats and OGD/R-treated PC12 cells, was a target gene of naringin. Naringin could alleviate OGD/R-induced injury via promoting the proliferation, and repressing the apoptosis of PC12 cells through regulating the expression of NFKB1 and apoptosis-associated proteins and ROS level. Besides, the depletion of NFKB1 was positive to cell proliferation but negative to cell apoptosis. Moreover, the depletion of NFKB1 enhanced the influences of naringin on cell proliferation and apoptosis as well as the expression of apoptosis-related proteins and ROS level. Western blotting indicated that both naringin treatment and depletion of NFKB1 could increase the expression of HIF-1α, p-AKT, and p-mTOR compared with OGD/R group. What's more, treatment by naringin and si-NFKB1 together could significantly increase these effects. Nevertheless, the expression of AKT and mTOR among each group was almost not changed. In conclusion, naringin could prevent the OGD/R-induced injury in PC12 cells in vitro by targeting NFKB1 and regulating HIF-1α/AKT/mTOR-signaling pathway, which might provide novel ideas for the therapy of cerebral ischemia-reperfusion (I/R) injury.

Evaluation of male infertility treatment following Rhus coriaria extract administration on rats exposed to morphine.

Morphine is the most common analgesic drug that is widely used in post-operative interventions. This drug causes free radical accumulation leading to spermatogenesis failure. Antioxidant agents like Sumach (Rhus coriaria) neutralize cellular free radicals. In this study, the properties of antioxidative, modulative of inflammatory cytokines, and apoptotic genes following Sumach extract administration on morphine-induced fertility destruction in male Wistar rats was evaluated. Sixty-four animals were grouped (n = 8) including; 1: control, 2: morphine, 3-5: Sumach (200, 400, 800 mg/kg), and 6-8: morphine + Sumach. Hydroalcoholic extract of Sumach seeds was prepared. Treatments with Sumach extract were applied orally and intraperitoneally daily for 8 weeks. The P53, Bcl2 and caspase-3 genes expression were measured by real-time PCR. Cytokines involved in inflammation were evaluated by ELISA. Sperm parameters, total antioxidant capacity (TAC), testosterone, and germinal layer height (GLH) were assessed. All parameters (investigated in this study) in Morphine group reduced significantly than the control group (P ˂ 0.01) (except P53 and caspase-3 genes expression and inflammatory cytokine which were improved). All factors in Sumach and Sumach + Morphine groups were significantly enhanced compared to the Morphine group (P ˂ 0.01) (except P53 and caspase-3 genes expression and inflammatory cytokine which were declined). Morphine disrupted the physiological function of male fertility system. Besides, all doses of Sumach showed no therapeutic changes compared to the control group. Sumach with anti-infertility features compensates the toxic effect of Morphine administration.

Rutin and Selenium Co-administration Reverse 3-Nitropropionic Acid-Induced Neurochemical and Molecular Impairments in a Mouse Model of Huntington's Disease.

Systemic administration of 3-nitropropionic acid (3-NPA) is commonly used to induce Huntington's disease (HD)-like symptoms in experimental animals. Here, the potential neuroprotective efficiency of rutin and selenium (RSe) co-administration on 3-NPA-induced HD-like symptoms model in mice was investigated. 3-NPA injection evoked severe alterations in redox status, as indicated via increased striatal malondialdehyde and nitric oxide levels, accompanied by a decrease in levels of antioxidant molecules including glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. Moreover, 3-NPA potentiated inflammatory status by enhancing the production of interleukin-1ß, tumor necrosis factor-α, and myeloperoxidase activity. Pro-apoptotic cascade was also recorded in the striatum as evidenced through upregulation of cleaved caspase-3 and Bax, and downregulation of Bcl-2. 3-NPA activated astrocytes as indicated by the upregulated glial fibrillary acidic protein and inhibited brain-derived neurotrophic factor. Furthermore, perturbations in cholinergic and monoaminergic systems were observed. RSe provided neuroprotective effects by preventing body weight loss, oxidative stress, neuroinflammation, and the apoptotic cascade. RSe inhibited the activation of astrocytes, increased brain-derived neurotrophic factor, and improved cholinergic and monoaminergic transmission following 3-NPA intoxication. Taken together, RSe co-administration may prevent or delay the progression of HD and its associated impairments through its antioxidant, anti-inflammatory, anti-apoptotic, and neuromodulatory effects.

Effect of cholestasis and NeuroAid treatment on the expression of Bax, Bcl-2, Pgc-1α and Tfam genes involved in apoptosis and mitochondrial biogenesis in the striatum of male rats.

Cholestasis means impaired bile synthesis or secretion. In fact, it is a bile flow reduction following Bile Duct Ligation (BDL). Cholestasis has a main role in necrosis and apoptosis. Apoptosis is a form of programmed cell death that has intrinsic and extrinsic pathways. The intrinsic pathway is mediated by Bcl-2 (B cell lymphoma-2) proteins which integrate death and survival signals. Bcl-2 has anti-apoptotic and Bax has pro-apoptotic effects. Also, striatum is one of the brain regions that has high expressions of Bcl-2 proteins. Moreover, Tfam and Pgc-1α are involved in mitochondrial biogenesis. On the other hand, NeuroAid, is a drug that has neuroprotective and anti-apoptosis effects. In this study, using quantitative PCR, we measured the expression of all these genes in the striatum of male rats following BDL and NeuroAid administration. Results showed, BDL increased the expression of Bax and Tfam and decreased the expression of Bcl-2. NeuroAid restored the effect of BDL on the expression of Bax, while did not alter the effect of BDL on Bcl-2. In addition, it increased the expression of Tfam that was previously elevated by BDL and raised the expression of Tfam in normal rats. Both BDL and NeuroAid, had no effect on Pgc-1α. In conclusion, cholestasis increased the expression of Bax and decreased the expression of Bcl-2, and this effect may have related to enhanced susceptibility of mitochondrial pathways following oxidative stress. Tfam expression was increased following cholestasis and this effect may have related to cellular compensatory mechanisms against high accumulation of free radicals or mitochondrial biogenesis failure. Furthermore, NeuroAid may play a role against apoptosis and can be used to increase mitochondrial biogenesis.

Analysis of In Vitro Cyto- and Genotoxicity of Barbatimão Extract on Human Keratinocytes and Fibroblasts.

Barbatimão (Stryphnodendron adstringens, Mart.) is a native Brazilian species used in traditional medicine and some commercial preparations owing to its strong wound-healing activity. However, controversy regarding its use due to safety concerns over the potential genotoxic effect of this plant remains. In order to clarify this issue, the effect of hydroalcoholic extract of barbatimão in vitro on cell viability, DNA damage, and induction of apoptosis in two commercial cell lines of keratinocytes (HaCaT) and fibroblasts (HFF-1) was evaluated. Barbatimão stem bark hydroalcoholic extract (70% ethanol) was obtained and lyophilized for subsequent use in all experiments. The main bioactive molecules quantified by HPLC were gallic acid, caffeic acid, quercetin, catechin, and epigallocatechin gallate (EGCG). Barbatimão (0.024 to 1.99 mg/mL) was found to decrease cellular mortality as compared to the control group. GEMO assay, a noncellular DNA protocol that uses H2O2-exposed calf thymus DNA, revealed not only a genotoxic effect of barbatimão, but also a potential genoprotective action against H2O2-triggered DNA fragmentation. These results indicated that barbatimão at concentrations of 0.49 and 0.99 mg/mL, which are near to the levels found in commercial preparations, exerted an in vitro genoprotective effect on cells by decreasing the levels of DNA oxidation quantified by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and reactive oxygen species (ROS) levels. Gene and protein apoptotic markers, quantified by qRT-PCR (BAX/Bcl-2 genes) and immunoassays (Caspases 3 and 8), respectively, also indicated a decrease in apoptotic events in comparison with control cells. Collectively, the results suggest that barbatimão could exert genoprotective and antiapoptotic effects on human keratinocytes and fibroblasts.

Trimetazidine ameliorates myocardial ischemia-reperfusion injury.

Aim of this study was to investigate the effects of trimetazidine attenuating the myocardial ischemia-reperfusion injury to myocardium in rats and the underlying mechanisms. A model of myocardial ischemia reperfusion was established via ligating the left anterior descending coronary artery in 30 rats, and then they were randomly assigned to model group (n=10), low dose group (n=10) and high dose group (n=10). Moreover, additional 10 rats were collected and allocated to sham operation group, which was served as control group. Then, rats in the low dose group and high dose group were given trimetazidine with the dose of 10mg/kg and 30mg/kg respectively by intragastric administration, while rats in the control group and model group were given the equivalent volume saline. The dose was given once a day for consecutive 4 weeks in all rats. Echocardiography was applied to evaluate cardiac function, including left ventricular end-systolic dimension (LVESD), left ventricular end diastolic dimension (LVEDD) and left ventricular ejection fraction (LVEF). Next, myocardial tissue was collected, and Bax and Bcl-2 mRNA and the protein levels in the four groups were detected by RT-PCR and Western blot respectively. The level of malonaldehyde (MDA) and super oxide dismutase (SOD) activity in rat myocaridum in each group were detected by colorimetric methods, while the variables of apoptosis were measured by TUNEL methods. In comparison with the control group, LVEDD, LVEDS of rats increased significantly, LVEF decreased obviously, as well as Bax level, MDA level and the apoptotic variables in myocardial tissue increased (P<0.05), but Bcl-2 level and SOD activity decreased significantly in low dose, high dose and model group (P<0.05). Compared with model group, LVEDD, LVEDS of rats decreased obviously, LVEF increased significantly, as well as Bax level, MDA level and the apoptotic variables in myocardial tissue decreased (P<0.05), but Bcl-2 level and SOD activity increased significantly in low dose group, high dose group (P<0.05). The regulatory role of trimetazidine on above indicators of rats was in a dose-dependent manner. Trimetazidine can ameliorate rat myocardium following ischemia-reperfusion injury by effectively attenuating the injury from myocardial cell apoptosis; meanwhile, it can resist cell apoptosis through regulating Bax and bcl-2 expression, which exhibits guiding significance for the treatment of myocardial ischemia and reperfusion.

Safflower polysaccharide inhibits the development of tongue squamous cell carcinoma.

BACKGROUND: Safflower polysaccharide (SPS) is one of the most important active components of safflower (Carthamus tinctorius L.), which has been confirmed to have the immune-regulatory function and antitumor effect. This study aimed to explore the effects of safflower polysaccharide (SPS) on tongue squamous cell carcinoma (TSCC). METHODS: HN-6 cells were treated with 5 µg/mL cisplatin and various concentrations of SPS (0, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28 mg/mL), and cell proliferation was measured. After treatment with 5 µg/mL cisplatin and 0.64 mg/mL SPS, the induction of apoptosis and the protein and mRNA expression of Bax, Bcl-2, COX-2, and cleaved caspase-3 in HN-6 cells were quantified. In addition, HN-6 cells were implanted into mice to establish an in vivo tumor xenograft model. Animals were randomly assigned to three groups: SPS treatment, cisplatin treatment, and the model group (no treatment). The body weight, tumor volume, and tumor weight were measured, and the expression of the above molecules was determined. RESULTS: SPS treatment (0.02-0.64 mg/mL) for 24-72 h inhibited HN-6 cell proliferation. In addition, 0.64 mg/mL SFP markedly induced apoptosis in HN-6 cells and arrested the cell cycle at the G0/G1 phase. Compared with the control group, the expression of Bcl-2 and COX-2 was markedly reduced by SPS treatment, whereas the expression of Bax and cleaved caspase-3 was increased. Moreover, SPS significantly inhibited the growth of the tumor xenograft, with similar changes in the expression of Bcl-2, COX-2, Bax, and cleaved caspase-3 in the tumor xenograft to the in vitro analysis. CONCLUSIONS: Our results indicated that SPS may inhibit TSCC development through regulation of Bcl-2, COX-2, Bax, and cleaved caspase-3 expression.

Mitigative role of garlic and vitamin E against cytotoxic, genotoxic, and apoptotic effects of lead acetate and mercury chloride on WI-38 cells.

BACKGROUND: Lead acetate (Led) and mercury chloride (Mer) represent important ecological and public health concerns due to their hazardous toxicities. Naturally found products play a vital role in chemopreventive agent innovation. The current study aimed to assess the modifying effect of garlic (Gar) and/or vitamin E (Vit E) against the half-maximal inhibitory concentration (IC50) Led and/or Mer-induced cytotoxic, genotoxic and apoptotic effects. METHODS: Human lung cells (WI-38) were pretreated with Gar and/or Vit E for 24h and then treated with Led and/or Mer either alone or with their combination for 24h. Cytotoxicity of Led and Mer and the viability of Gar and Vit E were assessed using MTT assay. The alkaline comet assay was used to assess DNA damage, whereas QRT-PCR was performed to evaluate p53, Bax, and Bcl2 mRNA-expression. RESULTS: The results of this study showed that IC50 of Led was (732.72µg/mL) and for Mer was (885.83µg/mL), while cell viability effective dose for Gar was (300µg/mL) and for Vit E was (26,800µg/mL). Treating cells with the IC50-concentration of Led or Mer or their combination using half IC50 of both of them induced severe DNA-damage. Bax-expression was increased, while p53 and Bcl2-expressions were decreased. Pretreatment of cells with Gar and/or Vit E ameliorated the previous alternations. CONCLUSIONS: Led and Mer can induce oxidative stress and change the expressions of apoptosis-related proteins in WI-38 cells. Gar and Vit E may be promising protective candidate agent against the toxic effect of heavy metals.

Tanshinone modulates the expression of Bcl-2 and Bax in cardiomyocytes and has a protective effect in a rat model of myocardial ischemia-reperfusion.

OBJECTIVE: To investigate the protective activity of tanshinone in a rat model of myocardial ischemia-reperfusion and determine its effect on the expression of Bcl-2 and Bax in cardiomyocytes. METHODS: We established a rat model of myocardial ischemia-reperfusion. Rats were randomly divided into blank (no surgery); saline; and low-dose (2 mg/ml), medium-dose (15 mg/ml), and high-dose (50 mg/ml) tanshinone groups. We measured heart rate and troponin (cTnI) levels, performed TUNEL to detect cardiomyocyte apoptosis, and detected LDH and CK-MB activities in serum by ELISA. We performed RT-qPCR and western blot to detect the expression of Bcl-2 and Bax mRNA and protein in cardiomyocytes. RESULTS: Rats treated with tanshinone experienced more stable heart rate after ischemia-reperfusion compared with those in the saline control group. cTnI decreased after ischemia-reperfusion in mice injected with tanshinone, while cTnI in saline-treated mice increased significantly compared with that in the blank control group. TUNEL staining showed that there were greater apoptotic cardiomyocytes in the saline group, but the tanshinone groups showed fewer apoptotic cardiomyocytes. LDH and CK-MB activities were significantly increased after reperfusion in the saline group (p<0.01) and also in the low- and medium-dose tanshinone groups (p<0.05). However, no significant differences were found in the high-dose tanshinone group (p>0.05). The expression levels of Bcl-2 mRNA and protein in cardiomyocytes of rats were higher in the three tanshinone groups in a dose-sensitive manner than those in the blank and saline groups (p<0.05). By contrast, the expression levels of Bax mRNA and protein were reduced in the three tanshinone groups in a dose-sensitive manner compared to those in the blank and saline groups (p<0.05). CONCLUSION: Tanshinone shows a protective effect in a dose-dependent manner in a rat model of myocardial ischemia-reperfusion, suggesting its potential therapeutic use.

Regulation of DMT1 on autophagy and apoptosis in osteoblast.

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 µmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Chemosensitizing Effect of Astragalus Polysaccharides on Nasopharyngeal Carcinoma Cells by Inducing Apoptosis and Modulating Expression of Bax/Bcl-2 Ratio and Caspases.

BACKGROUND Platinum-based chemotherapy is the most effective regimen for nasopharyngeal carcinoma, which presents highly invasive and metastatic activity. However, the dose-related toxicity of chemotherapy agents limits the dose administration. Astragalus polysaccharide (APS) is the major active ingredient extracted from Chinese herb Radix Astragali and is proven to be active against carcinomas. We aimed to assess the chemosensitizing effects of Astragalus polysaccharides on nasopharyngeal carcinoma in vitro and in vivo and to explore the underlying mechanism. MATERIAL AND METHODS We used BALB/c nu/nu mice and human nasopharyngeal carcinoma cell lines CNE-1, CNE-2, and SUNE-1. MTT, Annexin V/PI, Western blot analysis, and TUNEL assay were carried out. RESULTS APS significantly promoted anti-proliferative and apoptotic effects of cisplatin on nasopharyngeal carcinoma cells. APS also enhanced the anti-tumor effects and cisplatin-induced apoptosis in the xenograft model. The level of Bcl-2 decreased, while the levels of Bax, caspase-3, and caspase-9 increased in cisplatin combined with APS treatment compared to cisplatin only treatment. The ratio of Bax to Bcl-2 was significantly enhanced by the APS to cisplatin. CONCLUSIONS APS enhanced the anti-proliferative and apoptotic effect of cisplatin by modulating expression of Bax/Bcl-2 ratio and caspases on nasopharyngeal carcinoma cells and in the xenograft model.

Dioscin suppresses hepatocellular carcinoma tumor growth by inducing apoptosis and regulation of TP53, BAX, BCL2 and cleaved CASP3.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver, occurs frequently in the setting of chronic liver injury. Although multiple therapeutic approaches are available, the prognosis of patients with HCC remains poor. Dioscin is a natural steroid saponin that presents in various plants. The anti-cancer and anti-fibrotic effects have been extensively reported. However, the effect of dioscin on HCC remains unclear. We aimed to investigate the anti-HCC properties of dioscin in vitro and in vivo. METHODS: MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide) assay was used to analyze the growth inhibition activity of Dioscin in human cell lines, Bel-7402, HepG2, Lovo, and EAhy926. Antitumor activity through induction of apoptosis was evaluated by flow cytometry using Annexin-V and propidium iodide (PI) staining, laser scanning confocal microscopy (LSCM) analysis with Hochest33342 and PI labeling, and DNA fragmentation analysis. The expression of apoptosis-related proteins tumor protein p53 (TP53), BCL2-associated X protein (BAX), B-Cell CLL/Lymphoma 2 (BCL2) and Caspase 3 (CASP3) was measured by Western blot. Nude mice bearing Bel-7402 were administered intraperitoneally at different doses of dioscin and 5-FU (5-Fluorouracil) treatment was used as a control. Tumor volume and tumor weight of each mouse were then measured. RESULTS: We demonstrated that Dioscin inhibited proliferation of HCC cell lines in a dose-dependent manner. Dioscin also significantly induced morphological changes during death by apoptosis and increased DNA damage of Bel-7402 cells. Moreover, we demonstrated that Dioscin displayed anticancer activity via up-regulating expression of TP53, BAX and CASP3 protein, as well as down-regulating BCL2 in Bel-7402 cells. Notably, the in vivo anticancer activity of Dioscin was further assessed and achieved greater inhibition efficiency at the concentration increased to 24mg/kg/day than 5-FU at dose of 10mg/kg/day in nude mice bearing Bel-7402 cells. CONCLUSIONS: Dioscin inhibited tumor growth via inducing apoptosis, which was accompanied by altered expression of apoptotic pathway proteins, such as TP53, BAX, BCL2 and CASP3. Our findings indicate that further evaluation of dioscin as a novel therapeutic approach for HCC is warranted.

Growth inhibitory and apoptosis-inducing effects of allergen-free Rhus verniciflua Stokes extract on A549 human lung cancer cells.

Evidence suggests that Rhus verniciflua Stokes (RVS) or its extract has the potential to be used for the treatment of inflammatory and neoplastic diseases. However, direct use of RVS or its extract as a herbal medicine has been limited due to the presence of urushiol, an allergenic toxin. In the present study, we prepared an extract of the allergen­removed RVS (aRVS) based on a traditional method and investigated its inhibitory effect on the growth of various types of human cancer cells, including lung (A549), breast (MCF-7) and prostate (DU-145) cancer cell lines. Notably, among the cell lines tested, treatment with the aRVS extract strongly inhibited proliferation of the A549 cells at a 0.5 mg/ml concentration for 24 h that was not cytotoxic to normal human dermal fibroblasts. Furthermore, aRVS extract treatment largely reduced the survival and induced apoptosis of the A549 cells. At the mechanistic levels, treatment with the aRVS extract led to the downregulation of Bcl-2 and Mcl-1 proteins, the activation of caspase-9/-3 proteins, an increase in cytosolic cytochrome c levels, the upregulation of Bax protein, an increase in phosphorylated p53 protein but a decrease in phosphorylated S6 protein in the A549 cells. Importantly, treatment with z-VAD­fmk, a pan-caspase inhibitor attenuated aRVS extract-induced apoptosis in the A549 cells. These results demonstrate firstly that aRVS extract has growth inhibitory and apoptosis-inducing effects on A549 human lung cancer cells through modulation of the expression levels and/or activities of caspases, Bcl-2, Mcl-1, Bax, p53 and S6.

Supplementation freeze-thawed media with selenium protect adipose-derived mesenchymal stem cells from freeze-thawed induced injury.

Successful freezed-thaw of adipose-derived mesenchymal stem cells (ADMSCs) could be a major step in regenerative medicine as well as in the cloning of animal breeds. The aim of this study was to evaluate the efficacy of selenium on the optimizing of freezed-thaw media in the ADMSCs. ADMSCs were extracted from NMRI mice and purified with positive selection Monoclonal CD105 Antibody (PE) and negative selection Monoclonal CD31 and CD45 Antibody using MACS method as well as differentiation to adipose and bone tissue. ADMSCs were divided into four groups. ADMSCs were freezed-thaw under standard condition with or without the addition of 5 ng/ml selenium to both the cryopreservation and thawing solutions. Frozen cells were thawed after four months and viability and cytotoxicity of the cells were analyzed by the Trypan blue test and MTT assay respectively. RNA was extracted and cDNA was synthesized and the expression of apoptotic genes (P53, Fas, Bax, Caspase3, and Bcl2) was examined using Real time-PCR Rotor gene 2009. This study compares slow and rapid methods of cryopreservation. After thawing, viability of the cells treated with selenium was higher than the control group in rapid and slow cryopreserved ADMSCs. Also, the percentage of living cells in the slow cooling method was considerably more than with the rapid cooling method. After analysis of the results using Real time-PCR, the Bcl2 gene was shown to be expressed in both the rapid and slow cooling methods. In the rapid cooling group in addition to the BCL-2 gene, p53 was also expressed. It appears that selenium prevented the apoptotic genes from expression due to its anti-apoptotic effects. The slow cooling method is better and more optimized for ADMSCs protecting them from oxidative damage to a greater extent compared to the rapid cooling method.

Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling.

The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays critical roles in the pathogenesis and progression of various human cancers, including non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate the therapeutic potential of physalin A, a bioactive withanolide derived from Physalis alkekengi var. francheti used in traditional Chinese medicine, was evaluated in human NSCLC cells. Its and determined whether it effect oninhibited both constitutive and induced STAT3 activity, through repressing the phosphorylation levels of JAK2 and JAK3, resulting in anti-proliferation and pro-apoptotic effects on NSCLC cells was also determined, and. theThe antitumor effects of physalin A were also validated usingin an in vivo mouse xenograft models of NSCLC cells. Physalin A had anti-proliferative and pro-apoptotic effects in NSCLC cells with constitutively activated STAT3; it also suppressed both constitutive and induced STAT3 activity by modulating the phosphorylation of JAK2 and JAK3. Furthermore, physalin A abrogated the nuclear translocation and transcriptional activity of STAT3, thereby decreasing the expression levels of STAT3, its target genes, such as Bcl-2 and XIAP. Knockdown of STAT3 expression by small interfering RNA (siRNA) significantly enhanced the pro-apoptotic effects of physalin A in NSCLC cells. Moreover, physalin A significantly suppressed tumor xenograft growth. Thus, as an inhibitor of JAK2/3-STAT3 signaling, physalin A, has potent anti-tumor activities, which may facilitate the development of a therapeutic strategy for treating NSCLC.

Dietary Lactobacillus rhamnosus GG Supplementation Improves the Mucosal Barrier Function in the Intestine of Weaned Piglets Challenged by Porcine Rotavirus.

Lactobacillus rhamnosus GG (LGG) has been regarded as a safe probiotic strain. The aim of this study was to investigate whether dietary LGG supplementation could alleviate diarrhea via improving jejunal mucosal barrier function in the weaned piglets challenged by RV, and further analyze the potential roles for apoptosis of jejunal mucosal cells and intestinal microbiota. A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets: the basal diet and LGG supplementing diet. On day 11, all pigs were orally infused RV or the sterile essential medium. RV infusion increased the diarrhea rate, increased the RV-Ab, NSP4 and IL-2 concentrations and the Bax mRNA levels of jejunal mucosa (P<0.05), decreased the villus height, villus height: crypt depth, the sIgA, IL-4 and mucin 1 concentrations and the ZO-1, occludin and Bcl-2 mRNA levels of jejunal mucosa (P<0.05), and affected the microbiota of ileum and cecum (P<0.05) in the weaned pigs. Dietary LGG supplementation increased the villus height and villus height: crypt depth, the sIgA, IL-4, mucin 1 and mucin 2 concentrations, and the ZO-1, occludin and Bcl-2 mRNA levels of the jejunal mucosa (P<0.05) reduced the Bax mRNA levels of the jejunal mucosa (P<0.05) in weaned pigs. Furthermore, dietary LGG supplementation alleviated the increase of diarrhea rate in the weaned pigs challenged by RV (P<0.05), and relieve the effect of RV infection on the villus height, crypt depth and the villus height: crypt depth of the jejunal mucosa (P<0.05), the NSP4, sIgA, IL-2, IL-4, mucin 1 and mucin 2 concentrations of jejunal mucosa (P<0.05), the ZO-1, occludin, Bax and Bcl-2 mRNA levels of the jejunal mucosa (P<0.05), and the microbiota of ileum and cecum (P<0.05) in the weaned pigs challenged by RV. These results suggest that supplementing LGG in diets alleviated the diarrhea of weaned piglets challenged by RV via inhibiting the virus multiplication and improving the jejunal mucosal barrier function, which was possibly due to the decreasing apoptosis of jejunal mucosal cells and the improvement of intestinal microbiota.

Rhus javanica Linn protects against hydrogen peroxide­induced toxicity in human Chang liver cells via attenuation of oxidative stress and apoptosis signaling.

Rhus javanica Linn, a traditional medicinal herb from the family Anacardiaceae, has been used in the treatment of liver diseases, cancer, parasitic infections, malaria and respiratory diseases in China, Korea and other Asian countries for centuries. In the present study, the protective effects of R. javanica ethanolic extract (RJE) on hydrogen peroxide (H2O2)-induced oxidative stress in human Chang liver cells was investigated. The cell cytotoxicity and viability were assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activities of superoxide dismutase (SOD) and catalase (CAT) were measured using respective enzymatic kits. Cell cycle analysis was performed using flow cytometric analysis. The protein expression levels of p53, B-cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax) and caspase-3 were assessed by western blotting. Human Chang liver cells were treated with different concentrations (0.1, 0.3 or 0.5 mg/ml) of RJE, and were subsequently exposed to H2O2 (30 µM). Treatment with H2O2 (30 µM) significantly induced cytotoxicity (P<0.05) and reduced the viability of the Chang liver cells. However, pretreatment of the cells with RJE (0.1, 0.3 or 0.5 mg/ml) significantly increased the cell viability (P<0.001 at 0.5 mg/ml) in a concentration-dependent manner following H2O2 treatment. Furthermore, pretreatment with RJE increased the enzyme activities of SOD and CAT, and decreased the sub-G1 growth phase of the cell cycle in response to H2O2-induced oxidative stress (P<0.001 at 0.3 and 0.5 mg/ml H2O2). RJE also regulated the protein expression levels of p53, Bax, caspase-3 and Bcl-2. These results suggested that RJE may protect human Chang liver cells against oxidative damage by increasing the levels of antioxidant enzymes and regulating antiapoptotic oxidative stress mechanisms, thereby providing insights into the mechanism which underpins the traditional claims made for RJE in the treatment of liver diseases.