Neuro-protective effects of nano-formulated hesperetin in a traumatic brain injury model of Danio rerio.

Understanding the mechanism behind neuronal regeneration is critical for treating ischemic stroke and traumatic brain injury. The presence of neural stem cells in and around the sub-ventricular zone of human and also in zebrafish is evidenced. In this current study, the neuro-protective potential of nano-formulated hesperetin on injury-induced neurogenesis in zebrafish was assessed. Nanoformulation of hesperetin was prepared by anti-solvent precipitation technique using sodium dodecyl sulfate (SDS) as the stabilizing agent. The synthesized particles were characterized using SEM, DLS, XRD and FT-IR. Anti-oxidant capacity of nano hesperetin (nHST) in in vitro followed by in vivo studies in a traumatic brain injury (TBI) model of adult zebrafish (Danio rerio), catalase activity, histological analysis and gene expression studies for the genes Sox2, Nestin, Fabp7a and HuC were carried out. The synthesized particles were found to be in nanoscale and SDS had successfully integrated with hesperetin. Moreover, nHST had a significantly higher anti-oxidant capacity in vitro. Catalase levels in nHST treated group were significantly restored compared to other groups. Histological studies supported reduced tissue damage on oral administration of nano-hesperetin as compared to other groups. Gene expression studies showed that nano-hesperetin at a concentration of 10 µM when administered orally induced proliferation of neural stem cells without inducing cell death.

Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma.

MicroRNAs (miRNAs) are reported to function as a major component in the cellular signaling circuit, which regulates epithelial-mesenchymal transition (EMT). Dysregulation of the microRNA-200 (miR-200) family and EMT-associated genes enables tumor metastasis and resistance to therapy. The present study profiled miR-200 family members miR-200a, miR-200b, miR-200c, miR-141 and miR-429, and also several EMT-regulatory genes including zinc finger E-box-binding homeobox (ZEB)1, ZEB2, epithelial cadherin and vimentin in 40 oral primary tumors in order to understand their role(s) in oral squamous cell carcinoma (OSCC). The reverse transcription-quantitative polymerase chain reaction was used to analyze each sample. Results demonstrated a significant downregulation of miR-200 family members in tumors with a history of tobacco chewing/smoking (P<0.0006, P=0.0467, P=0.0014, P=0.0087 and P=0.0230, respectively) and undifferentiated pathology (miR-200a, P=0.0067; miR-200c, P=0.0248). EMT markers ZEB2 (P=0.0451) and vimentin (P=0.0071) were significantly upregulated in the oral tumors. Furthermore, ZEB2 antisense RNA1 was overexpressed in 50% of OSCC samples (P=0.0075). EMT-regulatory genes did not exhibit any association with clinical outcome. The present study also analyzed the expression of EMT-regulatory genes in 523 head and neck squamous cell carcinoma (HNSCC) samples from The Cancer Genome Atlas (TCGA) database, and the association with treatment outcome. Analysis of TCGA datasets also demonstrated no significant association in the expression of EMT markers with disease recurrence and treatment outcome. The results of the present study revealed dysregulation of miR-200 family miRNAs and EMT-regulatory genes in OSCC without any significant effect on treatment outcome.

Transient gestational exposure to drinking water containing excess hexavalent chromium modifies insulin signaling in liver and skeletal muscle of rat progeny.

Chromium (Cr), an essential micronutrient potentiates insulin action, whereas excess hexavalent Cr (CrVI) acts as an endocrine disruptor. Pregnant mothers living in areas abutting industries using the metal and chromite ore dumps are exposed to ground water contaminated with Cr. Nevertheless, the impact of prenatal exposure to excess CrVI on insulin signaling in the progeny remains obscure. We tested the hypothesis "transient gestational exposure to drinking water containing excess CrVI may modify insulin signaling during postnatal life". Pregnant Wistar rats were given drinking water containing 50, 100 and 200 ppm CrVI (K2Cr2O7) from gestational day 9-14 encompassing the period of organogenesis; the male progenies were tested at postnatal day 60. Neither fasting blood glucose nor oral glucose tolerance was altered in CrVI treated progeny. Nevertheless, western blot detection pointed out attenuated expression level of insulin receptor (IR), its downstream signaling molecules (IRS-1, pIRS-1Tyr632, Akt and pAktSer473) and organ specific glucose transporters (GLUT2 in liver and GLUT4 in gastrocnemius muscle), along with a significant increase in serum insulin level in male progenies exposed to CrVI. While 14C-2-deoxy glucose uptake increased in the liver, the same decreased in the skeletal muscle whereas, 14C-glucose oxidation recorded a consistent decrease in both tissues of CrVI exposed rats. These findings support our hypothesis and suggest that transient gestational exposure to excess CrVI may affect insulin signaling and glucose oxidation in the progeny, predictably rendering them vulnerable to insulin resistance.

Cissus quadrangularis prevented the ovariectomy induced oxidative stress in the femur of adult albino rats.

UNLABELLED: The increasing evidence suggesting the role of free radicals in bone resorption and bone loss prompted us to explore whether the consumption of antioxidant rich medicinal plant C. quadrangularis modifies antioxidant status in ovariectomized rats. METHODS: Twenty four female adult rats, 90days old showing regular estrous cycles were used for the present study. The animals were divided into two groups. The Group-1 rats (n=6) were sham operated and Group-II rats were bilaterally ovariectomized (n=18) and treated with C. quadrangularis for sixty days (100mg/kg body weight and 250mg/kg body weight). After sixty days, the rats were killed, femora were dissected out, minced and homogenized in Tris-HCl buffer (pH 7.4) and the supernatant was collected and used for biochemical assays. RESULTS: Ovariectomy registered a decrease (p<0.05) in the activities of SOD, GPx, GST, ALP, collagen content and increased (p<0.05) the activities of TRAP and lipid peroxidation. Simultaneous administration of C. quadrangularis maintained the enzyme activities in ovariectomized rats. CONCLUSION: C. quadrangularis, a natural herb may be used to treat the estrogen deficiency/menopause onset and ovariectomy induced oxidative stress.

Age-related changes in serum levels of insulin-like growth factor-II and its binding proteins correlate with calcaneal bone mineral density among post-menopausal South-Indian women.

BACKGROUND: Insulin-like growth factor (IGF) system components are important regulators of bone metabolism, which have a predominant role in determining bone mineral density (BMD). While the serum levels of IGF-I are regulated by various systemic hormones and growth factors, IGF-II levels reflect the skeletal production relative to physical activity, mechanical loading, aging, race etc. Though various studies have been carried out among women of different ethnic groups to understand the relationship between serum levels of IGF-II and BMD, the results seem to be quite inconclusive. METHODS: We evaluated the same, recruiting South-Indian women who engage themselves in a wide variety of physical activities pertaining to their profession and life style. RESULTS: Serum levels of IGF-II and IGF binding protein (IGFBP)-3 showed positive correlation with calcaneal BMD, whereas IGFBP-4 showed negative correlation. These IGF system components exhibited similar correlations with serum bone formation markers and opposite trend with bone resorption marker. While both IGF-II and IGFBP-3 levels were observed to be decreased with aging and menopause, IGFBP-4 levels increased. CONCLUSIONS: The alterations in serum levels of IGF-II and its binding proteins due to aging and menopause could be some of the major contributors of decreased calcaneal BMD observed among elderly women.

Effects of Cissus quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells.

Osteoporosis is a public health problem which is associated with significant morbidity and mortality. The repair of bone defect is still a big challenge for orthopedic surgeons. Traditional use of Cissus quadrangularis (C. quadrangularis) in the treatment of bone disorders has been documented. The present study was employed to delineate the effects of ethanolic extract of C. quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells. Lactate dehydrogenase assayed in the conditioned medium of control and C. quadrangularis treated cells did not differ significantly indicating that ethanolic extract of C. quadrangularis is nontoxic to osteoblastic cells. [(3)H] Thymidine incorporation assay revealed that C. quadrangularis treatment has increased the DNA synthesis of human osteoblastic SaOS-2 cells indicating increased proliferation of these cells. The data on alizarin red and ALP staining revealed increased matrix mineralization of human osteoblast like SaOS-2 cells. The study also revealed that the anabolic actions of ethanolic extract of C. quadrangularis in human osteoblast like cells are mediated through increased mRNA and protein expression of Runx2, a key transcription factor involved in the regulation of bone matrix proteins. Chromatin immunoprecipitation analysis revealed increased transcriptional activity of Runx2 on the promoter of osteocalcin after C. quadrangularis treatment. These results indicate positive regulation of C. quadrangularis on the proliferation, differentiation, and matrix mineralization of human osteoblast like SaOS-2 cells.

Dihydrotestosterone is a determinant of calcaneal bone mineral density in men.

Male osteoporosis is an increasingly important health problem worldwide. Though androgen deficiency leads to bone loss in men, information on the relative contribution of aromatizable and non-aromatizable androgens in maintaining bone mineral density (BMD) and the mechanisms involved are unclear. This cross-sectional study was designed to explore the same. Hundred osteoporotic men with age matched normal were studied for serum levels of sex steroids, PTH, IGF system components, cytokines and bone turnover markers. Our findings show that serum DHT, IGF-I, IGF-II and IGFBP-3 levels were significantly decreased while IL-1beta and bone turnover markers were significantly increased in osteoporotic men compared to normal. Pearson correlation analysis revealed that serum DHT, IGF-I, IGF-II and IGFBP-3 levels were positively and strongly correlated with BMD, while serum IL-1beta levels were negatively correlated with BMD. Serum PTH, testosterone, estradiol, IGFBP-4, TNF-alpha, IL-4 and IFN-gamma levels were similar between the two groups. We observed that DHT levels significantly declined with age. However, the significant difference in DHT between the osteoporotic and normal groups is the same regardless of age. A multiple regression model adjusted for age demonstrated that DHT/BMD association is fairly stronger among those with osteoporosis than the normal. Our findings for the first time point out that DHT is an important determinant of BMD in men. Most importantly, the strong positive correlation of serum DHT with BMD offers new perspectives in understanding the role of non-aromatizable androgen in regulating bone metabolism in men, and might serve as a potential clinical marker in the diagnosis of male osteoporosis.

Differential response of Leydig cells in expressing 11beta-HSD type I and cytochrome P450 aromatase in male rats subjected to corticosterone deficiency.

Emerging evidence suggests that the glucocorticoid and estradiol are important for Leydig cell steroidogenesis and are regulated via aromatase for estradiol production and 11beta-HSD for oxidatively inactivating glucocorticoid. Although it is known that corticosterone deficiency impaired Leydig cell steroidogenesis, its effect on the expression of Leydig cell 11beta-HSD type I and aromatase are yet to be recognized. Following metyrapone-induced corticosterone deficiency, serum corticosterone and testosterone levels decrease, whereas serum estradiol remains unaltered. 11beta-HSD type I mRNA and its activity was decreased by corticosterone deficiency, whereas the activity and mRNA of aromatase remains unaltered. Simultaneous administration of corticosterone prevented its deficiency-induced changes of 11beta-HSD type I in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell 11beta-HSD enzyme activity and 11beta-HSD type I mRNA expression, and the Leydig cells need to maintain their intracellular concentration of corticosterone for a normal function.

Modulation of antioxidant defense system by the environmental fungicide carbendazim in Leydig cells of rats.

Carbendazim (methyl-2-benzimidazole carbamate, MBC) a metabolite of benomyl is one of the most widespread environmental contaminant of major concern to human and animal reproductive health. The present investigation was undertaken to study the impact of carbendazim exposure on Leydig cell functions. Adult albino male rats of the Wistar strain were administered with carbendazim (25 mg/(kg (body weight)/day)) orally for 48 days. The control animals received vehicle (corn oil) alone. Another group of rats were treated with carbendazim and the same was withdrawn for a further period of 48 days. After the treatment period, rats were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), prolactin (PRL), testosterone and estradiol. Testes were immediately removed and Leydig cells were isolated in aseptic condition. Purified Leydig cells were used for quantification of steroidogenic enzymes such as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GT), glucose-6-phosphate dehydrogenase (G6PDH) and non-enzymatic antioxidants such as reduced glutathione (GSH), alpha-tocopherol (vitamin E), ascorbic acid (vitamin C) and beta-carotene (vitamin A) were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also quantified. Carbendazim exposure had no effect on body weight, serum LH and prolactin. However, testis weight, serum testosterone and estradiol were significantly decreased. In addition to this, Leydig cellular activities of steroidogenic enzymes such as 3beta-HSD, 17beta-HSD, antioxidant enzymes SOD, CAT, GPx, GR, GST, gamma-GT, G-6-PDH and non-enzymatic antioxidants such as GSH, vitamins E, C and A were significantly diminished, whereas LPO and ROS were markedly elevated. All these above-mentioned parameters from the animals after withdrawal of MBC treatment were similar to those of the control group. Thus, the present study suggests that chronic low dose treatment of MBC is capable of inducing reproductive toxicity through increased oxidative stress, but is transient and reversible upon withdrawal of treatment.