Iranian Journal of Basic Medical Sciences
Year:2017 | Volume:20 | Issue:10|Papers Count:15

The up-regulation and down-regulation of gonadotropin-releasing hormone (GnRH) in central precocious puberty is not yet known. However, recent advances in neuroendocrinology have shown the controlling role of arginine-phenylalanine RF-amide-related peptides (RFRPs) on GnRH secretion in different phenomenon of reproduction such as estrus cycle and pregnancy, but the exact role of RFRPs in puberty and its related pathologic condition, precocious puberty, is not clear yet. This paper hypothesizes that RFRP is a regulatory peptide of puberty and might prevent the precocious puberty. On the basis of previous studies on hormonal fluctuations at the time of puberty, RFRP might have a role on controlling of premature secretion of GnRH and avoiding central precocious puberty.

Objective(s): Chronic diabetes impedes cardioprotection in reperfusion injury and hence protecting the diabetic heart would have important outcomes. In this study, we evaluated whether combined postconditioning with ischemia and cyclosporine-A can restore oxidative stress and histopathological changes in reperfusion injury of the diabetic myocardium. Materials and Methods: Streptozocin-induced diabetic rats’ hearts and nondiabetic controls in eight subgroups (with or without receiving ischemic-postconditioning (IPostC), cyclosporine-A, an inhibitor of mitochondrial permeability transition, or both of them) suffered from 30 min regional ischemia followed by 45 min reperfusion on an isolated-heart Langendorff system. The levels of lactate dehydrogenase (LDH) in the coronary effluent, and the levels of oxidative stress markers including 8-isoprostane, superoxide dismutase (SOD), glutathione peroxidase (GPX), and total antioxidant capacity (TAC) in myocardial supernatant prepared from the ischemic zone were measured using specific kits, spectrophotometrically. Histopathological studies were performed through the hematoxylin-eosin staining method. Results: Administration of IPostC and cyclosporine-A (alone or together) in nondiabetic hearts potentially reduced the severity of histological changes and level of LDH release as compared with untreated-controls (P<0. 05). Administration of any lone procedures in diabetic hearts did not show significant cardioprotective effects (P>0. 1). However, the combined postconditioning with ischemia and CsA exerted significant protective effects in diabetic hearts (P<0. 05). Conclusion: By augmenting the protective effects of IPostC and CsA through their combined application, reperfusion injury and related oxidative stress are reduced in diabetic hearts similar to non-diabetics.

Objective(s): Low penetration depth of light is the main defect of photodynamic therapy (PDT), which could be improved by sonodynamic therapy (SDT). In this study, a combination of PDT and SDT known as sonophotodynamic therapy (SPDT) was investigated using two reverse arrangements in CT26 tumor model. Materials and Methods: The liposomal zinc phthalocyanine was synthesized and characterized. It was then administered to CT26 tumor models as a sensitizer. The animal models were subjected to PDT, SDT, and the combined treatment in different groups. The doubling time for the survival of tumors and animals was considered as a measure to evaluate treatments efficacy. Results: In all treatment groups there was a significant decline in tumor volume 15 days after treatment compared to the main control group, but the optimum response was observed in the group receiving a combined treatment with the priority of PDT. 120 days after treatment, in the groups treated by PDT and SDT, the tumor shrank by 20%, while in the group receiving SPDT with PDT priority, 80% of tumors was recovered. No case of complete tumor progression was observed in SPDT group with SDT priority. This could be due to the pores created in cell membranes during ultrasound irradiation of the tumor, which removed the sensitizer molecules from the cells and reduced PDT efficacy in SPDT group with SDT priority. Conclusion: It seems that SPDT with PDT priority offers a more efficient alternative than each of PDT, SDT individually or SPDT with the reverse arrangement.

Objective(s): This study was aimed to investigate the effect of Berberis integerrima (B. integerrima) extract on insulin sensitivity in high-fructose-fed insulin-resistant rats. Materials and Methods: Experimental rats were randomly divided into two groups: the control group was fed a regular chow diet while other group fed with a high-fructose diet for 8 weeks. After the first six weeks, the animals were treated with B. integerrima extract or pioglitazone for two weeks. Insulin and adiponectin levels were measured by ELISA. Additionally, Insulin resistance was calculated using a Homeostasis Model Assessment of Insulin resistance (HOMA-IR). The plasma free fatty acid (FFA) profile was obtained by gas chromatography. PPARγ and GLUT4 gene expression were assessed by real-time polymerase chain reaction (PCR) and western-blotting. Results: Comparing the B. integerrima treated group with the control group, weight gain (P=0. 009) and levels of insulin (P=0. 001), blood glucose (P<0. 0001), and HOMA-IR (P<0. 0001) were significantly reduced. Additionally, the adiponectin concentration was significantly increased (P<0. 0001). Among the FFA fractions, the mean concentration of palmitoleic acid and stearic acid in the B. integerrima group were significantly higher than the control group (P<0. 0001 and P=0. 005, respectively). However, there was no significant difference at the mRNA and protein level of GLUT4 and PPAR-γ between B. integerrima treated group and control group. Conclusion: The study findings revealed that B. integerrima might be a protective candidate against Type 2 diabetes/insulin resistance through direct insulin-like effect and an increase in adiponectin levels. However, the mechanism of B. integerrima was independent of GLUT4 and PPARγ .

Objective(s): In this study, we aimed to investigate the effects of anti-Fas ribozyme on the apoptosis of T lymphocytes (T cells) in mice model with chronic obstructive pulmonary disease (COPD). Materials and Methods: Male 6-week-old C57BL/6 mice were used to establish the COPD model by exposure to cigarette smoke. The COPD mice were sacrificed for spleen dissection and T cell isolation. T cells were randomly divided into four groups (n=10 per group). Group A was used as the control. B, C, and D groups were transfected with empty lentivirus, anti-Fas ribozyme, and an anti-Fas ribozyme mutant, respectively. The expression of Fas mRNA and protein in the T cells were evaluated using qPCR and Western blot, respectively. Flow cytometry was used to evaluate the apoptosis of CD4＋ T cells and calculate the ratio of CD4+ to CD8+ T cells (CD4＋ /CD8＋ ). Results: Anti-Fas ribozyme significantly inhibited the expression of Fas in the T cells of COPD mice. In addition, the number of apoptotic CD4＋ T cells and CD4＋ /CD8＋ of the C and D groups were significantly lower and higher than those of group A, respectively (P<0. 05). The apoptotic CD4＋ T cells and CD4＋ CD8＋ of the C group were significantly lower and higher than those of group D, respectively (P<0. 05). Conclusion: Anti-Fas ribozyme significantly inhibited the expression of Fas, increased CD4+/ CD8+, and inhibited the apoptosis of T cells in COPD mice.

Objective(s): Here, a reporter cell line containing two reporter vectors were developed, in order to monitor the Human T-Lymphotropic Virus type1(HTLV-1) infectivity and the cell viability simultaneously. Materials and Methods: The reporter cell line was constructed by stably transfected baby hamster's kidney cell line (BHK-21), with the genomes expressing two different reporters in separate plasmids. The first reporter gene is transactivated by the HTLV-1 tax protein, while the second reporter is continuously expressed when introduced into a mammalian cell. In order to show its functionality, the effect of the drug mix on HTLV-1 was assayed by this system and was compared to the results obtained by other methods. Results: HTLV-1 reporter cell line was found to produce high level of luciferase when co-cultured with MT-2 and Hut-102 cells but not with Jurkat cell. Moreover, the combination therapy against HTLV-1 can reduce luciferase expression of the cell when co-cultured with MT-2 and Hut-102 comparable to the ELISA (R=0. 932, P-value =0. 002). In addition, the results revealed the superiority of the present system over the molecular methods. Conclusion: The results demonstrated that the biological assay system is a beneficial tool for the medium-throughput anti-HTLV-1 drug screening and inhibitory effect.

Objective(s): Tuberculosis (TB) has still remained a global health issue. One third of the world's population is infected with tuberculosis and the current BCG vaccine has low efficiency; hence, it is necessary to develop a new vaccine against TB. The aim of the current study was to evaluate the efficiency of a novel DNA vaccine encoding Mtb32C-HBHA antigen in inducing specific immune responses against Mycobacterium tuberculosis. Materials and Methods: A DNA plasmid vaccine expressing Mtb32C-HBHA fusion protein was constructed and its ability in protein expression was examined by RT-PCR and Western blot methods. Female BALB/c mice were vaccinated with 100 μ g of purified recombinant vector in an attempt to assess its immunogenicity and protective efficacy. Further, the cytokines, IFN-γ , IL-12, IL-4, IL-10, and TGF-β were assessed. Results: The levels of all the studied cytokines were significantly increased (P<0. 05) compared with the control group. IFN-γ production in the group receiving DNA vaccine plus BCG was increased compared with those receiving only DNA vaccine or BCG (P<0. 001). Conclusion: The immunogenicity of the new chimeric DNA vaccine was confirmed alone and in combination with BCG. Based on the results of the current study, the constructed DNA vaccine induced the expression of Mtb32C-HBHA fusion protein efficiently in vitro. Furthermore, high levels of the specific cytokines were induced in mice. By using this DNA vaccine as a booster after BCG, higher amounts of IFN-γ will be produced.

Objective(s): Autophagy-related 4B (ATG4B) plays an important role in the process of autophagy induction. However, the molecular events that govern the expression of ATG4B in this process are not well known. Materials and Methods: Human ATG4B 3'-UTR region (1377 nt) containing miR-34a/miR-34c-5p binding site was amplified by PCR. Luciferase assay was used to assess the activity of reporter genes. Real-time PCR was used to detect the levels of miR-34a and miR-34c-5p. Western blot was used to analyze the protein levels of ATG4B, LC3 and p62. Results: Both miR-34a and miR-34c-5p could directly target the 3'-UTR of ATG4B mRNA at same site. Overexpression of either miR-34a or miR-34c-5p significantly down-regulated ATG4B at both mRNA and protein levels and this effect can be reversed by ATG4B overexpression. Moreover, Rapamycin-induced autophagy is accompanied with the upregulation of ATG4B and the downregulation of miR-34a/miR-34c-5p. Ectopic expression of either miR-34a or miR-34c-5p markedly suppressed rapamycin-triggered autophagy. Conclusion: In the present study, we found that miR34/ATG4B signaling axis involves in rapamycin-triggered autophagy. This study may provide a new insight for understanding the mechanisms of ATG4B regulation and autophagy induction.

Objective(s): Autologous bone transplantation known as the “ gold standard” to reconstruction of osseous defects has known disadvantages. This study was designed to explore the effects of hydroxy-apatite/tricalcium-phosphate (HA/TCP) and platelet-rich plasma (PRP) on the osteogenesis ability of human adipose-derived mesenchymal stem cells (hAdMSCs) in vitro and in vivo. Materials and Methods: hAdMSCs were incubated with HA/TCP granules and/or PRP in vitro and then, cell proliferation and differentiation was assessed by MTT assay, AZR S staining and SEM examination. In vivo, four cylindrical defects were drilled in the mandibular bones of 5 mongrel dogs and divided randomly into the following groups: I-autologous crushed bone, II-no filling material, III-HA/TCP and PRP, IV-PRP-enriched hAdMSCs seeded on HA/TCP granules. Inserted hAdMSCs were labeled to trace their contribution to bone tissue regeneration. Finally, cell tracing and tissue regeneration were evaluated by immunohistochemistry and histomorphometry methods, respectively. Results: In vitro, co-incubation with HA/TCP granules significantly reduced proliferation and osteogenic differentiation ability of hAdMSCs; while PRP application promoted these capacities (P<0. 05). In vivo, PRP-enriched hAdMSCs seeded on HA/TCP granules induced considerable bone formation in osseous defects (P<0. 05). It was obviously shown that hAdMSCs were incorporated into the newly-formed bone. Conclusion: Based on this study, application of stem cells could offer a helpful therapeutic tool in bone tissue regeneration. Although inserted hAdMSCs were identifiable throughout the newly-formed bone tissue, their few number could be an indicator of indirect role of hAdMSCs in tissue regeneration.

Objective(s): This study was to investigate the antihyperlipidemic and antioxidant effect of total flavonoid extract from Actinidia kolomikta (TFAK) in hyperlipidemia induced by a high-fat diet. Materials and Methods: Male SD rats were randomly divided into 6 groups: normal group, model (hyperlipidemic diet) group, hyperlipedemic diet supplemented with TFAK (50, 100 and 200 mg/kg) and simvastatin (30 mg/kg) groups. The rats were administrated TFAK by oral for 28 days. Body weight, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured. The atherogenic index (AI) and coronary risk index (CRI) were calculated. The activity of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in hepatic tissue was examined. Histopathologic changes were also checked. Results: The levels of TC, TG and LDL-c were increased in model group. Compared to the model group, TFAK reduced significantly the body weight, TC, TG, LDL-c, AI, CRI and elevated the level of HDL-c. Moreover, the activity of SOD was elevated significantly, whereas the content of MDA decreased. The activity of HMG-CoA reductase was also decreased. Morphological evaluation found that rats in model group developed a severe steatosis, but the severity of liver steatosis was ameliorated in TFAK treated groups. The possible mechanism may be associated with decrease HMG-CoA reductase activity. Conclusion: Our results suggest that TFAK exerts strong antioxidant and lipid-lowering effects, prevents hepatic fatty deposition and regulates the HMG-CoA reductase.

Objective(s): As the second cause of cancer death, gastric cancer (GC) is one of the eminent dilemmas all over the world, therefore investigating the molecular mechanisms involved in this cancer is pivotal. Unrestricted proliferation is one of the characteristics of cancerous cells, which is due to deficiency in cell regulatory systems. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of the epigenome. lncRNA extra coding CEBPA (ecCEBPA) is involved in DNA methylation. This lncRNA reduces CEBPA promoter methylation by interacting with DNA methyltransferase 1. lncRNA UCA1 (urothelial carcinoma-associated 1) elevates cell proliferation through the PI3K/Akt signaling pathway which has a critical role in cell growth and apoptosis. The aim of this study was to examine the expression of ecCEBPA and UCA1 genes in GC tissues as well as their clinical significance. Materials and Methods: Total RNA extraction, cDNA synthesis, and quantitative real-time PCR were performed for cells and 80 paired GC tissues. Furthermore, clinical relevance of UCA1 expression was investigated in TCGA cohort data. Results: Our results showed ecCEBPA and UCA1 over-expression in GC tissues. Furthermore, lncRNAs associations with clinicopathological features were demonstrated both in the current and TCGA cohort. Kaplan-Meier analysis indicated that patients with higher UCA1 expression had a worse overall survival in the case of pancreatic and lung adenocarcinomas but not other solid cancer types including GC. Conclusion: These data demonstrate UCA1 and ecCEBPA involvement in GC and suggest that these lncRNAs might be useful as diagnostic/ prognostic biomarkers in cancer.

Objective(s): Epidemiological and biochemical studies conducted over the past two decades have established a strong link between type 2 diabetes mellitus (T2DM) and Alzheimer’ s disease (AD). However, the exact mechanisms through which aberrations in insulin signaling associated with T2DM contribute to cognitive decline are not yet known. Materials and Methods: In an effort to explore possible molecular links between T2DM and AD, the present study investigated the status of neurodegeneration, adult hippocampal neurogenesis, and nitrosative stress induced protein S-nitrosylation in streptozotocin (STZ) induced mice models of T2DM. Morris water maze task and subsequent histological and immunohistochemical assessment were conducted. Expression of neurogenesis markers (Ki67, DCX, and NeuN) and APP 770 was determined by qRT-PCR. Results: A significant decline in spatial learning and reference memory was observed with consequent neurodegeneration in brain cortex and hippocampus in the diabetic group as compared to the control group. A subsequent increase in expression of APP 770 was also observed in T2DM brain regions. Moreover, a significant decrease in transcriptional expression of Ki67, DCX, and NeuN was also evident in T2DM brain regions, which indicated possible aberrations in adult hippocampal neurogenesis in T2DM. Furthermore, an increased immunohistochemical signal for S-nitrosylation was also observed in T2DM, which also suggested its potential contribution in T2DM associated neuronal deterioration. Conclusion: It is suggested that these identified aberrations in the diabetic brain may communally increase the susceptibility of developing AD in patients with T2DM. Further studies of the underlying molecular mechanisms may help to strategize a combination therapy for these debilitating disorders.

Objective(s): We intended to determine whether the ovarian varicose which is one of the common etiologies of the pelvic congestion syndrome, has the ability to interfere with the DNA methylation reprogramming in the oocyte and thereby affect the oocyte quality or not. Materials and Methods: Varicose model was induced according to the Turner’ s method in the rats. Briefly, a 20-gauge needle was placed on the left renal vein and a thread was tied over both the needle and the renal vein medial to the insertion of the ovarian vein, and then the needle was removed. Evaluation of prooxidant-antioxidant balance (PAB) was assessed using specific kits and the expression level of the DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3L was assessed by Real-time PCR. Immunofluorescent staining for 5-methylcytosine in the oocytes evaluated the global DNA methylation. Results: A significant PAB increase in the ovaries from varicose group was seen. Real-time PCR demonstrated a remarkable decrease in the expression of the Dnmt3a and Dnmt3L which are responsible for de novo DNA methylation in the oocytes. Immunofluorescent staining for 5-mC showed a reduction in the fluorescence intensity in the oocytes collected from the varicose group. Conclusion: Our findings from Real-time PCR and immunocytochemistry suggest that the epigenetic parameters in the oocyte could be affected by varicose induction and these epigenetic alteration has the potential to affect the oocyte quality. We suggest that the epigenetic changes could happen in the oocytes after the induction of ovarian varicose and lead to the oocyte quality reduction or even infertility.

Objective(s): The objectives of the current study were to evaluate the effects of hepatic ischemia/reperfusion (IR) injury on the activity of antioxidant enzymes, biochemical factors, and histopathological changes in rat kidney, and to investigate the effect of crocin on IR-related changes. Materials and Methods: Thirty-two male Wistar rats were randomly allocated into four groups (n=8). They were sham-operated, IR, crocin pre-treatment, and crocin pretreatment+IR groups. Sham-operated and Crocin pre-treatment groups received normal saline (N/S, 2 ml/day) and crocin (200 mg/kg) for seven consecutive days intraperitoneally (IP), respectively, then rats underwent laparotomy, only. IR and crocin pretreatment+IR groups received N/S and crocin with the same dose, time, and route, respectively, then rats underwent partial (70%) ischemia for 45 min that was followed by reperfusion for 60 min. At the end of the experiment, kidney specimens were taken for histopathological and antioxidant evaluations and also blood samples were obtained for biochemical analysis. Results: The results of the present study showed that crocin pre-treatment significantly increased the activity of antioxidants, decreased the serum levels of liver enzymes and blood urea nitrogen following IR-induced hepatic injury. Crocin also ameliorated kidney´ s histopathological disturbance beyond IR-induced hepatic injury. Conclusion: Crocin as an antioxidant agent protected renal insult following liver IR injury by increasing the activity of antioxidant enzymes, reducing serum levels of liver enzymes, and improving histopathological changes.

Objective(s): Metastasis is the main cause of death in patients with melanoma. Cannabis-based medicines are effective adjunctive drugs in cancer patients. Tau and Stathmin proteins are the key proteins in cancer metastasis. Here we have investigated the effect of a standardized Cannabis sativa extract on cell migration and Tau and Stathmin gene expression in the melanoma cell line. Materials and Methods: In the treatment group, melanoma (B1617) was treated 48 hr with various concentrations of standardized C. sativa extract. Cells with no treatment were considered as the control group, then study was followed by Quantitative RT-Real Time PCR assay. Relative gene expression was calculated by the Δ Δ ct method. Migration assay was used to evaluate cancer metastasis. Results: Tau and stathmin gene expression was significantly decreased compared to the control group. Cell migration was also significantly reduced compared to controls. Conclusion: C. sativa decreased tau and stathmin gene expression and cancer metastasis. The results may have some clinical relevance for the use of cannabis-based medicines in patients with metastatic melanoma.