فهرست مطالب

Cell Journal - Volume:22 Issue: 4, 2020
  • Volume:22 Issue: 4, 2020
  • تاریخ انتشار: 1399/02/10
  • تعداد عناوین: 21
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  • Sima Aghajari, Sayed Mohammad Javad Mortazavi, Mehdi Kalani, Samaneh Nematolahi, Parham Habibzadeh, Shirin Farjadian* Page 401
    Objective

    Astronauts are exposed to a wide range of environmental stresses during spaceflights that reduce their immune responses and make them more susceptible to infections and malignancies. Exposure to a low dose of a certain stress induces an adaptive response, which leads to resistance to higher doses of the same or other types of stress. We designed this study to investigate the effect of radiofrequency electromagnetic field (RF-EMF)-induced adaptive response on immune system modulation in a mouse model of hindlimb unloading (HU) as a ground-based animal model of spaceflight conditions.

    Materials and Methods

    In this experimental study, serum levels of T helper (Th)-mediated cytokines were determined by the multiplex cytometric bead assay in four groups of mice (n=10 per group): HU mice, RF-EMF-treated mice, HU mice pre-exposed to RF-EMF; and untreated controls. Mice were exposed to 2450 MHz RF-EMF with SAR 0.478 W/ kg for 12 hours/day for three successive days.

    Results

    Tumor necrosis factor-alpha (TNF-α), interleukin-9 (IL-9) and IL-22 were significantly decreased in HU mice. Comparison between HU mice and RF-EMF-treated mice showed an opposite change in IL-6, while IL-9, IL-22, IFN-γ and TNF-α decreased in both groups. However, just interferon gamma (IFN-γ) was significantly decreased in HU mice that were pre-exposed to RF-EMF compared to the control group.

    Conclusion

    The effect of RF-EMF in elevating IL-6 and reducing IL-9 in opposite directions in HU mice suggest a modulating effect of RF-EMF on HU-induced changes in these cytokines, as Th2 and Th9 eventually returned to normal levels and balances in cytokine ratios were also restored in HU mice pre-exposed to RF-EMF.

    Keywords: Cytokine, Flow Cytometry, Hindlimb Suspension, Radio Wave
  • Siyavash Mirzaei, Hamid Mobedi*, Hamid Gourabi, Mohammad Hosein Sanati, Sakine Khezli, Hamid Omidian, Masoume Ighaeie Page 406
    Objective

    It is so difficult to formulate human growth hormone (hGH) in a solution with high stability and new drug delivery system (NDDs) due to physiochemical instability. The purpose of this study was to investigate the possibility of using Tris as a hGH stabilizer.

    Materials and Methods

    In this experimental study, the role of tris(hydroxymethyl)aminomethane (Tris) was evaluated as a hGH stabilizing agent in phosphate buffer, as a practical aqueous solution and a media to release NDDs. Highperformance liquid chromatography (HPLC) and enzyme-linked immune sorbent assay (ELISA) were applied to investigate the stability of hGH in solutions and dynamic light scattering (DLS) was used to measure the effect of Tris on the hydrodynamic size of hGH in aqueous solutions. Ultra violet (UV) spectrophotometry was used to check the hGH spectrum. In computational study, formation of ligand-protein complex of the Tris-hGH, and the intermolecular interactions between Tris and hGH were studied by molecular docking modeling.

    Results

    The results demonstrated that Tris at the optimum concentration, increases hGH stability in aqueous solutions. Also, molecular docking modeling confirmed that amino acid residues such as tyrosine (Tyr), proline (Pro), glutamic acid (Glu), aspartic acid (Asp), leucine (Leu), and phenylalanine (Phe) in hGH structure, were linked with Tris as a ligand.

    Conclusion

    It seems that interactions between hGH and Tris are the most important reason for increment of the physicochemical stability of hGH in aqueous solutions containing Tris.

    Keywords: Human Growth Hormone, Molecular Modeling, Protein Stability, Tris(hydroxymethyl)aminomethane
  • Florina Popovska Perčinić, Milica Manojlović Stojanoski*, Lazo Pendovski, Suzana Dinevska Kjovkarovska, Biljana Miova, Jasmina Grubin, VericaMilošević, Vladimir Ajdžanović Page 415
    Objective

    As a consequence of global warming, the increase in the average annual temperature is observed, while the living organisms actively adapt to these changes. High environmental temperature initiates numerous physiological, autonomic, and behavioral responses, and activates the stress response. Thus, the aim of the study was to investigate effect of a moderate increase in ambient temperature on the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis by determining histological changes in adrenal glands and hormonal levels in adult male rats.

    Material and Methods

    In this experimental study, the morpho-functional state of adrenal glands was estimated by stereological evaluation of parameters, including the adrenal volume, adrenocortical cell/nuclear size and number, and the volume density of vascular tissues after four days of exposure to a moderate increase in ambient temperature of 35 ± 1˚C. Novelli histochemical and vascular endothelial growth factor (VEGF) immunohistochemical staining provided insight into the adrenal gland vascular network. Additionally, the adrenal levels of aldosterone, corticosterone, and pituitary adrenocorticotropic hormone (ACTH) were determined as crucial indicators of the hypothalamic-pituitaryadrenocortical (HPA) axis activity.

    Results

    Prolonged exposure to a moderate increase in ambient temperature for four days resulted in a significant increase in ACTH level up to 24%, which altered adrenal glands both structurally and functionally. The adrenocortical volume and number of cells in all cortical zones were markedly increased (P<0.05). A statistically significant increase was shown in the level of aldosterone (16%) and corticosterone (25%) in serum levels of individuals.

    Conclusion

    Increased activity of the HPA axis reflects the response to a moderate increase in ambient temperature during four days, showing the capacity of the HPA axis to adapt the organism to daily temperature changes.

    Keywords: Adrenal Glands, Adrenocorticotropic Hormone, Corticosterone, Pituitary, Temperature
  • Hamed Rezaei Nasab*, Abdolhamid Habibi, Masoud Nikbakht, Mohammad Rashno, Saeed Shakerian Page 425
    Objective

    Physical activity leads to changes in the level of gene expression in different kinds of cells, including changes in mitochondrial biogenesis in the myocardium in diabetic patients. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a gene that plays an important role in regulating mitochondrial biogenesis. The purpose of this study was to investigate changes in serum levels and cardiac muscle expression of PGC-1α in diabetic rats in response to the administration of dichloroacetate (DCA) and endurance training.

    Materials and Methods

    In this experimental study, 64 male Wistar rats were selected and randomly divided into eight groups after induction of diabetes with streptozotocin (STZ). The endurance training protocol was performed on a treadmill for 6 weeks. Intraperitoneal injection of DCA of 50 mg/ kg body weight was used for the inhibition of Pyruvate Dehydrogenase Kinase 4 (PDK4) in the myocardium. Gene expression were measured using real-time polymerase chain reaction (PCR). One-way ANOVA and Tukey’s test were used to statistically analyze the data.

    Results

    The results of the study showed thatPDK4gene expression in the endurance training group, diabetes+endurance training group, diabetes+endurance training+DCA group and endurance training+DCA group was higher compared to the control group. Expression of PGC-1α was higher in the endurance training group compared to the control group but was lower compared to the control group in diabetes+endurance training+DCA group and diabetes+DCA group (P<0.05).

    Conclusion

    Considering that PGC-1α plays an important role in mitochondrial biogenesis, it is likely that by inhibiting PDK4 and subsequently controlling oxidation of fatty acid (FA) in the heart tissue, oxidative stress in the heart tissue of diabetic patients will be reduced and cardiac efficiency will be increased.

    Keywords: Endurance Training, Diabetes, Dichloroacetate, Mitochondrial Biogenesis
  • Zahra Fazeli*, Masoumeh Rajabibazl, Sepideh Faramarzi, Mir Davood Omrani, Sayyed Mohammad Hossein Ghaderian, Niloufar Safavi Naini Page 431
    Objective

    In the recent years, mesenchymal stem cells (MSCs) were considered as the suitable source of cells for transplantation into the damaged tissues in regenerative medicine. There was low number of these cells in different organs and this characteristic was the main drawback to use them in treatment of diseases. Cellular senescence of the stem cells has been demonstrated to be dependent to the telomerase activity. The aim of present experimental study was to evaluate correlation of the expression of telomerase components and WNT signaling pathway in MSCs derived from human peripheral blood (PB-MSCs).

    Materials and Methods

    In this experimental study, following the isolation of MSCs from peripheral blood mononuclear cells, RNA was extracted from these cells in the early culture (8-9th days) and late culture (14-17th days). Then, expression of TERT, TERC, TCF4, GSK and CTNNB1 was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) based on SYBR Green.

    Results

    Our data indicated that there was a significantly reduced expression of TERT in the late culture of human MSCs derived from peripheral blood (P<0.05). Although a negative correlation was observed between GSK and TERC expression levels in the early culture of MSCs, spearman analysis showed that there was no significant correlation between the expression of telomerase components (TERC and TERT) and WNT signaling pathway (P>0.05).

    Conclusion

    The obtained results suggested that WNT signaling pathway likely plays a minor role in the maintenance of telomere length and proliferation potential of MSCs.

    Keywords: Cellular Senescence, Mesenchymal Stem Cells, Regenerative Medicine, WNT Signaling Pathway
  • Yeon Hee Lee, Ashish Ranjan Sharma*, Supriya Jagga, Sang Soo Lee, Ju Suk Nam Page 437
    Objective

    Rspondins (RSPOs) are regarded as the significant modulators of WNT signaling pathway and they are expressed dynamically during developmental stages. Since in osteoarthritis (OA) both cartilage and subchondral bone suffer damages and WNT signaling pathway has a crucial role in their maintenance, the objective of the study was to analyze expression profile of RSPO family and its receptors [leucine-rich repeat-containing G-protein coupled receptors (LGRs)] in OA tissue samples as well as in differentiating chondrocytes and osteoblasts.

    Material and Methods

    In this experimental study, human early and advanced stage of OA tissue samples were analyzed for the morphological changes of articular cartilage by hematoxylin and eosin (H&E) staining, safranin-O staining and lubricin immunostaining. RSPOs and LGRs expression were confirmed by immunohistochemistry. Human primary chondrocytes and human osteoblast cell line, SaOS-2, were cultured in differentiation medium till day 14 and they were analyzed in terms of expression of RSPOs, LGRs and specific marker for chondrogenesis and osteogenesis by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR).

    Results

    Advanced stage OA tissue samples showed increased expression of RSPO1 and LGR6 in a region close to subchondral bone. While RSPO2 and LGR5 expression were seen overlapping in the deep region of articular cartilage. Differentiating chondrocytes demonstrated elevated expression of RSPO2 and LGR5 from day 7 to day 14, whereas, osteoblasts undergoing differentiation showed enhanced expression of RSPO1 and LGR6 from day 2 to day 14. Under tumor necrosis factor alpha (TNFα) stimulatory conditions, RSPO2 and RSPO1 recovered the suppressed expression of inflammatory, chondrogenic and osteogenic markers, respectively. A recovery in the stability of β-catenin was also noticed in both cases.

    Conclusions

    Spatial expression of RSPOs during progression of OA might be dynamically controlled by cartilage and subchondral bone. Interplay amid chondrocytes and osteoblasts, via RSPOs, might provide probable mechanisms for treating inflammatory pathogenic conditions like OA.

    Keywords: Chondrocyte, LGR, Osteoarthritis, Osteoblast, R-spondin
  • Zahra Chekini, Maryam Shahhoseini, Reza Aflatoonian*, Parvaneh Afsharian Page 450
    Objective

    Endometriosis is a common gynecological and inflammatory disorder. Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that is secreted by accumulated active macrophages in ectopic endometrial tissues. Two promoter polymorphisms of MIF [-794(CATT)5–8 /-173G/C] were identified to susceptibility and severity of several immune and inflammatory diseases. We aimed to evaluate the possible association between MIF promoter polymorphisms and susceptibly to endometriosis and its corolation with mRNA level.

    Materials and Methods

    This case-control study was performed in Royan Institute from 2015 to 2017. Polymorphisms were evaluated in 106 endometriosis patients and 110 controls. For 17 endometrioma tissues, gene expression studies were conducted during secretory phase of menstrual cycle. Restriction fragment length polymorphism (RFLP) analysis was performed to determine -173G/C polymorphism and -794(CATT)5-8 were detected by sequencing. Quantitative polymerase chain reaction (Q-PCR) was carried out to determine MIF expression level.

    Results

    Homozygote of CATT7 was observed only in endometriosis whilst we did not detect the significant allele and genotype variation in both groups. The homozygotes for -794(CATT)5–8 and -173G/C polymorphisms were obtained to estimate the haplotype frequencies. Significantly higher haplotype frequencies were observed for CATT5/G in controls [global P value=0.044]. Additionally, the CATT5/C and CATT7/G haplotypes were not detected in any groups. Expression level of mRNA in ectopic tissue of endometriosis patients with CATT6,7/CC haplotype, were significantly higher compared to other haplotypes including CATT5,5/GG (2.91 fold, P=0.007), CATT5,5/GC (2.48 fold, P=0.047) and CATT 6,6/GG (2.08 fold, P=0.046).

    Conclusion

    We report, for the first time, a strong linkage between the decreased repetition of CATT and G allele in control and CATT 6/C and CATT7/C haplotypes in endometriosis patients. Increased MIF expression is affected by genetic variants in the MIF promoter in ectopic endometrial tissues. This promoter haplotype might play an important role in the development and establishment of endometriosis.

    Keywords: Endometriosis, Gene Expression, Haplotype, Macrophage Migration Inhibitory Factor, Polymorphism
  • Tamouchin Moharrami, Jafar Ai, Somayeh Ebrahimi Barough, Mohammad Nouri, Maryam Ziadi, Hossein Pashaiefar, Fatemeh Yazarlou, Mohammad Ahmadvand, Soheil Najafi, Mohammad Hossein Modarressi* Page 457
    Objective

    Endometrial receptivity plays a key role in pregnancy success in assisted reproduction cycles. Recent evidence suggests that seminal plasma (SP) and follicular fluid (FF) influence the uterine endometrium to improve implantation of the embryo and the establishment of pregnancy. In this study, we attempt to assess the influence of FF and SP on the expression levels of main endometrial receptivity genes (HOXA10, HOXA11, ITGAV, ITGB3 and LIF) in endometrial stromal cells.

    Materials and Methods

    In this experimental study, SP and FF were collected from 15 healthy fertile men and 15 healthy fertile women, respectively. Tissue specimens of the endometrium were obtained from 12 women undergoing hysterectomy for benign conditions. After endometrial stromal cell isolation and culture, dose- and time-dependent cytotoxic effects of pooled FF and SP on 3D-cultured endometrial cells were evaluated. A second independent set of 12 endometrium samples was treated under determined optimum conditions and evaluated for gene expression analysis using quantitative real-time polymerase chain reaction (qRT–PCR).

    Results

    The results of this study indicated that exposure of endometrial stromal cells to FF resulted in the elevated expression of HOXA10 (fold change=2.6, P=0.02), HOXA11 (fold change=3.3, P=0.002), LIF (fold change=4.6, P=0.0003), ITGB3 (fold change=3.5, P=0.012), and ITGAV (fold change=2.8, P=0.001) compared to untreated cells. In addition, we found that SP-treated endometrial cells showed increased mRNA levels of only the LIF gene (fold change=2.5, P=0.008) compared to untreated cells.

    Conclusion

    Human SP and FF may modulate the endometrial receptivity and improve the implantation rate in assisted reproduction cycles through the up-regulation of endometrial receptivity genes.

    Keywords: Endometrium, Follicular Fluid, Implantation, Seminal Plasma
  • Mehdi Totonchi, Babak Babaabasi, Hadi Najafi, Mojtaba Rezazadeh Valojerdi, Poopak Eftekhari Yazdi, Lila Karimian, Navid Almadani, AnahitaMohseni Meybodi, Morteza Kimiai, Mehri Mashayekhi, Tahereh Madani, Hamid Gourabi* Page 467
    Objective

    In vitro fertilization (IVF) is one of the most efficient approaches within the context of assisted reproductive technology (ART) to treat infertility. High pregnancy rates have become the major index of successful IVF in clinical studies. It is not clear yet which factors are certainly responsible for IVF success, as various outcomes were obtained in different IVF centers with different settings. In this study, we aimed to address controversies in the interpretation of promising results of IVF with respect to preimplantation genetic screening (PGS).

    Materials and Methods

    In this retrospective case series study, we built a dataset containing data from 213 IVF patient candidates for PGS (654 embryos) with blastomere biopsy at day 3 and trophectoderm biopsy in day 5, referred to Royan Institute, Tehran, Iran from 2015 to 2018. Next, the data were analyzed to find influential factors affecting success rate of ART cycles.

    Results

    Data analyses showed that regardless of PGS indications (ART failures, recurrent miscarriage, chromosomal abnormalities, etc.), the pregnancy rate is influenced by maternal and embryonic factors such as the age of mother as well as quantity and quality of transferred embryos. Furthermore, genotyping of embryos using array comparative genomic hybridization (aCGH) depicted the highest rate of chromosomal aberrations for chromosomes 1, 16 and 19 while the lowest frequency for chromosomes 11 and 17. Similarly, we detected 463 genetically abnormal embryos by aCGH, among which only 41.9% could be detected by classical fluorescent in situ hybridization (FISH) method.

    Conclusion

    This study not only highlighted the advantages of aCGH over the FISH method in detection of chromosomal abnormalities, but also emphasized the importance of genetic abnormality as an indication for determination of IVF success rate.

    Keywords: Array Comparative Genomic Hybridization, Assisted Reproductive Technology, In Vitro Fertilization, Preimplantation Genetic Screening
  • Khodaberdi Kalavi, Ogholniaz Jorjani, Mohammad Ali Faghihi, Seyed Javad Mowla* Page 476
    Objective

    Leishmaniasis is caused by members of the Leishmania species and constitute a group of infective diseases that range from cutaneous lesions to lethal visceral forms. In infected persons, macrophages recognize and eliminate the parasites via phagocytosis. In order to change a hostile environment into an environment adequate for survival and reproduction, the engulfed Leishmania species needs to modulate the function of its host macrophage. The expression patterns of cytokine genes such as interleukin-12 (IL-12), tumour necrosis factor-alpha (TNF-α), IL-1, and interferon-gamma (IFNγ) represent the immune response. In this study, we employed an RNA-seq approach for human monocyte-derived macrophages infected with Leishmania major (L. major) to decipher cytokine gene expression alterations in host macrophages.

    Materials and Methods

    In this descriptive study, human monocytes were isolated by magnetic activated cell sorting (MACS) and cultured in the presence of monocyte colony stimulating factor (M-CSF) to obtain the macrophages. Monocyte-derived macrophages were then co-cultured with metacyclic promastigotes of L. major for 4 hours. RNA isolation was performed using TRIzol reagent. RNA sequencing was performed using the Illumina sequencing platforms. Gene expression analysis was performed using a Bioconductor DESeq2 package.

    Results

    Our data revealed significant changes in immune response gene expressions in macrophages infected with L. major, with an up-regulation of cytokines and mostly down-regulation of their receptors.

    Conclusion

    The obtained data could shed more light on the biology of L. major and how the host cell responds to leishmaniasis.

    Keywords: Chemokines, Cytokines, Leishmania major, Macrophages, RNA Sequencing
  • Atefeh Rezaeian, Mohammad Karimian*, Abasalt Hossienzadeh Colagar Page 482
    Objective

    In this study, we evaluated the effects of promoter methylation of MTHFR on oligozoospermia risk, followed by an in silico analysis.

    Materials and Methods

    In a case-control study, semen samples were collected from infertile and healthy control men. MTHFR promoter region was amplified by methylation-specific polymerase chain reaction (PCR). Finally, the promoter region of MTHFR was analyzed by bioinformatics software.

    Results

    Our data revealed significant associations of CpG island promoter methylation with oligozoospermia in a case-control study. In silico analysis showed that promoter contains a strong nucleosome exclusion region, a bonafide CGIs, six PROSITE motifs without a defined TATA box and 14 transcription factor (TF) binding sites, which are directly involved in spermatogenesis

    Conclusion

    Based on our findings, methylation of the MTHFR gene promoter region may be a risk factor for oligozoospermia. However, this is a preliminary report representing data for future comprehensive studies to make a clinical conclusion on the potential biomarker role of methylation of this promoter in elevating susceptibility to oligozoospermia.

    Keywords: Bioinformatics, DNA Methylation, Male Infertility, Methylenetetrahydrofolate Reductase, Oligozoospermia
  • Mohammad Mahmoudi Asl, Reza Rahbarghazi, Rahim Beheshti, Alireza Alihemmati, Mohammad Reza Aliparasti, Ali Abedelahi* Page 491
    Objective

    Many attempts have been made to preserve fertility by improving the cryopreservation of the ovarian tissue. This current studyaimed to improve of direct cover vitrification (DCV) protocol on follicular preservation and angiogenesis in autografted ovarian tissue.

    Materials and Methods

    In this experimental study, sixty five female Balb/c mice (5-6 week-old) were anesthetized and their ovaries were dissected. The left ovaries were vitrified by DCV solution, thawed by descending concentrations of sucrose, and then autografted subcutaneously. The right ovaries were autografted with no vitrification procedure prior to transplantation. The animals were sacrificed under anesthesia on the 7th day after transplantation to obtain ovarian tissue. Follicular quality was assessed by histological and ultrastructure observations, and angiogenesis was examined by immunohistochemical staining and real-time polymerase chain reaction (PCR) analysis.

    Results

    The histological and ultrastructure features of the follicles preserved well after vitrification of the ovarian tissue by 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO). Revascularizationwas manifested prominently in the DCV1-vitrified/grafted ovaries by von Willebrand factor (vWF) and alpha smooth muscle actin (α-SMA) immunostaining. The ovarian tissue vitrified in DCV1 protocol had higher expression levels of angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) 7 days after autotransplantation (P<0.01).

    Conclusion

    These findings suggest that DCV with 10% of both EG and DMSO, is an effective cryopreservation solution for preservation of good quality follicles as well an upregulation of angiogenic factors after ovarian tissue transplantation.

    Keywords: Angiogenesis, Cryopreservation, Graft, Mouse, Ovary
  • Alireza Rajabzadeh, Fatemeh Rahbarizadeh, Davoud Ahmadvand, Maryam Kabir Salmani, Amir Ali Hamidieh* Page 502
    Objective

    Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospect in cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety, which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of less immunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likely overcome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a third generation lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation and cytotoxicity of redirected human T cells.

    Materials and Methods

    In this experimental study, we established a second generation novel CAR (VHH-based antiMUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembrane domain and signalling endodomains of CD28 and CD3ζ. Next, we constructed lentiviral vectors that contained this CAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cell surface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3+ T cells.

    Results

    The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CAR showed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxic activity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) compared with A431 (MUC1-negative) or untransduced T cells.

    Conclusion

    Our results suggested that, given the unique properties of VHHs to prevent immunogenic responses and tonic signalling, our novel VHH-based anti-MUC1 CAR might be effective for clinical purposes in cancer immunotherapy

    Keywords: Immunotherapy, Single-Chain Antibodies, T-Lymphocytes
  • Maryam Mohammad Sadeghipour, Mehdi Mahmoodi, Mojgan Noroozi Karimabad, Mohammad Reza Mirzaei, Mohammad Reza Hajizadeh* Page 514
    Objective

    Diosignin and 4-hydroxy-L-isulosine (4-OH-Ile) are the two active ingredients of Fenugreek (Trigonella foenumgraecum). Thus, in this study, we examined the effects of hydroalcoholic extract of fenugreek seeds (HEFS), diosgenin and 4-OH-Ile on the expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), peroxisome proliferator-activated receptor gamma (PPARγ) and low-density lipoprotein (LDL) receptor (LDLR) which are involved in lipid metabolism in SW480 cell line.

    Materials and Methods

    In this experimental study, SW480 cells were cultured in RPMI-1640 medium and treated with HEFS, diosignin, 4-OH-Ile or orlistat for 24 and 48 hours. Inhibitory concentration of 20% (IC20) was calculated using MTT method and cells were then pre-treated with the IC20 concentrations for 24 and 48 hours before RNA extraction and cDNA synthesis. Changes in the expression of ACC, FAS, PPARγ and LDLR genes were assayed by employing the real time-polymerase chain reaction (PCR) method.

    Results

    Our results showed a significant down-regulation in the expression of ACC (P<0.001 and P<0.001 after 24 and 48 hours, respectively) and FAS genes (P<0.001 and P<0.001 after 24 and 48 hours, respectively) in SW480 cells treated with HEFS, diosignin, 4-OH-Ile, or orlistat, but significant up-regulation in the expression of PPARγ (P<0.001 and P<0.001 after 24 and 48 hours, respectively) and LDLR (P=0.005 and P=0.001 after 24 and 48 hours, respectively).

    Conclusion

    According to the results of the present study, HEFS, diosgenin and 4-OH-Ile up or down-regulate the expression of some predominant genes involved in lipid metabolism pathway, similar to that observed for orlistat. These types of regulatory effects are presumably proper for the treatment of obesity and overweight.

    Keywords: Trigonella, Diosgenin, Orlistat, Obesity
  • Xiangjing Min, Yanling Guo, Yishan Zhou, Xiuping Chen* Page 523
    Objective

    Ulcerative colitis (UC) is a long-lasting inflammatory disease of the colon. Epidemiological studies showed that the
    prevalence and incidence of UC are increasing worldwide in recent years. Neferine is a natural alkaloid isolated from Nelumbo nucifera Gaertn that exerts a variety of biological activities. This study was designed to evaluate the protective effect of neferine on dextran sulfate sodium (DSS)-induced experimental UC in mice.

    Materials and Methods

    In this experimental study, 4% DSS was used to induce a mice model of UC. Neferine (5 and
    10 mg/kg) was administered by intraperitoneal injection (ip). Clinical symptoms and disease activity index (DAI) scores
    were recorded and calculated. Pathological changes of colon tissues were detected by Hematoxylin and Eosin (H&E)
    staining. The levels of inflammatory mediators were detected by ELISA kits. Western blotting and immunohistochemical
    analysis were used for the evaluation of protein expressions.

    Results

    Neferine treatment significantly alleviated DSS-induced UC by inhibiting weight loss, decreasing DAI scores,
    and alleviating the pathological changes in colon tissues. Furthermore, neferine significantly decreased serum levels
    of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 and
    increased serum levels of anti-inflammatory cytokine IL-10. The increased myeloperoxidase (MPO) activity and nitric
    oxide (NO) in colon tissues were also inhibited. In addition, neferine significantly down-regulated inducible NO synthase
    (iNOS), cyclooxygenase-2 (COX-2), and intercellular cell adhesion molecule-1 (ICAM-1) expression in colon tissues.

    Conclusion

    These results provided evidence that neferine could protect against DSS-induced UC symptoms in an
    experimental mice model. This effect might be mediated through inhibition of inflammation.

    Keywords: Dextran Sulfate Sodium, Inflammation, Neferine, Ulcerative Colitis
  • Shima Ebadollahi, Mahdi Pouramir*, Ebrahim Zabihi, Monireh Golpour, Mohsen Aghajanpour, Mir Page 532
    Objective

    Arbutin (p-hydroxyphenyl-β-D-glucopyranoside) possesses beneficial functions including antioxidant, antiinflammatory, and anti-tumoral activities. Due to the important role of oxidative stress and apoptosis in the successful treatment of cancer, understanding mechanisms that lead to apoptosis in cancer cells, is essential. The purpose of the current study was to evaluate the effect of arbutin on tert-butyl hydroperoxide (t-BHP)-induced oxidative stress and the related mechanisms in fibroblast and Lymph Node Carcinoma of the Prostate (LNCaP) cells.

    Materials and Methods

    In this experimental study, the LNCaP and fibroblast cell lines were pre-treated with arbutin (50, 250 and 1000 μM). After 24 hours, t-BHP (30 and 35 μM) was added to the cells. Viability was measured (at 24 and 48 hours) using MTT assay. The antioxidant effect of arbutin was measured by FRAP assay. The mRNA expression of P53 and BAX/BCL-2 ratio were measured using quantitative polymerase chain reaction (PCR). The percentage of apoptotic or necrotic cells was determined using a double staining annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit.

    Results

    Arbutin pre-treatment increased the total antioxidative power and cell viability in the MTT assay and reduced BAX/BCL-2 ratio, P53 mRNA expression and necrosis in fibroblasts exposed to the oxidative agent (P<0.001). In addition, our results showed that arbutin can decrease cell viability, induce apoptosis and increase BAX/BCL-2 ratio in LNCaP cells at some specific concentrations (P<0.001).

    Conclusion

    Arbutin as a potential functional β-D-glucopyranoside has strong ability to selectively protect fibroblasts against t-BHP-induced cell damage and induce apoptosis in LNCaP cells.

    Keywords: Arbutin, Fibroblast, LNCaP, Oxidative Stress, Tert-Butyl Hydroperoxide
  • Ali Rajabi, Zohreh Sharifi, Fatemeh Yari, Mohammadreza Deyhim, Mohammadali Jalili Page 542
    Objective

    MicroRNAs (miRNAs) are short, noncoding RNAs that play vital roles in gene regulation. It has been shown that storage has an effect on platelet miRNAs. MiR-16 is highly expressed in platelets and it appears to target the genes involved in cell death. It has been shown that platelets could be stored in Composol for a longer period of time. The aim of the present study was to assess and compare the expression pattern of miR-16 in platelet concentrates (PCs) in plasma and Composol media.

    Materials and Methods

    In an experimental study, ten PC bags were collected and each bag was divided into two separate bags, one with the 70% Composol and the other with only plasma. Both bags were stored for 7 days at 22˚C and tested on days 1, 3, 5, and 7 of storage. For each sample, we performed quantitative real-time polymerase chain reaction (qRT-PCR). The water-soluble tetrazolium salt-1 (WST-1) test was used to assess platelet viability in all of the samples. Statistical analysis was done by SPSS and REST software. A P<0.05 was considered statistically significant.

    Results

    miR-16 was significantly elevated during the storage days, with fold changes of 3.47 (plasma) and 2.77 (Composol). The Composol group had significantly decreased miR-16 expression compared with the plasma group. Results of the WST-1 test showed less decrease in optical density (OD) in the Composol group (0.93 ± 0.4) during the storage days compared with the plasma group (0.75 ± 0.3).

    Conclusion

    Our finding supported results from previous studies that reported an increase in miR-16 expression during platelet storage. In addition, miR-16 down-regulation in Composol medium implied that Composol might be a good solution for long-term platelet storage because it has the potential to elevate the shelf-life of platelets stored at 22˚C.

    Keywords: Blood Platelets, MicroRNAs, miR-16, Platelet Storage
  • Nafissa Telailia*, Sylvain Fisson, Hacène Frih Page 548
    Objective

    von Frey Filament (vFF) is an aesthesiometer to measure paw withdrawal thresholds. Our aim was to validate the manually von Frey test technique for assessing neuropathic pain behavioral signs in a sciatic nerve ligation model.

    Materials and Methods

    In this experimental study, peripheral neuropathic pain associated with sciatic nerve chronic ligation (SN-CL) was induced. Filaments used against posterior pad mid-plantar region using a simplified up-down method (SUDO). In addition to baseline withdrawal thresholds, the behavioral test was repeated after surgery thrice more with an interval of ten days. vFF (2 to 26 g) were used in ascending order for hyperalgesia assessment.

    Results

    In SN-CL rats, the results validate a loss of pain sensation, resulted in, long-lasting ipsilateral allodynia with the development of contralateral allodynia later and an extraterritorial development of neuropathic signs. Variability for the development of ipsilateral and contralateral allodynia over time was noted in sham (SH) control rats. SN-CL group showed a contralateral hyperalgesia development just at the 16th-day after surgery with an absence of ipsilateral hyperalgesia development at the different days of paw withdrawal thresholds measurements.

    Conclusion

    Manually vFF test technique was successfully used for assessing neuropathic pain behavioral signs in sciatic a nerve ligation model with the absence of ipsilateral hyperalgesia development.

    Keywords: Filaments, Injury, Mechanical Allodynia, Mechanical Hyperalgesia Neuropathic Pain
  • Samaneh Sharif*, Mohammad Hossein Ghahremani, Masoud Soleimani Page 556
    Objective

    Neuroblastoma (NB) is one of the frequently observed malignant solid tumors of childhood and infancy, accounting for 15% of pediatric cancer deaths. Recently, the approach of differentiation therapy has shown considerable promise in effective treatment of NB patients. MiR-124 belongs to the nervous system-specific miRNAs that is increased during neuronal differentiation and may be one of the potential therapeutic targets for the treatment of NB. However, despite its well-established therapeutic potential, its efficient delivery to the targeted tumor cells is a challenging task. Mesenchymal stem cells (MSCs) are multipotent adult progenitor cells that have antitumor properties, and they can migrate to cancer cells and tumors. This study aimed to assess whether human adipose tissue-derived MSCs (hADMSCs) have the potential to deliver exogenous miRNAs to NB cells to induce differentiation and decrease proliferation of cancer cells.

    Materials and Methods

    In this experimental study, hAD-MSCs were isolated, cultured, and differentiated. The M17 human NB cell line were also cultured. A specific type of miRNAs, i.e., miR-124 was successfully delivered to M17 NB cells with the aid of hAD-MSCs using the direct or indirect (exosome-based) contacts.

    Results

    It was shown that indirect delivery of miR-124 considerably decreased the proliferation of NB cells and induced their differentiation.

    Conclusion

    The results suggest the use of delivered exogenous miRNAs by the derived exosomes from hAD-MSCs as a novel cell-free stem cell-based therapy for NB cancer.

    Keywords: Differentiation, Exosome, Mesenchymal Stem Cells, MiR-124, Neuroblastoma
  • Alireza Lotfi, Mitra Soleimani, Nazem Ghasemi* Page 565
    Objective

    Astaxanthin (AST) is a carotenoid with anti-oxidative, anti-inflammatory, and anti-apoptotic properties. It has also been reported that AST exerts protective effects against neurodegenerative diseases and reduces oxidative stress-induced the central nervous system (CNS) injury. In this study, we aimed to evaluate the protective potential of AST in inhibiting demyelination and oligodendrocyte death in a rat model of multiple sclerosis (MS).

    Materials and Methods

    In this experimental study, forty Wistar rats were randomly assigned to four experimental groups: control group (with normal feeding), cuprizone (CPZ group) that daily received 0.6% CPZ for 4 weeks, sham group that daily received 0.6% CPZ plus dimethyl sulfoxid (DMSO) for 4 weeks, and AST group that daily received 0.6% CPZ and after 12 hours were treated with AST (3 mg/kg), for 4 weeks. Muscle strength was evaluated by the behavioral basket test at the end of every week for 4 weeks. Luxol Fast Blue (LFB) staining was utilized for the identification of myelination and demyelination. Myelin density was evaluated by the ImageJ software. The expression of A2B5 (oligodendrocyte precursor protein) and myelin oligodendrocyte protein (MOG) were assessed by immunohistochemistry (IHC) and the expression of myelin basic protein (MBP), MOG, and platelet-derived growth factor-alpha (PDGFR-α) genes was examined by the real-time polymerase chain reaction (RT-PCR) technique.

    Results

    The administration of AST reduced the oligodendrocyte damage and myelin sheath disruption in a rat model of MS. The basket behavioral test showed the improvement of muscle strength in the AST group compared with CPZ and sham groups. Besides, the results of real-time PCR and IHC indicated the beneficial effects of AST in declining demyelination and oligodendrocyte death in a rat model of MS.

    Conclusion

    AST reduces damages to the myelin sheath and oligodendrocyte death in a rat model of MS.

    Keywords: Astaxanthin, Cuprizone, Multiple Sclerosis, Oligodendrocyte
  • Jose Luis Royo* Page 572

    Recently, it has been proposed the association of a common deletion affecting toll-like receptor 2 promoter (-196 to -177) to type 2 diabetes mellitus risk. However, genotyping results show a significant deviation from the HardyWeinberg Equilibrium (HWE). The law of Hardy-Weinberg shows that for an autosomal biallelic marker with allele frequencies fA=p and fa=q, the proportion of subjects with genotypes AA, Aa, and aa should follow the following: fAA=p2, f Aa=2pq, and faa=q2. Departure from HWE or Hardy-Weinberg Disequilibrium (HWD) in a human control population can be caused by natural factors such as selective pressure against a certain genotype. However their prevalence is scarce and magnitude of effect over the HWE are small. Other factors such as inbreeding caused by consanguinity, population stratification, or technical problems in genotyping are more usual. Nevertheless, if the control population follows a perfect HWE, the presence HWD among patients might be explained by the genetic association and evidencing a real link between the locus and the trait under study. However, HWD affecting both cases and controls, such as the one reported might be explained by one of the aforementioned issues.