Iranian Journal of Basic Medical Sciences
Year:2018 | Volume:21 | Issue:3|Papers Count:15

Objective (s): Antrodia cinnamomea (AC) is found with anti-inflammatory and immunomodulatory biological activities. In this study, we investigated the anti-hepatitis effect of the emulsified AC extract from RO water or supercritical fluid CO2 with ethanol co-solvent extract methods of AC preparations.Materials and Methods: Five groups of eight to ten weeks male rats with a count of ten for each group were studied to evaluate the protection of two kinds of AC extract from hepatic injury. Acute liver injury of rats was induced by injecting 40% carbon tetrachloride (CCl4) 1 mg/kg intraperitoneally. Positive and negative control groups rats were perfused with CCl4 or isotonic saline, respectively. Experimental groups received oral administration once/day of AC preparations before CCl4 treatment: water AC extract (WAE group), or emulsified AC extract from supercritical fluid extraction (EAE group) for 5 days, and sacrificed on the 6th day and the blood and liver samples were collected under chloral hydrate anesthesia. The anti-inflammatory, antioxidant markers, and relevant signaling pathways were measured (AST, ALT, ROS, IL-1, IL-6, NO, and COX-2, MAPKs, and caspase-3).Results: EAE at 50 mg/kg significantly decreased the serum AST, ALT, IL-1, IL-6, NO, and ROS levels. Both extracts reduced the activation of p-ERK in the liver samples, but EAE inhibited COX-2 and caspase-3 protein expression better than WAE. The EAE ameliorated CCl4-induced hepatic injury significantly; as compared with WAE and the positive control.Conclusion: The hepatoprotection of EAE could be attributed to the antioxidant and anti-inflammatory effects of Antrodia.

Objective (s): When the nerve is injured near its entrance to the muscle belly, we cannot perform conventional methods. One useful method in such a situation is neurotization surgery. In this study, Bone marrow mesenchymal stem cells (BMSCs) implanted into the paralyzed muscle after neurotization surgery. These cells can stimulate axon growth and motor endplate formation, also prevent muscle atrophy.Materials and Methods: Thirty-six adult male Sprague-Dawley rats were randomized into six groups: intact group, sham surgery group, control group, DMEM group, cell+DMEM group, denervated group. The motor nerve of the lateral head of gastrocnemius muscle was cut, and the proximal portion of the severed nerve was transplanted to the proximal third of the muscle paralysis. BMSCs with/or DMEM was injected into the site of injury. All animals were evaluated by withdrawal reflex latency (WRL), electromyography, muscle weight, histology and immunohistochemistry.Results: The WRL difference between the control and cell+DMEM groups at weeks 4 and 12 post-operation was statistically significant (P<0.05). The mean number of motor end plates in cell+DMEM group was more than control group (P<0.05). At 12 weeks post-operation, the difference of the mean nerve conduction velocity (NCV) between cell treated group and sham surgery groups were not statistically significant (P>0.05). In weeks 4 and 12 post-operation, the mean fiber diameters in cell+DMEM group were more than control group (P<0.05).Conclusion: The results of this study demonstrate that transplantation of BMSCs after neurotization surgery, prevent muscle atrophy and improve muscle function.

Objective (s): Inflammation is involved in various forms of pulmonary arterial hypertension (PAH). Although the pathophysiology of PAH remains uncertain, NF-kB and p38 mitogen-activated protein kinase (p38 MAPK) has been reported to be associated with many inflammatory mediators of PAH. This study aimed to evaluate the effect of chronic intermittent hypobaric hypoxia (CIHH) on pulmonary inflammation and remodeling in monocrotaline (MCT) induced PAH in rats.Materials and Methods: An in vivo model of PAH induced by MCT was employed. Statistical analyses were done using one-way analysis of variance (ANOVA) or Fisher's LSD test for multiple comparisons.Results: Four weeks of CIHH exposure following MCT injection resulted in significant reduction of mean pulmonary artery pressure (mPAP) level and improvement of right ventricular hypertrophy (RVH). Morphometric analyses showed decreased wall thickness of pulmonary arterioles in MCT+CIHH treated rats. These findings are consistent with the decrease in Ki-67 immunostaining. Following CIHH treatments, apoptotic analysis showed a consistent decrease in T lymphocytes together with lower levels of CD4+ T cell subset as measured in spleen and blood samples. Furthermore, CIHH treatment resulted in markedly reduced expression of TNF-a and IL-6 via the inhibition of NF-kB and p38 MAPK activity in rat lungs.Conclusion: Altogether, these results provide new evidence relating to the mode of action of CIHH in the prevention of PAH induced by MCT.

Objective (s): Cucurbitacins exhibit a range of anti-cancer functions. We investigated the effects of cucurbitacins D, E, and I purified from Ecballium elaterium (L.) A. Rich fruits on some apoptotic and autophagy genes in human gastric cancer cell line AGS.Materials and Methods: Using quantitative reverse transcription PCR (qRT-PCR), the expression of LC3, VEGF, BAX, caspase-3, and c-MYC genes were quantified in AGS cells 24 hr after treatment with cucurbitacins D, E, and I at concentrations 0.3, 0.1 and 0.5 mg/ml, respectively. Cell cycle and death were analyzed by flowcytometry.Results: Purified cucurbitacins induced sub-G1 cell-cycle arrest and cell death in AGS cells and upregulated LC3 mRNA effectively, but showed a very low effect on BAX, caspase-3, and c-MYC mRNA levels. Also after treatment with cucurbitacin I at concentration 0.5 mg/ml, VEGF mRNA levels were increased about 4.4 times. Pairwise comparison of the effect of cucurbitacins D, E, and I on LC3 mRNA expression showed that the cucurbitacin I effect is 1.3 and 1.1 times that of cucurbitacins E and D, respectively; cucurbitacin D effect is 1.2 times that of cucurbitacin E (P-value <0.05). In silico analysis showed that among autophagy genes, LC3 has an important gastric cancer rank relation.Conclusion: Cucurbitacins D, E, and I purified from E. elaterium fruits upregulate LC3 and induce sub-G1 cell-cycle arrest and cell death in human gastric cancer cell line AGS. Cucurbitacin I effect on LC3 mRNA expression is significantly more than that of cucurbitacins E and D.

Objective (s): Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody.Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

Objective (s): Scutellarin (Scu) is the main effective constituent of Erigeron breviscapus which has anti-oxidant, anti-apoptotic, anti-inflammatory and other therapeutic properties. The purpose of this study was to investigate the protective effect of Scu on myocardial infarction (MI) induced by isoprenaline (ISO).Materials and Methods: The rats were subcutaneously injected with ISO (45 mg/kg) on the first day, then single tail-intravenously injected with different doses of Scu (10 mg/kg, 20 mg/kg, 40 mg/kg) for 7 consecutive days. The protective effect of Scu on ISO-induced MI was evaluated by measuring markers of heart injury in serum, levels of lipid peroxidation, and antioxidants in heart tissue, observing pathological changes of tissue, and detecting quantified expression of apoptotic-related family members and inflammation.Results: Compared with the model group, the concentration of troponin T (CTn-T) and troponin I (CTn-I), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in the serum all decreased in the Scu high dose group. The activities of superoxide dismutase (SOD), catalase (CAT), and the content of reduced glutathione (GSH) in heart increased, and the content of malondialdehyde (MDA) and inducible nitric oxide synthase (iNOS) decreased. In addition, the histopathologic aspects showed that pathological heart change was found in the model group, and was reduced to varying degrees in the Scu group. Moreover, the expression of Bax, P53, Caspase3, Caspase9, cytochrome C, NGAL, NFkB, IL-1b and IL-6 in the heart decreased, while the expression of Bcl2 increased.Conclusion: Scu could reduce the degree of MI induced by ISO by improving the antioxidant, anti-apoptotic, and anti-inflammatory capacities of the body.

Objective (s): Multiple sclerosis (MS) is considered as a chronic type of an inflammatory disease characterized by loss of myelin of CNS. Recent evidence indicates that Interleukin 17 (IL-17)-producing T helper cells (Th17 cells) population are increased and regulatory T cells (Treg cells) are decreased in MS. Despite extensive research in understanding the mechanism of Th17 and Treg differentiation, the role of microRNAs in MS is not completely understood. Thereby, as a step closer, we analyzed the expression profile of miR-9-5p and miR-106a-5p, and protein level of retinoic acid receptor (RAR)-related orphan receptor C (RORC; Th17 master transcription factor) as direct target of miR-106a-5p and forkhead box P3 (FOXP3; Treg master transcription factor) as indirect target of miR-9-5p in CD4+ T cells in two groups of relapsing and remitting in our relapsing-remitting MS (RR-MS) patients.Materials and Methods: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression of miRNAs and mRNAs, in 40 RR-MS patients and 11 healthy individuals. Thus, FOXP3 and RAR-related orphan receptor gt (RORgt) was assessed in CD4+T-cells by flow cytometry. We also investigated the role of these miRNAs in Th17/Treg differentiation pathway through bioinformatics tools.Results: An up-regulation of miR-9-5p and down-regulation of miR-106a-5p in relapsing phase of MS patients were observed compared to healthy controls. RORC and FOXP3 were up-regulated in relapsing and remitting phases of MS, respectively.Conclusion: Expression pattern of miR-9-5p and miR-106a-5p and their targets suggest a possible inducing role of miR-9-5p and suppressing role of miR-106a-5p in differentiation pathway of Th17 cells during MS pathogenesis.

Objective (s): Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in humans, particularly in children under 5 years and travelers in developing countries.To our knowledge, no vaccine is licensed yet to protect against ETEC infection. Like many Gram-negative pathogens, ETEC can secrete outer membrane vesicles (OMVs). These structures contain various immunogenic virulence proteins such as LT and therefore can be used as vaccine candidates. In this study we attempted to isolate the OMVs of ETEC cultivated at different temperatures and evaluate their immunogenicity and protective efficacy in a murine model of infection.Materials and Methods: OMVs was purified from bacterial supernatant by ultracentrifugation. OMVs were encapsulated in chitosan nanoparticles prepared by ionic gelation method within a layer of Eudragit L100 for oral delivery. Female BALB/c mice of 9 weeks’ old were immunized by parenteral injection and oral administration with free and encapsulated OMVs obtained from bacteria cultivated at 37oC and 42oC. The serum samples were collected and the antibody titers were measured by an enzyme-linked immunosorbent assay (ELISA).Results: The protein concentrations of OMVs were 3.47 mg/ml and 2.46 mg/ml for bacteria grown at 37oC and 42oC respectively. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 532 nm. The encapsulation efficiency of NP was 90%. Mice immunized with OMVs, inhibited the ETEC colonization in their small intestine and induced production of antibodies against LT toxin.Conclusion: The results obtained in this research place OMVs among promising candidates to be used for vaccination.

Objective (s): Recent studies revealed that microRNAs (miRNAs) may play crucial roles in the responses and pathologic processes of spinal cord injury (SCI). This study aimed to investigate the effect and the molecular basis of miR-103 on LPS-induced injuries in PC12 cells in vitro and SCI rats in vivo.Materials and Methods: PC12 cells were exposed to LPS to induce cell injuries to mimic the in vitro model of SCI. The expression of miR-103 and SOX2 in PC12 cells were altered by transient transfections. Cell viability and apoptotic cell rate were measured by CCK-8 assay and flow cytometry assay. Furthermore, Western blot analysis was performed to detect the expression levels of apoptosis- and autophagy- related proteins, MAPK/ERK pathway- and JAK/STAT pathway-related proteins. In addition, we also assessed the effect of miR-103 agomir on SCI rats.Results: LPS exposure induced cell injuries in PC12 cells. miR-103 overexpression significantly increased cell viability, reduced cell apoptosis and autophagy, and opposite results were observed in miR-103 inhibition. miR-103 attenuated LPS-induced injuries by indirect upregulation of SOX2. SOX2 overexpression protected PC12 cells against LPS-induced injuries, while SOX2 inhibition expedited LPS-induced cell injuries. Furthermore, miR-103 overexpression inhibited MAPK/ERK pathway and JAK/STAT pathway through upregulation of SOX2. We also found that miR-103 agomir inhibited cell apoptosis and autophagy in SCI rats.Conclusion: This study demonstrates that miR-103 may represent a protective effect against cell apoptosis and autophagy in LPS-injured PC12 cells and SCI rats by upregulation of SOX2 expression.

Objective (s): Traumatic brain injury (TBI) is one of the most common causes of death and disability in modern societies. The role of steroids and melatonin is recognized as a neuroprotective factor in traumatic injuries. This study examined the role of melatonin receptors in the neuroprotective effects of estrogen.Materials and Methods: Seventy female ovariectomized Wistar rats were divided into five groups and two subgroups. All animals underwent brain trauma. The groups were as follow: 1) trauma, 2) melatonin receptor antagonist vehicle + estrogen, 3) MT1 melatonin receptor antagonist + estrogen, 4) MT2 melatonin receptor antagonist+ estrogen, 5) MT3 melatonin receptor antagonist+ estrogen. Brain edema (24 hr), intracranial pressure (ICP) (-1, 0, 1, 4 and 24 hr) and blood–brain barrier (BBB) permeability (5 hr) and aquaporin (AQP4) expression (24 hr) were evaluated after TBI.Results: MT1, MT2 and MT3 melatonin receptors had anti-edema effects while MT1 and MT2 have a role in protecting BBB by estrogen. Furthermore, the activity of MT3 and MT2 melatonin receptors weakened the effect of estrogen on ICP. However, melatonin receptors had no role in the effect of estrogen on AQP4 protein.Conclusion: Based on the above results, it seems that melatonin receptors appear to influence the effect of estrogen in TBI without altering AQP4 expression. The role of the receptors is different in this interaction.

Objective (s): Therapeutic effect of many selectable methods applied in clinical practice for treating hypertrophic scar (HS) is not still so satisfactory. Meanwhile, a few medicines may lead to several undesirable complications. The traditional Chinese medicine, Rg3, has been reported for multiple antitumor effects previously. We have conducted series of animal experiments and confirmed the inhibitory effect of Rg3 in HS before. The aim of this study was to further verify the conclusions of previous studies and reveal the specific functional mechanisms of Rg3.Materials and Methods: The HS specimens were obtained from the patients aged from 15 to 36 years without systemic diseases and the primary cultured cells were isolated from the scar tissue and expanded in vitro. In every experiment, hypertrophic scar fibroblasts (HSFs) were divided into three groups and respectively cultured in medium with or without different Rg3 concentrations (50, 100 mg/ml). Cell viability assay, flow cytometry analysis (FCM), quantitative PCR, cell migration assay, immunofluorescence staining, western blot and ELISA were employed.Results: The outcomes demonstrated that Rg3 could suppress cell proliferation, vascularization and extracellular matrix (ECM) deposition of HSFs in vitro by TGF-b /SMAD and Erk signaling pathways. Significant statistical differences were between control group and Rg3-treated groups (P<0.05).Conclusion: This study provides sufficient in vitro evidences for Rg3 as a promising drug in the treatment of human HS.

Objective (s): To investigate the effect of miR-103 on the angiogenesis of ischemic stroke rats via targeting vascular endothelial growth factor (VEGF) at the molecular level.Materials and Methods: Rat models had received the middle cerebral artery occlusion (MCAO) or sham operation before grouping, and cell models of oxygen-glucose deprivation (OGD) were performed. FITC-dextran, matrigel, and Trans-well assays were used to evaluate the vascular density, tube formation, and cell migration respectively. The expression levels of miR-103 and VEGF were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Dual-luciferase assay was used for analyzing the targeting relationship between miR-103 and VEGF.Results: We found the reduced miR-103 in rats after MCAO. Down-regulating miR-103 with the miR-103 inhibitor enhanced VEGF, ameliorated the neurological scores, decreased infarct volume, and increased vascular density in rats after MCAO. Besides, in OGD human umbilical vein endothelial cells (HUVECs), inhibition of miR-103 could promote the increase of tube length and the migration of cells. Additionally, we found that miR-103 could directly target VEGF and thereby lead to the down-expression of VEGF. Meanwhile, si-VEGF could reverse the effect of miR-103 inhibitor on angiogenesis in rats subjected to MCAO.Conclusion: Inhibition of miR-103 could promote ischemic stroke angiogenesis and reduce infarction volume via enhancing VEGF, which provides a new target for the clinical treatment of ischemic stroke.

Objective (s): Radiotherapy is one of the most effective modalities of cancer therapy, but clinical responses of individual patients varies considerably. To enhance treatment efficiency it is essential to implement an individual-based treatment. The aim of present study was to identify the mechanism of intrinsic apoptosis pathway on radio sensitivity and normal tissue complications caused by the radiotherapy.Materials and Methods: Peripheral blood mononuclear cells from ten breast cancer patients were exposed to 6MV X-rays to deliver 1 and 2 Gy. Expression levels of Bax, Bcl-2, and Bax/Bcl-2 ratio were examined by relative quantitative RT-PCR. All the patients received similar tangential irradiation of the whole breast and conventional fractionation. Skin dosimetry was done by GAF Chromic EBT-3 film and clinical radio sensitivity was determined using the acute reactions to radiotherapy of the skin according to Radiation Therapy Oncology Group score. All statistical analyses were performed using Graph Pad Prism, version 7.01.Results: In the in-vitro experiment, Bax and Bax/Bcl-2 ratios were significantly increased with 1 and 2 Gy doses (P<0.001 and P<0.0001, respectively). Herein, the notable result was a significant correlation between dose-response curve slope (as an in-vitro radio sensitivity index) and acute skin toxicity score following irradiation (as a clinical radio sensitivity index). There was no significant relationship between skin dose and reactions (P>0.05 for all patients).Conclusion: Significant correlation between Bax/Bcl-2 ratio determined before radiation therapy and clinical response in the patients, can be used as a biomarker to identify radiosensitive individuals. However, further studies are required to validate radiation-induced apoptotic biomarkers.

Objective (s): Targeted next-generation sequencing (NGS) provides a consequential opportunity to elucidate genetic factors in known diseases, particularly in profoundly heterogeneous disorders such as non-syndromic hearing loss (NSHL). Hearing impairments could be classified into syndromic and non-syndromic types. This study intended to assess the significance of mutations in these genes to the autosomal recessive/dominant non-syndromic genetic load among Iranian families.Materials and Methods: Two families were involved in this research and two patients were examined by targeted next-generation sequencing. Here we report two novel mutations in the MYO7A and EYA1 genes in two patients detected by targeted NGS. They were confirmed by Sanger sequencing and quantitative real-time PCR techniques.Results: In this investigation, we identified a novel mutation in MYO7A, c.3751G>C, p.A1251P, along with another previously identified mutation (c.1708C>T) in one of the cases. This mutation is located in the MYTH4 protein domain which is a pivotal domain for the myosin function. Another finding in this research was a novel de-novo deletion which deletes the entire EYA1 coding region (EX1-18 DEL). Mutations in EYA1 gene have been found in branchiootorenal (BOR) syndrome. Interestingly the patient with EYA1 deletion did not show any other additional clinical implications apart from HL. This finding might argue for the sole involvement of EYA1 function in the mechanism of hearing.Conclusion: This investigation exhibited that the novel mutations in MYO7A, c.3751G>C, p.A1251P, and EYA1, EX1-18 DEL, were associated with NSHL. Our research increased the mutation spectrum of hearing loss in the Iranian population.

Objective (s): Diabetes is a metabolic syndrome which is associated with the worldwide major public health problems. There are many natural compounds from the sea-market, as a valuable aquatic source, along with the variety of health and therapeutic benefits. In the present research, with respect to the traditional and ethnic uses of Sargassum oligocystum algae for healing of some diseases which have similar metabolic mechanism to the diabetes, its anti-diabetic effects in animal model was proposed.Materials and Methods: The animals (rat) were divided into the normal control, diabetic control, positive control and, the test groups. The test groups were gavaged with oral doses of 150 and 300 mg/kg of algae hydroalcoholic extracts. After 30 days of intervention the serum glucose, cholesterol, triglyceride, HDLC, LDLC, insulin, insulin resistance, b-cells function and, the histopathology of pancreatic tissue were evaluated.Results: In animals that were fed with algae extracts a significant decrease in the fasting blood glucose, triglyceride and HOMA-IR and an increase in the HOMA-B with no significant impacts on the insulin, cholesterol and HDL were observed. Also, the histopathology evaluations in the groups which were treated with algae extract revealed the regeneration and reconstitution of damaged pancreatic b-cells.Conclusion: The results give evidence that, the S. oligocystum algae extract has a healing effect on diabetes which can be considered as a new research prospect for the natural therapy of diabetes.