Expression of the Apoptotic Proteins in Glioblastoma U87-MG Cell Line Treated by Botulinum Toxin

Background: Glioblastoma multiforme (GBM) is the most type of brain malignancy in adults. Radical excision surgery, chemotherapy, and radiotherapy in some cases are still unsuccessful, and most patients with GBM die within three to six months following diagnosis. Botulinum toxin type A (BtxA) has cellular toxin effects and suppresses the cell division of certain types of cancer cell lines in vivo and in vitro study. The present study designed to evaluate the apoptotic effect of BtxA on the GBM cell line.Material & methods: U87-MG GBM cell line cultured according to the routing protocols, divided into two groups including, trial (BtxA treated) and control groups. Cells of the trial group exposed to different doses of BtxA. The cell viability, cycle arrest, and pro-apoptotic proteins evaluated respectively by MTT assay, SubG1, and Western blotting.Results: MTT assay showed that the viability of the BtxA treated cells at doses of 1.45 Unit and other doses after 24 to 48 hours, significantly decreased (p<0.001) compared to the control groups. Apoptosis percentage of the SubG1 test also indicated that 1.45 Unit dose significantly increased in the cells exposed to BtxA compared to the control group in 24 hours. The expression of P53 and Caspase 3 proteins indicated a significant increase.Conclusion: BtxA induces apoptosis in U87- MG cell line via p53 and caspase three pathways and could have clinical applications. In vivo studies need to confirm the clinical application of the present findings

Neurotoxin Botulinum Inserts Apoptotic Effects on Certain Cancer Cell Lines Via Neural Niche of Tumors: A Molecular Study

Background: Background: Botulinum toxin A (BtxA) is a powerful neurotoxin reported to be effective as a cancer adjuvant therapy with fewer side effects. Previously we showed the apoptotic effects of BtxA on the GBM cell line (U87-MG). In order to confirm the positive neurotoxicity of BtxA on other cancer cell lines, including SK-OV-3, CHO, MCF-7, and PC-3, the present research has designed.Methods: The cell lines prepared, cultured, and exposed to different concentrations of BtxA for 24 and 48 hours. Using MTT, Annexin V/PI assays and western blotting, cell viability, and apoptosis studied.Results: Our results showed that different BtxA Botox concentrations led to significant cell death in each cell line in a dose-dependent manner (P < 0.001) but did not for PC-3 cells. The results of the Annexin V/PI staining indicated that BtxA induced apoptosis after 24 hours. The 1.45U, 1.75U, and 1.65U of BtxA respectively induced more than 50% apoptosis in MCF 7, SK-OV-3, and CHO cell lines. The results of caspase 3 showed more protein expression in the treated group compares to the control group.Conclusion: BtxA, as a neurotoxin, can insert therapeutic anti-cancer and apoptotic effects on various types of cancerous cells; further studies need to illuminate the possible mechanisms

Neuroprotective Effect of Chalcone on P53, Caspase III Expression and D2-Like Dopaminergic Receptor Up-Regulation in In-vitro Parkinson's Model

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder of the central nervous system (CNS). Several studies indicated abnormal cell death in neurodegenerative diseases. Chalcone is a compound of natural origin with various properties such as antioxidant, anti-inflammatory, and inhibition of apoptosis. We investigated the impact of chalcone in an in-vitro model of PD. Materials and Methods: PC12 cells were divided into four groups. Negative control, 6-hydroxy dopamine (6-OHDA) group (treatment with 75µM 6-OHDA), sham (treated with dimethyl sulfoxide), and the experimental groups with different dosages of chalcone treatment. Cell viability and reactive oxygen species (ROS) were assessed by MTT and ROS kit, respectively. The expressions of D2-like receptors, P53, and caspase III were evaluated by Western blotting. Results: We found that 6-OHDA induced cytotoxicity and ROS production. The viability results showed that all doses of chalcone significantly increased viability after 48 hours compared to the control group (P<0.01). The western blotting results showed that caspase III and P53 expression decreased significantly in the experimental groups compared to the 6-OHDA group. However, D2-like receptor expression did not significantly differ between the experimental and the 6-OHDA group.Conclusion: Complementary therapies, such as the use of antioxidants and the chalcone family, along with standard treatments for neurodegenerative diseases such as Parkinson's, may reduce the symptoms of the disease

Method of Housing and Transfer and Experimental Autoimmune Encephalomyelitis: An Experimental Study on C57BL/6

Background: Multiple Sclerosis (MS) is an autoimmune inflammatory demyelinating disease of human central nervous system. Although experimental autoimmune encephalomyelitis (EAE) is the most commonly used method to induce MS, there are unexpected results in the modeling outcomes, which led to inappropriate clinical score scaling. Recent studies focused on the possible factors that may affect the final outcome of EAE modeling. Some of these factors were observed and discussed in our experiment on C57BL/6 model. Objectives: The present research was carried out to find the possible effects of environmental factors, including transfer, handling, housing, and dark-light cycle on EAE modeling scoring. Materials and Methods: Twenty female mice (C57BL/6) were used that divided into two groups (n = 10) by random. The routine method of MS induction in mammals was used in both groups. Following induction, animals of group one were placed in a separated room with the least local translocation and handling, whereas animals of the second group were placed in the same room as the other animals with normal local allocation as others. The animals were observed and scored using routine clinical scoring for EAE. Results: Our data showed that the EAE induction in group one was significantly more successful than group two (with the mean score > 3). Conclusions: Although the EAE is still a scientific method to induce MS in rodents, it requires more attention to environmental factors that might influence the result. The mechanisms of these factors are unknown, but it seems that the role of housing environment should be taken into consideration

Berberine Supplement and Resistance Training May Ameliorate Diazinon Induced Neural Toxicity in Rat Hippocampus Via the Activation of the TrkB and ERK Signaling Pathway

Background: Diazinon is an organophosphate pesticide that is broadly applied to control insectswhich causes oxidative damages in hippocampus tissue. We aimed to examine whether resistancetraining and berberine supplementation can protect the hippocampus against berberine-inducedneural toxicity.Methods: Fifty-six male Wistar rats were assigned randomly into eight groups of seven including:Control (Ctrl), Sham (normal saline), T1 (diazinon + berberine chloride (2 mg/kg) + resistancetraining), T2 (diazinon + berberine chloride [15 mg/kg] + resistance training), T3 (diazinon), T4(diazinon +resistance training), T5 (diazinon + berberine chloride [2 mg/kg]), and T6 (diazinon+ berberine chloride [15 mg/kg]). In the experimental groups, diazinon was intraperitoneallyadministered at a dose of 1.5 mg/kg. In the training groups, rats were trained every three days for sixweeks and 8-12 dynamic movements (repetitions) during each climb (six climbs for two sets). Theexpression of hippocampus PI3K and CDK genes and TrkB and ERK protein levels were evaluated inthe brain of diazinon-treated rats.Results: The protein expression of ERK and TrkB were increased following the treatment of diazinonintoxicated rats with berberine and resistance training (P=0.001). The administration of berberineat a dose of 15 mg/kg in combination with resistance training significantly (P=0.001) decreasedthe cell death rate in the hippocampus. Diazinon treatment caused extensive apoptosis in thehippocampus region of the rats’ brain (P=0.001). The gene expression of PI3K and CDK wassignificantly increased and the cell death rate significantly decreased (P=0.001) in the hippocampusfollowing the treatment of rats with berberine and resistance training.Conclusion: Six weeks of resistance training in combination with berberine treatment significantlyreduced apoptosis in the hippocampus region of diazinon-intoxicated rats. It seems theneuroprotection effects of berberine and resistance training are mediated by the stimulation of theexpression of enzymes responsible for the antioxidant defense within neuronal cell

The Effect of Intrathecal Administration of Muscimol on Modulation of Neuropathic Pain Symptoms Resulting from Spinal Cord Injury; an Experimental Study

Introduction: Neuropathic pain can be very difficult to treat and it is one of the important medical challenging about pain treatments. Muscimol as a new agonist of gamma-Aminobutyric acid receptor type A (GABAA) have been introduced for pain management. Thus, the present study was performed to evaluate the pain alleviating effect of intrathecal injection of different doses of muscimol as GABAA receptor agonist in spinal cord injury (SCI) model of neuropathic pain. Methods: In the present experimental study male Wistar rats were treated by muscimol 0.01, 0.1 or 1 µg/10ul, intrathecally (i.t.) three weeks after induction of spinal cord injury using compression injury model. Neuropathic pain symptoms were assessed at before treatment, 15 minutes, one hour and three hours after muscimol administration. The time of peak effect and optimum dosage was assessed by repeated measures analysis of variance and analysis of covariance, respectively. Results: Muscimol with the dose of 0.01 µg in 15 minutes caused to improve the thermal hyperalgesia (df: 24, 5; F= 6.6; p<0.001), mechanical hyperalgesia (df: 24, 5; F= 7.8; p<0.001), cold allodynia (df: 24, 5; F= 6.96; p<0.001), and mechanical allodynia (df: 24, 5; F= 15.7; p<0.001). The effect of doses of 0.1 µg and 1 µg were also significant. In addition, the efficacy of different doses of muscimol didn't have difference on thermal hyperalgesia (df: 24, 5; F= 1.52; p= 0.24), mechanical hyperalgesia (df: 24, 5; F= 0.3; p= -0.75), cold allodynia (df: 24, 5; F= 0.8; p= -0.56), and mechanical allodynia (df: 24, 5; F= 1.75; p= 0.86). Conclusion: The finding of the present study revealed that using muscimol with doses of 0.01µg, 0.1µg, and 1 µg reduces the symptoms of neuropathic pain. Also the effect of GABAA agonist is short term and its effectiveness gradually decreases by time

Neurogenesis in the rat neonate's hippocampus with maternal short‐term REM sleep deprivation restores by royal jelly treatment

Abstract Background Numerous studies have shown the effects of rapid eye movement sleep deprivation (REM‐SD) on behavior and brain structures. The impact of REM‐SD on learning and memory, thus neurogenesis, has been reported in previous studies. Royal jelly (RJ) is known as the wealthiest biological nutrient with various physiological properties. This study aimed to study the possible effect of RJ on neurogenesis of the rat hippocampus neonates following exposure of mother to REM‐SD during pregnancy. Methods Thirty neonate rats from 15 pregnant Wistar rats were used. To induce REM‐SD, the flowerpot method was used. The pregnant rats were divided into five groups (n = 3): group 1, no treatment; group 2, REM‐SD; groups 3, 4, and 5, REM‐SD +RJ. The former group received 72 h REM‐SD during pregnancy (days 7, 14, 21), and the latter group received REM‐SD + RJ (three trial groups). At week 4, the rat neonates of all groups were sacrificed (n = 6 each group). Their brains were fixed, removed, and prepared for Nissl and Hoechst 33342 staining. By using real time polymerase chain reaction methode the brain‐derived neurotrophic factor BDNF gene expression was studied (RT‐PCR), brain‐derived neurotrophic factor (BDNF) gene expression was studied. The results were analyzed statistically, and the Pv  < .05 was considered significant. Results The results showed a significant decrease in the number of neurons in the hippocampus of neonatal rats of REM‐SD mothers compared to the neonates of the mother with REM‐SD + RJ. REM‐SD also led to an increase in apoptosis reaching the neonates from the REM‐SD + RJ animals. High expression of BDNF was observed in the hippocampus of the neonates from REM‐SD + RJ treated mothers. Conclusion RJ acts as a neuroprotective agent that could compensate for the effects of REM‐SD on learning and memory via restoring neurogenesis

5-HT2A Serotonin Receptor Density in Adult Male Rats’ Hippocampus after Morphine-based Conditioned Place Preference

Introduction: A close interaction exists between the brain opioid and serotonin (5-HT)&nbsp;neurotransmitter systems. Brain neurotransmitter 5-HT plays an important role in the regulation&nbsp;of reward-related processing. However, a few studies have investigated the potential role of&nbsp;5-HT2A receptors in this behavior. Therefore, the aim of the present study was to assess the influence of morphine and Conditioned Place Preference (CPP) on the density of 5-HT2A receptor&nbsp;in neurons of rat hippocampal formation. Methods: Morphine (10 mg/kg, IP) was injected in male Wistar rats for 7 consecutive days&nbsp;(intervention group), but control rats received just normal saline (1 mL/kg, IP). We used a&nbsp;hotplate test of analgesia to assess induction of tolerance to analgesic effects of morphine on days&nbsp;1 and 8 of injections. Later, two groups of rats were sacrificed one day after 7 days of injections,&nbsp;their whole brains removed, and the striatum and PFC immediately dissected. Then, the NR1&nbsp;gene expression was examined with a semi-quantitative RT-PCR method. Results: Our data showed that the maximum response was obtained with 2.5 mg/kg of morphine.&nbsp;The density of 5-HT2A receptor in different areas of the hippocampus increased significantly at&nbsp;sham-morphine and CPP groups (P<0.05). On the other hand, the CPP groups had more 5-HT2A&nbsp;receptors than sham-morphine groups and also the sham-morphine groups had more 5-HT2A&nbsp;receptors than the control groups. Conclusion: We concluded that the phenomenon of conditioned place preference induced by&nbsp;morphine can cause a significant increase in the number of serotonin 5-HT2A receptors in neurons&nbsp;of all areas of hippocampus

The Effect of Resistance Training and Berberine Chloride on the Apoptosis-related Unfolded Protein Response Signaling Pathway in the Hippocampus of Diazinon-poisoned Rats

Introduction: Diazinon is one of the most widely-used organophosphate pesticides in the world. This toxin enters the body in various ways and induces oxidative stress in various tissues. It has been proved that activation of Unfolded Protein Response (UPR) under oxidative stress is a steady mechanism for maintaining cell function and survival. Therefore, the present study aimed to review the effect of Resistance Training (RT) and Berberine Chloride (BC) on the apoptosis-related UPR signaling pathway in the hippocampus of diazinon-poisoned rats. Methods: In this experimental study, 40 male Wistar rats weighing 250 ±50 g were randomly divided into eight groups of five rats of 1) diazinon + 2 mg/kg BC + RT, 2) diazinon + 15 mg/kg BC + RT, 3) diazinon, 4) diazinon + RT, 5) diazinon + 2 mg/kg BC, 6) diazinon + 15 mg/ kg BC, 7) healthy control, and 8) sham. The groups were treated for 5 weeks. At the end of the fifth week, ATF-4, ATF-6, and CHOP gene expression in hippocampus tissue were measured by quantitative real-time RT-PCR. Results: Diazinon significantly increased the expression of ATF-4, ATF-6, and CHOP in the hippocampus tissue of rats. Administrating 15 mg/kg BC with RT significantly decreased these genes, indicating a decrease in the rate of apoptosis in the hippocampus. Conclusion: This study showed that RT and BC have a protective effect against diazinon-induced toxicity in the hippocampus

Association of Age with the Expression of Denervation Marker in the Neuromuscular Junction of Male Wistar Rats

Background and Objectives: One of the major problems during aging is sarcopenia, which one of its important involved mechanisms is loss of motor neurons. The aim of this study was to investigate the association of age with the expression of denervation marker in the neuromuscular junction of male Wistar rats.   Methods: In this study, 7 young adult male rats of 4 to 6 months of age and 7 old male rats of 24 to 26 months of age, were tested. after a two-week familiarization period, the soleus and plantaris muscles of the rats were removed, embedded, and subjected to immunohistochemically staining to determine the neuromuscular junction site and neural cell adhesion molecule (NCAM) expression rate. The results were analyzed by analysis of variance at the significant level of p ≤ 0.01.   Results: The results showed that the expression of NCAM was significantly higher in the aged group compared to the young adult group, and in both groups, the expression rate of this protein in the fast plantaris muscle was more than slow soleus muscle.   Conclusion: With age, the peripheral nerve degradation process occurs in both slow contraction muscles and fast contraction muscles, but the severity of this denervation is higher in the fast contraction muscles compared to the slow contraction muscles