Aging exaggerates pulpal pain sensation by increasing the expression levels of nociceptive neuropeptides and inflammatory cytokines.

BACKGROUND: Dental pain is a main clinical problem in the elderly population and its assessment and treatment make special challenges for health care services. However, the age-induced alteration in dental pain perception and the underlying molecular mechanism(s) has not yet been fully clarified. METHODS: Here, the effect of aging on nociceptive behaviors following inflammatory dental pulp pain was evaluated. Since prostaglandins, nociceptive neuropeptides, and inflammatory cytokines have critical roles in the development of aging as well as pain signaling, the expression levels of COX-2, CGRP, IL-1ß, IL-6, TNF-α and its converting enzyme TACE were assessed in the trigeminal ganglion of young and aged rats with dental pain. Dental pulp pain was induced by intradental application of capsaicin (100 µg). The immunofluorescence (COX-2 and CGRP) and western blot techniques were used. RESULTS: The data showed that aged animals have different pattern of pain. So that, the mean of nociceptive scores was significantly greater in aged rats at 10 and 15 min after capsaicin injection. In aged rats, dental pain was persisting over 7 h, while it was disappeared at 300 min in young rats. Molecular data showed that dental pain significantly increased the expression of COX-2, CGRP, IL-1ß, IL-6, TNF-α and TACE in the trigeminal ganglion of the young and aged rats. In addition, the amount of those parameters, except TACE, in capsaicin-treated aged animals were significantly (p < 0.05) greater than those in capsaicin-treated young rats. CONCLUSION: It seems that the induction of pro-inflammatory cytokines in an acute inflammatory pulpal pain model may contribute, at least in part to the increased nociceptive behaviors and pain perception in aged rats.

Apelin signalling in the periaqueductal grey matter alleviates capsaicin-evoked pulpal nocifensive behaviour and capsaicin-induced spatial learning and memory impairments in rat.

AIM: Pulpal pain is a common orofacial health issue that has been linked to cognitive impairment. Because of its prominent role in pain modulation and cognitive impairment, apelin (Apl) is regarded as a promising target for clinical pain management. The role of Apl in orofacial pain, however, is unknown. The purpose of this study was to determine the effects of intra-periaqueductal grey matter (PAG) administrations of Apl-13 on capsaicin-evoked pulpal nocifensive behaviour and capsaicin-induced spatial learning and memory impairments in rats. METHODOLOGY: Forty-nine male Wistar rats (200-250 g) were randomly divided into seven groups (n = 7 per group). The groups included: untreated intact, capsaicin (Caps) only, three Caps+Apl groups that received different dosages of intra-PAG injection of Apl-13 (1, 2 and 3 µg/rat) 20 min prior to capsaicin application, and two Apl+antagonist groups that received Apl receptor antagonist or naloxone (a µ opioid receptor) 20 min before Apl injection. Learning and memory were assessed using the Morris water maze test. One-way analysis of variance followed by Tukey post hoc tests was used for statistical analysis. RESULTS: Intra-PAG administration of Apl-13 significantly reduced the capsaicin-induced nocifensive behaviour (p < .01). This antinociception effect was inhibited by F13A and naloxone. Apl-13 inhibited nociception-induced learning and memory deficits (p < .01). The cognitive effects were also blocked by pre-treatment administration of F13A (3 µg/rat). CONCLUSIONS: These findings indicated that Apl-13, via Apl receptors (AR or APJ) and µ opioid receptors, alleviated capsaicin-induced dental nocifensive behaviour and protected against nociception-induced learning and memory impairments. As a result of our findings, Apl appears to be a promising analgesic option for further research in orofacial pain models and clinical trials.

Effects of short environmental enrichment on early-life adversity induced cognitive alternations in adolescent rats.

Early-life experiences, including parental care, affect cognitive performance later in life. Being exposed to early-life maternal separation (MS) increases susceptibility to stress-related psychopathology. Previous studies suggest that MS could induce learning and memory impairments. Since enriched environment (EE) provides more opportunities for exploration and social interaction, in the present study we evaluated the effects of a short EE paradigm with a duration of 13 days on cognitive abilities of maternally separated rats (MS; 180 min/day, postnatal day (PND) 1-21) during adolescence in four experimental groups: Control, Control+EE, MS, and MS+EE. Plasma corticosterone (CORT) and brain-derived neurotrophic factor (BDNF) levels were also measured in experimental animals. We also studied the induction of long-term potentiation (LTP) in the slices of hippocampal CA1 area. The behavioral and electrophysiological assessments were started at PND 35. MS caused higher basal CORT levels in plasma and impaired spatial learning, memory, and social interaction. LTP induction was also impaired in MS rats and plasma BDNF levels were reduced in these animals. MS also induced more anxiety-like behavior. Short EE reduced plasma CORT levels had the potential to improve locomotor activity and anxiety-like behavior in MS+EE rats and reversed MS-induced impairments of spatial learning, memory, and social behavior. LTP induction and plasma BDNF levels were also enhanced in MS+EE rats. We concluded that short EE might be considered as a therapeutic strategy for promoting cognition.

Activation of L-type calcium channels and attenuation of oxidative stress are involved in the improving effect of methyl jasmonate on learning and memory and its anxiolytic property in rats.

The present study was designed to evaluate the effect of plant bioactive compound methyl jasmonate on learning and memory, anxiety-like behaviors, and brain oxidative stress in rats. It has been indicated that methyl jasmonate stimulates calcium-binding protein expression and increases intracellular calcium (Ca2+). Therefore, we investigated the potential role of L-type calcium channel on methyl jasmonate effects. The animals were intracerebroventriculary (i.c.v.) injected with different doses of methyl jasmonate (0.5, 2.5, and 5 µg/rat). L-type calcium channel blocker (nifedipine 5 µg/rat, i.c.v.) was injected 30 min before methyl jasmonate (5 µg/rat). Shuttle box apparatus was used to evaluate passive avoidance memory. Anxiety-like behaviors were assessed by open field and elevated plus maze tests. Lastly, oxidative stress-related indices were assessed in hippocampus and prefrontal cortex. The data showed that methyl jasmonate dose-dependently could improve passive avoidance learning and memory and reduce anxiogenic behaviors. The methyl jasmonate effects were significantly prevented by nifedipine. Furthermore, central microinjection of methyl jasmonate significantly decreased hydrogen peroxide concentration, and increased reactive oxygen species scavenger activity (catalase and peroxide enzymes) in rats' hippocampus as well as prefrontal cortex. Indeed, the results indicated that the beneficial effects of methyl jasmonate on learning and memory and anxiety might be partly associated with L-type calcium channel and partly on the inhibition of oxidant indices.

Pain influences food preference and food-related memory by activating the basolateral amygdala in rats.

The amygdala has been demonstrated to contribute to pain-related behavior and food preference. Here, the effect of pain on food preference and food-matched visual-cue memory, in the presence or absence of a basolateral amygdala (BLA) lesion, has been evaluated using a novel innovative apparatus and protocol. Forty adult male Wistar rats were randomly divided into five groups (n = 8) as follows: control, pain, ibuprofen + pain, BLA lesion, BLA lesion + pain groups. Bilateral lesions of the BLA were produced by passing a current of 1.5 mA for 7 s. Pain was induced on the right hind paw of the rats by sub-plantar injection of 50 µl of 2.5% formalin. The animals were encountered with four different meals including wholemeal, wholemeal + sugar, white flour, and biscuit. Each test session consisted of six trials with inter-trial intervals of 15 min. The number of visits to each meal zone and port, the amount of time spent in each food zone and port, traveled distance in each food zone, food consumption per each visit and the total food consumption were recorded. The control group showed a high biscuit preference and low white flour preference. Rats suffering BLA lesion and rats in the BLA lesion + pain group exhibited a shifted preference curve. They had a bias toward eating wholemeal + sugar rather than white flour and biscuit. This group also showed an impaired spatial memory. In conclusion, our findings suggest that the BLA may be involved in pain-induced food preference and food-matched visual-cue memory.

Focal unilateral mechanical lesion in barrel cortex impairs rat's abilities to discriminate textures.

PURPOSE/AIM OF THE STUDY: Whiskers are important sensory organs that play a key role in rodents' discriminative and exploration behaviours and unilateral injuries of the somatosensory cortex related to whisker barrel cortex can change the activity of neurons in the intact contralateral barrel cortex. We evaluated the effects of unilateral mechanical lesion of right barrel cortex on novel texture discrimination in behavioural test and neuronal responses of left barrel cortex. MATERIALS AND METHODS: Ten days after a unilateral mechanical lesion in the right barrel cortex, adult male rats were experimented regarding three paired different textures in novel texture discrimination test dependent on whiskers. In addition, responses of left barrel cortical neurons to controlled deflections of right whiskers were recorded using extracellular single-unit recordings technique. RESULTS: Data analysis showed that the discrimination ratio and preference indexes as criteria to find a novel texture significantly decreased in the lesion group compared to the intact rats (p < .05). In electrophysiological level, the barrel neural cortical spontaneous activity and the ON and OFF response magnitude of intact barrel cortex neurons in the lesion group decreased compared to the intact group (p < .05). CONCLUSIONS: The present study showed that unilateral mechanical lesion in the rats' barrel cortex cause a decrease in their abilities for discriminating textures, as well as, the anaesthetized rats whose response properties of intact barrel cortical area changed to whisker deflection, too. These changes can influence on the ability of rats to differentiate textures.

The citrus flavanone naringenin prevents the development of morphine analgesic tolerance and conditioned place preference in male rats.

BACKGROUND: Opioids are effective analgesics in the management of chronic pain. However, their clinical use is hindered by adverse side effects such as addiction and analgesic tolerance. Naringenin is a common polyphenolic constituent of the citrus fruits and is one of the most commonly consumed flavonoids within our regular diet. However, its influences on opioid tolerance and addiction have not yet been clarified. OBJECTIVES: To examine the effect of different doses of naringenin on analgesic tolerance, conditioned place preference and neuroinflammation in morphine-exposed rats. METHODS: Analgesic tolerance was induced by the injection of 10 mg/kg morphine twice daily for 8 days in 70 male Wistar rats. To evaluate the effect of naringenin on the development of morphine tolerance, different doses (10, 25 and 50 mg/kg i.p.) were injected 15 min before morphine. The tail-flick test was used to assess nociceptive threshold. Conditioned place preference test was used to evaluate morphine-seeking behaviors. The lumbar spinal cord was assayed to determine glial fibrillary acidic protein (GFAP) and cyclooxygenase-2 (COX-2) levels by Western blotting. RESULTS: The data showed that naringenin could significantly prevent morphine tolerance (p < .001) and conditioned place preference. In addition, chronic morphine-induced GFAP and COX-2 overexpression was significantly reversed by 50 mg/kg naringenin (p < .05 and p < .01, respectively). CONCLUSION: Our results suggest that naringenin may have a potential anti-tolerant/anti-addiction property against chronic morphine misuse and that this preventive effect is associated with its anti-neuroinflammatory effects.

Mode of delivery alters dental pulp nociception and pain-induced changes in cognitive performance in adults male rats.

This study examined the effects of delivery mode on the response to inflammatory pulpal pain and pain-induced changes in cognitive performance in adult rats. Experiments were done on rats born by vaginal or caesarean section (C-section) delivery. Dental pulp was irritated by intradental capsaicin (100 µg) application and then nociceptive scores were recorded for 40 min. Spatial and passive avoidance learning and memory were assessed using the Morris water maze (MWM) and shuttle box tools, respectively. Additionally, in vivo recording of field excitatory postsynaptic potential (fEPSP) in the CA1 of the hippocampus was used to verify synaptic plasticity. Capsaicin produced more significant nociceptive behavior in vaginally delivered rats compared to C-section rats (P < 0.01). C-section-delivered rats show better performance in both MWM and shuttle box tests. Likewise, C-section rats had greater fEPSP slopes compared to the vaginally delivered group (P < 0.05). Capsaicin impairs cognitive performance in rats born by each delivery route. However, capsaicin effects were more significant in rats delivered vaginally than by C-section. Overall, C-section-delivered rats show lower sensitivity to capsaicin-evoked pulpal nociception and better cognitive performance than vaginally delivered rats. These effects are in part mediated by reduced neuroinflammation and enhanced neuronal synaptic plasticity following C-section delivery.

Central microinjection of phytohormone abscisic acid changes feeding behavior, decreases body weight, and reduces brain oxidative stress in rats.

Objectives: Natural products have a potential role on food intake in mammals. It has been reported that phytohormone abscisic acid (ABA) has a regulatory role on metabolic processes. Here, the effects of ABA on feeding behavior and brain oxidative stress were investigated in male Wistar rats. Methods: ABA was injected intracerebroventricularly. Experimental groups were included (n = 9): control (received no injection), ABA vehicle (received normal saline), and ABA-treated groups were injected with different doses of ABA (2.5, 5, and 10 µg/rat for 7 days). Daily cumulative daytime and nighttime food consumption, meal frequency, meal duration, and alteration in body weight were recorded. At the end of behavioral experiment, catalase and peroxidase activity and malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels were assayed. Results: The results showed that ABA (5 and 10 µg) increased the meal frequency. Moreover, ABA could decrease body weight and MDA and H2O2 levels and increased the catalase and peroxidase activities in diencephalon. It also decreased the MDA concentration in the brain stem. Discussion: Taken together, ABA has an important effect on feeding behavior and body weight in rats likely via increasing antioxidant capacity. However, further studies are still required to determine the underlying mechanisms of ABA on the feeding behavior.

Phytohormone abscisic acid ameliorates cognitive impairments in streptozotocin-induced rat model of Alzheimer's disease through PPARß/δ and PKA signaling.

Aim: Alzheimer's disease (AD) is characterized by oxidative stress, neuroinflammation and progressive cognitive decline. Abscisic acid (ABA) is produced in a variety of mammalian tissues, including brain. It has anti-inflammatory and antioxidant effects and elicits a positive effect on spatial learning and memory performance. Here, the possible protective effect of ABA was evaluated in streptozotocin (STZ)-induced AD rat model which were injected intracerebroventriculary (i.c.v.) with STZ (3 mg/kg). Material and Methods: The STZ-treated animals received ABA (10 µg/rat, i.c.v.), ABA plus PPARß/δ receptor antagonist (GSK0660, 80 nM/rat) or ABA plus selective inhibitor of PKA (KT5720, 0.5 µg/rat) for 14 d. Learning and memory were determined using Morris water maze (MWM) and passive avoidance (PA) tests. Results: The data showed that STZ produced a significant learning and memory deficit in both MWM and PA tests. ABA significantly prevented the learning and memory impairment in STZ-treated rats. However, ABA effects were blocked by GSK0660 and KT5720. Conclusion: The data indicated that ABA attenuates STZ-induced learning and memory impairment and PPAR-ß/δ receptors and PKA signaling are involved, at least in part, in the ABA mechanism.

Induction of cross-tolerance between protective effect of morphine and nicotine in 6-hydroxydopamine-induce neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells.

PURPOSE: Parkinson's disease is a progressive neurodegenerative disease characterized by progressive and selective death of dopaminergic neurons. It has been reported that nicotine and morphine have protective roles during neuronal damage in Parkinson's disease. In addition, the induction of cross-tolerance between their biological effects has been shown in numerous reports. METHODS: Here, we investigated the effects of nicotine and morphine on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson's disease. Cell damage was induced by 150 µM 6-OHDA and the cells viability was examined by MTT assay. Intracellular reactive oxygen species, calcium level, and mitochondrial membrane potential were determined by fluorescence spectrophotometer method. Biochemical markers of apoptosis were also evaluated by immunoblotting. RESULT: The data showed that morphine and nicotine prevent 6-OHDA- induced cell damage and apoptosis. However, the protective effects of nicotine were not observed in chronic morphine-pretreated cells. Morphine had no protective effects in chronic nicotine-incubated cells. CONCLUSION: A cross-tolerance between protective effects of morphine and nicotine was occurred in 6-OHDA-induced SH-SY5Y cell toxicity.

DNA Domino-Based Nanoscale Logic Circuit: A Versatile Strategy for Ultrasensitive Multiplexed Analysis of Nucleic Acids.

In recent years, the analytical application of logical nanodevices has attracted much attention for making accurate decisions on molecular diagnosis. Herein, a DNA domino-based nanoscale logic circuit has been constructed by integrating three logic gates (AND-AND-YES) for simultaneous analysis of multiple nucleic acid biomarkers. In the first AND gate, a chimeric target DNA comprising of four biomarkers was hybridized to three biomarker-specific oligonucleotides (TRs) via their 5'-end regions and to a capture probe-magnetic microparticle. After harvesting the complex, 3' overhang regions of the TRs were labeled with three distinct monolayer double-stranded (ds) DNA-gold nanoparticles (DNA-AuNPs). Upon gleaning the complex and addition of initiator oligonucleotide, a series of toehold-mediated strand displacement reactions, which are reminiscent of a domino chain, spontaneously occurred between the confined dsDNAs on the nanoparticles' surface in the second AND gate. The output of the second gate entered into the last gate and triggered an exponential hairpin assembly to form four-way junction nanostructures. The resulting nanostructures bear split parts of DNAzyme at each end of the four arms which, in the presence of hemin, form catalytic hemin/G-quadruplex DNAzymes with peroxidase activity. The smart biosensor has exhibited a turn-on signal when all biomarkers are present in the sample. In fact, should any of the biomarkers be nonexistent, the signal remains turned-off. The biosensor can detect the biomarkers with a LOD value of 100 aM and a noticeable capability to discriminate single-nucleotide substitutions.

Human Dental Pulp Stem Cells Differentiate into Oligodendrocyte Progenitors Using the Expression of Olig2 Transcription Factor.

The helix-loop-helix transcription factor Olig2 is essential for lineage determination of oligodendrocytes. Differentiation of stem cells into oligodendrocytes and transplanting them is a novel strategy for the repair of different demyelination diseases. Dental pulp stem cells (DPSCs) are of great interest in regenerative medicine due to their potential for repairing damaged tissues. In this study, DPSCs were isolated from human third molars and transfected with the human Olig2 gene as a differentiation inducer for the oligodendrogenic pathway. Following the differentiation procedure, the expression of Sox2, NG2, PDGFRα, Nestin, MBP, Olig2, Oct4, glial fibrillary acidic protein and A2B5 as stage-specific markers was studied by real-time RT-qPCR, immunocytochemistry and Western blot analysis. The cells were transplanted into a mouse model of local sciatic damage by lysolecithin as a model for demyelination. Oligodendrocyte progenitor cells (OPCs) actively remyelinated and recovered the lysolecithin-induced damages in the sciatic nerve as revealed by treadmill exercise, the von Frey filament test and hind paw withdrawal in response to a thermal stimulus. Recovery of behavioral reflexes occurred 2-6 weeks after OPC transplantation. The results demonstrate that the expression of Olig2 in DPSCs reduces the expression of stem cell markers and induces the development of oligodendrocyte progenitors as revealed by the emergence of oligodendrocyte markers. DPSCs could be programmed into oligodendrocyte progenitors and considered as a simple and valuable source for the cell therapy of neurodegenerative diseases.

Mode of delivery alters sensitivity to thermal and chemical stimuli in adult rats: An experimental study.

Background: The mode of delivery might prompt a long-lasting alteration in physiological and behavioral responsiveness in offspring. Objective: This study was intended to evaluate if the mode of delivery could alter sensitivity to thermal and chemical stimuli in female rats. Materials and Methods: 56 adult female Wistar rats (200-220 gr) that were born by vaginal or cesarean section (C-section) were used (n = 28/each). Inflammatory pain was induced by subcutaneous injection of formalin into the hind paw. The thermal nociceptive threshold was determined by tail-flick and hot plate tests. Besides, the Western blot test was used to evaluate the spinal cord levels of c-Fos and c-Jun proteins. Results: Formalin-induced inflammation was significantly decreased in C-section group as compared to vaginally born rats (p < 0.001). The baseline nociceptive threshed and morphine-induced analgesia were significantly increased in C-section groups in comparison to vaginally born rats. In addition, the levels of c-Fos and c-Jun proteins were significantly decreased in the spinal cord of C-section rats as compared to vaginally born animals (p < 0.01). Morphine treatment could decrease the expression of c-Fos and c-Jun in the C-section group (p < 0.05). Conclusion: Overall, C-section rats showed lower spinal nociceptive processing and neuronal activity later in life, compared to the vaginal born rats.

Methyl jasmonate ameliorates pain-induced learning and memory impairments through regulating the expression of genes involved in neuroinflammation.

OBJECTIVE: Orofacial pain with high prevalence is one of the substantial human health issues. The importance of this matter became more apparent when it was revealed that orofacial pain, directly and indirectly, affects cognition performances. Currently, researchers have focused on investigating pharmaceutics to alleviate pain and ameliorate its subsequent cognitive impairments. DESIGN: In this study, the rats were first treated with the central administration of methyl jasmonate (MeJA), which is an antioxidant and anti-inflammatory bio-compound. After 20 min, orofacial pain was induced in the rats by the injection of capsaicin in their dental pulp. Subsequently, the animals' pain behaviors were analyzed, and the effects of pain and MeJA treatments on rats learning and memory were evaluated/compared using the Morris water maze (MWM) test. In addition, the expression of tumor necrosis factor-α (TNF-α), IL-1ß, BDNF, and COX-2 genes in the rats' hippocampus was evaluated using real-time polymerase chain reaction. RESULTS: Experiencing orofacial pain resulted in a significant decline in the rats learning and memory. However, the central administration of 20 µg/rat of MeJA effectively mitigated these impairments. In the MWM, the performance of the MeJA-treated rats showed a two- to threefold improvement compared to the nontreated ones. Moreover, in the hippocampus of pain-induced rats, the expression of pro-inflammatory factors TNF-α, IL-1ß, and COX-2 significantly increased, whereas the BDNF expression decreased. In contrast, MeJA downregulated the pro-inflammatory factors and upregulated the BDNF by more than 50%. CONCLUSIONS: These findings highlight the notable antinociceptive potential of MeJA and its ability to inhibit pain-induced learning and memory dysfunction through its anti-inflammatory effect.

Evidence for the induction of analgesic cross-tolerance between opioid and apelin/APJ systems in male rats.

BACKGROUND: Opioids are potent pain relievers for managing severe pain. However, their effectiveness is hindered by tolerance, which causes the need for higher doses and leads to adverse effects. In a previous study, we found that prolonged use of apelin, similar to opioids, results in a tolerance to its analgesic effects. It remains unclear whether there is a cross-tolerance between morphine and apelin, meaning if the analgesic effects of one can reduce the effectiveness of the other. METHODS: The tail-flick test was used to assess the nociceptive threshold. All experiments were carried out on 63 male Wistar rats, which received intrathecal apelin (3µg/rat) or morphine (15µg/rat) for 7 days. To determine cross-tolerance between the analgesic effect of morphine and apelin, the analgesic property of apelin or morphine was assessed in chronic morphine- or apelin-treated groups, respectively. To determine the role of apelin and opioid receptors signaling on the development of analgesic cross-tolerance, F13-A and naloxone, as apelin and opioid receptor antagonists, were injected simultaneously with morphine or apelin. At the end of the tests, the expression levels of apelin and mu-opioid receptors were evaluated by western blotting. RESULTS: The data indicated that chronic apelin or morphine produced tolerance to the antinociceptive effects of each other. F13-A and naloxone could inhibit the induction of such cross-tolerance. The molecular data showed that there was a significant downregulation of apelin receptors in chronic morphine-treated rats and vice versa. CONCLUSION: Chronic administration of apelin or morphine induces analgesic cross-tolerance that may, in part, be mediated through receptor interactions and downregulation. The demonstrated efficacy of F13-A in these experiments highlights its potential as a novel target for improving pain management through the inhibition of the apelin/APJ signaling pathway, meriting further investigation.

Bunium persicum Seeds Extract in Combination with Vincristine Mediates Apoptosis in MCF-7 Cells through Regulation of Involved Genes and Proteins Expression.

BACKGROUND: Bunium persicum seeds, a member of the Apiaceae family, have historically been consumed as part of the Iranian diet. OBJECTIVE: While many of this herb's biological properties have been fully investigated, there is currently no reliable information about its anticancer/cytotoxic properties. METHODS: Herein, we first determined the major bioactive compounds of B. persicum seed extract (BPSE) via GC-Mass analysis. We evaluated the cytotoxicity of the extract alone as well as in combination with vincristine (VCR), a commonly used chemotherapy drug, using MTT assays on two breast cancer cell lines, MCF-7 and MDA-MB-231, as well as a normal breast cancer cell line, MCF-10A. Moreover, these compounds were evaluated in vitro for their anticancer activity using ROS assays, Real-Time PCR, Western blots, flow cytometry, and cell cycle assays. RESULTS: As a result of our investigation, it was determined that the extract significantly reduced the viability of cancerous cells while remaining harmless to normal cells. The combination of BPSE and VCR also resulted in synergistic effects. BPSE and/or BPSE-VCR treatment increased the intracellular ROS of MCF-7 cells by over twofold. Moreover, the IC30 of BPSE (100 µg/ml) significantly increased the BAX/BCL-2 and P53 gene expression while reducing the expression of the MYC gene. Moreover, treated cells were arrested in the G2 phase of the cell cycle. The BPSE-VCR combination synergistically reduced the NF-κB and increased the Caspase-7 proteins' expression. The percent of apoptosis in the cells treated with the extract, VCR, and their combination was 27, 11, and 50, respectively. CONCLUSIONS: The present study demonstrated the anticancer activity of the BPSE and its potential for application in combination therapy with VCR.

Direct Interaction of Minocycline to p47phox Contributes to its Attenuation of TNF-α-Mediated Neuronal PC12 Cell Death: Experimental and Simulation Validation.

Minocycline, a repurposed approved medication, shows promise in treating neurodegeneration. However, the specific pathways targeted by minocycline remain unclear despite the identification of molecular targets. This study explores minocycline's potential protective effects against TNF-α-mediated neuronal death in PC12 cells, with a focus on unraveling its interactions with key molecular targets. The study begins by exploring minocycline's protective role against TNF-α-mediated neuronal death in PC12 cells, showcasing a substantial reduction in cleaved caspase-3 expression, DNA fragmentation, and intracellular ROS levels following minocycline pretreatment. Subsequently, a comprehensive analysis utilizing pull-down assays, computational docking, mutation analysis, molecular dynamics simulations, and free energy calculations is conducted to elucidate the direct interaction between minocycline and p47phox-the organizer subunit of NADPH oxidase-2 (NOX2) complex. Computational insights, including a literature survey and analysis of key amino acid residues, reveal a potential binding site for minocycline around Trp193 and Cys196. In silico substitutions of Trp193 and Cys196 further confirm their importance in binding with minocycline. These integrated findings underscore minocycline's protective mechanisms, linking its direct interaction with p47phox to the modulation of NOX2 activity and attenuation of NOX-derived ROS generation. Minocycline demonstrates protective effects against TNF-α-induced PC12 cell death, potentially linked to its direct interaction with p47phox. This interaction leads to a reduction in NOX2 complex assembly, ultimately attenuating NOX-derived ROS generation. These findings hold significance for researchers exploring neuroprotection and the development of p47phox inhibitors.

CRISPR/Cas9-Mediated Disruption of ZNF543 Gene: An Approach Toward Discovering Its Relation to TRIM28 Gene in Parkinson's Disease.

Genetic studies of familial forms of Parkinson's disease (PD) have shown that the ZNF543 gene is a candidate gene that operates relevant to this disease. However, until now, there is no evidence for ZNF543 gene function in PD, and mechanisms resulting from its mutation have not been elucidated. Given the same genetic location of the ZNF543 gene with TRIM28 and their effects on PD pathogenesis, we surmised that ZNF543 might act as a transcription factor for TRIM28 gene expression. By knocking out the ZNF543 gene via the CRISPR/Cas9 editing platform, we assessed the functional effect of loss of expression of this gene on TRIM28 gene expression. Four sgRNAs with different PAM sequences were designed against two parts of the regulatory region of ZNF543 gene, and highly efficient disruption of ZNF543 expression in human neuroblastoma cell line was evaluated by polymerase chain reaction and T7 endonuclease assay. Moreover, evaluation of TRIM28 gene expression in ZNF543-knocked-out cells indicated a significant increase in TRIM28 gene expression, suggesting that ZNF543 probably regulates the expression of TRIM28. This approach offers a window into pinpointing the mechanism by which ZNF543 gene mutations mediate PD pathogenicity.

Repeated gentle handling or maternal deprivation during the neonatal stage increases adult male rats' baseline orofacial pain responsiveness.

OBJECTIVE: Early life experiences have been found to have a long-lasting effect on brain development in adult life. The purpose of this study was to determine whether neonatal manipulation could alter orofacial pain responsiveness in adult rats METHODS: In the first 21 days of life, male rats were exposed to gentle handling or maternal deprivation (MD) procedures to establish models of handled and MD rats, respectively. The rats were assigned to three of the following experimental groups at the age of two months: intra-dental capsaicin (100 µg), intra-lip formalin (50 µL), and repeated nitroglycerin (NTG) (5 mg/rat/ip) infusion. In addition, there were three drug vehicle groups and three groups that received capsaicin, formalin, or NTG without prior handling or MD procedures. The behaviors were recorded following the pain induction. RESULTS: Spontaneous pain behaviors in the first phase of formalin test was significantly increased in MD (p < 0.01) and handled rats in comparison with the vehicle group (p < 0.05). The second-phase data showed that formalin-induced spontaneous pain behaviors was increased in rats- treated with MD as compared to either vehicle or handled+formalin groups (p < 0.001). Capsaicin-induced dental pulp nociception was increased in the MD group in comparison with the capsaicin (p < 0.001) and capsaicin+handled (p < 0.001) groups. Moreover, NTG-induced migraine-like behaviors symptoms were increased in the MD group as compared to control and handled groups (p < 0.05). CONCLUSIONS: In this study neonatal gentle handling or MD treatment increased orofacial pain in adulthood, showing early life experiences permanent effects on the development of trigeminal circuits in the brain.