Ionized Calcium Binding Adaptor Molecule Research Articles

Median nerve electrical stimulation improves traumatic brain injury by reducing TACR1 to inhibit nuclear factor-κB and CCL7 activation in microglia.

The existing report elucidates that median nerve electrical stimulation (MNS) plays a role in treating traumatic brain injury (TBI). Herein, we explored the mechanism of MNS in TBI. A TBI-induced coma model (skull was hit by a cylindrical impact hammer) was established in adult Sprague-Dawley rats. Microglia were isolated from newborn Sprague-Dawley rats and was injured by lipopolysaccharide (LPS; 10 ng/mL). Consciousness was assessed by sensory and motor functions. Brain tissue morphology was detected using hematoxylin-eosin staining assay. Ionized calcium binding adapter molecule 1, NeuN and tachykinin receptor 1 (TACR1) level were detected by immunohistochemical assay. Levels of pro-inflammatory and anti-inflammatory factors were measured by enzyme linked immune sorbent assay (ELISA). Levels of TACR1, C-C motif chemokine 7 (CCL7), phosphorylation (p)-P65 and P65 were assessed by quantitative real time polymerase chain reaction (qRT-PCR) and western blot. M1 markers (inducible nitric oxide synthase and CD86) and M2 markers (arginase-1 (Arg1) and chitinase 3-like 3 (YM1)) of microglia as well as the transfection efficiency of short hairpin TACR1 (shTACR1) were assessed by qRT-PCR. Immunofluorescence and flow cytometry assay were used to detect microglia morphology and neuron apoptosis. MNS reduced neuron injury and microglia activation in the TBI-induced rat coma model. MNS reversed the effects of TBI on levels of inflammation-related factors, M1/M2 microglia markers, TACR1, p-P65/P65 and CCL7 in rats. shTACR1 reversed the effects of LPS on inflammation-related factors, M1/M2 microglia markers, microglia activation, neuron apoptosis, p-P65/P65 value and CCL7 level. Our results revealed that MNS improved TBI by reducing TACR1 to inhibit nuclear factor-κB (NF-κB) and CCL7 activation in microglia.

Protective effect of melatonin against metabolic disorders and neuropsychiatric injuries in type 2 diabetes mellitus mice

BackgroundType 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia and progressive cognitive dysfunction, and our clinical investigation revealed that the plasma concentration of melatonin (Mlt) decreased and was closely related to cognition in T2DM patients. However, although many studies have suggested that Mlt has a certain protective effect on glucose and lipid metabolism disorders and neuropsychiatric injury, the underlying mechanism of Mlt against T2DM-related metabolic and cognitive impairments remains unclear. PurposeThe aim of the present study was to investigate the therapeutic effect of Mlt on metabolic disorders and Alzheimer's disease (AD)-like neuropsychiatric injuries in T2DM mice and to explore the possible underlying molecular mechanism involved. MethodsA T2DM mouse model was established by a combination of a high-fat diet (HFD) and streptozotocin (STZ, 100 mg/kg, i.p.), and Mlt (5, 10 or 20 mg/kg) was intragastrically administered for six consecutive weeks. The serum levels of glycolipid metabolism indicators were measured, behavioral performance was tested, and the protein expression of key molecules involved in the regulation of synaptic plasticity, circadian rhythms, and neuroinflammation in the hippocampus was detected. Moreover, the fluorescence intensities of glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule 1 (IBA-1), amyloid β-protein (Aβ) and phosphorylated Tau (p-Tau) in the hippocampus were also observed. ResultsTreatment with Mlt not only improved T2DM-related metabolic disorders, as indicated by increased serum concentrations of fasting blood glucose (FBG), glycosylated hemoglobin (HbAlc), insulin (INS), total cholesterol (TC) and triglyceride (TG), improved glucose tolerance and liver and pancreas function but also alleviated AD-like neuropsychiatric injuries in a HFD/STZ-induced mouse model, as indicated by decreased immobility time in the tail suspension test (TST) and forced swimming test (FST), increased preference indices of novel objects or novel arms in the novel object recognition test (NOR) and Y-maze test (Y-maze), and improved platform positioning capability in the Morris water maze (MWM) test. Moreover, treatment with Mlt also improved the hyperactivation of astrocytes and microglia in the hippocampus of mice, accompanied by reduced expression of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor (TNF-α), Aβ, and p-Tau and increased expression of brain-derived neurotrophic factor (BDNF), Synapsin I, Synaptotagmin I, melatonin receptor 1B (MT1B), brain muscle arnt-like protein 1 (Bmal1), circadian locomotor output cycles kaput (Clock), period 2 (Per2), and cryptochrome 2 (Cry2). ConclusionMlt alleviated T2DM-related metabolic disorders and AD-like neuropsychiatric injuries in a HFD/STZ-induced mouse model, possibly through a mechanism involving the regulation of glial activation and associated neuroinflammation and the balancing of synaptic plasticity and circadian rhythms in the hippocampus.

Du-moxibustion ameliorates depression-like behavior and neuroinflammation in chronic unpredictable mild stress-induced mice

BackgroundNeuroinflammation is involved in the advancement of depression. Du-moxibustion can treat depression. Here, we explored whether Du-moxibustion could alleviate neuroglia-associated neuro-inflammatory process in chronic unpredictable mild stress (CUMS) mice. MethodsC57BL/6J mice were distributed into five groups. Except for the CON group, other four groups underwent CUMS for four consecutive weeks, and Du-moxibustion was given simultaneously after modeling. Behavioral tests were then carried out. Additionally, Western blot was conducted to measure the relative expression levels of high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB). Immunofluorescence was employed to evaluate the positive cells of ionized calcium binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP). Furthermore, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) were analyzed using an ELISA assay. ResultsWe found that CUMS induced depression-like behaviors, such as reduced sucrose preference ratio, decreased locomotor and exploratory activity, decreased the time in open arms and prolonged immobility. Furthermore, versus the CON group, the expression of HMGB1, TLR4, MyD88, NF-κB, positive cells of Iba-1, IL-1β and TNF-α were increased but positive cells of GFAP were decreased in CUMS group. However, the detrimental effects were ameliorated by treatment with CUMS+FLU and CUMS+DM. LimitationsA shortage of this study is that only CUMS model of depression were used, while other depression model were not included. ConclusionsDu-moxibustion alleviates depression-like behaviors in CUMS mice mainly by reducing neuroinflammation, which offers novel insights into the potential treatment of depression.

Plasma exosomes impair microglial degradation of α-synuclein through V-ATPase subunit V1G1.

Microglia are the main phagocytes in the brain and can induce neuroinflammation. Moreover, they are critical to alpha-synuclein (α-syn) aggregation and propagation. Plasma exosomes derived from patients diagnosed with Parkinson's disease (PD-exo) reportedly evoked α-syn aggregation and inflammation in microglia. In turn, microglia internalized and released exosomal α-syn, enhancing α-syn propagation. However, the specific mechanism through which PD-exo influences α-syn degradation remains unknown. Exosomes were extracted from the plasma of patients with PD by differential ultracentrifugation, analyzed using electron microscopy (EM) and nanoparticle flow cytometry, and stereotaxically injected into the unilateral striatum of the mice. Transmission EM was employed to visualize lysosomes and autophagosomes in BV2 cells, and lysosome pH was measured with LysoSensor Yellow/Blue DND-160. Cathepsin B and D, lysosomal-associated membrane protein 1 (LAMP1), ATP6V1G1, tumor susceptibility gene 101 protein, calnexin, α-syn, ionized calcium binding adaptor molecule 1, and NLR family pyrin domain containing 3 were evaluated using quantitative polymerase chain reaction or western blotting, and α-syn, LAMP1, and ATP6V1G1 were also observed by immunofluorescence. Small interfering ribonucleic acid against V1G1 was transfected into BV2 cells and primary microglia using Lipofectamine® 3000. A PD mouse model was established via injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into mice. A lentiviral-mediated strategy to overexpress ATP6V1G1 in the brain of MPTP-treated mice was employed. Motor coordination was assessed using rotarod and pole tests, and neurodegeneration in the mouse substantia nigra and striatum tissues was determined using immunofluorescence histochemical and western blotting of tyrosine hydroxylase. PD-exo decreased the expression of V1G1, responsible for the acidification of intra- and extracellular milieu. This impairment of lysosomal acidification resulted in the accumulation of abnormally swollen lysosomes and decreased lysosomal enzyme activities, impairing lysosomal protein degradation and causing α-syn accumulation. Additionally, V1G1 overexpression conferred the mice neuroprotection during MPTP exposure. Pathogenic protein accumulation is a key feature of PD, and compromised V-type ATPase dysfunction might participate in PD pathogenesis. Moreover, V1G1 overexpression protects against neuronal toxicity in an MPTP-based PD mouse model, which may provide opportunities to develop novel therapeutic interventions for PD treatment.

Therapeutic efficacy of cinnamein, a component of balsam of Tolu/Peru, in controlled cortical impact mouse model of TBI

Traumatic brain injury (TBI) remains a major health concern which causes long-term neurological disability particularly in war veterans, athletes and young adults. In spite of intense clinical and research investigations, there is no effective therapy to cease the pathogenesis of the disease. It is believed that axonal injury during TBI is potentiated by neuroinflammation and demyelination and/or failure to remyelination. This study highlights the use of naturally available cinnamein, also chemically known as benzyl cinnamate, in inhibiting neuroinflammation, promoting remyelination and combating the disease process of controlled cortical impact (CCI)-induced TBI in mice. Oral delivery of cinnamein through gavage brought down the activation of microglia and astrocytes to decrease the expression of inducible nitric oxide synthase (iNOS), glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) in hippocampus and cortex of TBI mice. Cinnamein treatment also stimulated remyelination in TBI mice as revealed by PLP and A2B5 double-labeling, luxol fast blue (LFB) staining and axonal double-labeling for neurofilament and MBP. Furthermore, oral cinnamein reduced the size of lesion cavity in the brain, improved locomotor functions and restored memory and learning in TBI mice. These results suggest a new neuroprotective property of cinnamein that may be valuable in the treatment of TBI.

A M1 muscarinic acetylcholine receptor-specific positive allosteric modulator VU0486846 reduces neurogliosis in female Alzheimer’s mice

Alzheimer's disease (AD) is the most prevalent type of dementia, disproportionately affecting females, who make up nearly 60% of diagnosed cases. In AD patients, the accumulation of beta-amyloid (Aβ) in the brain triggers a neuroinflammatory response driven by neuroglia, worsening the condition. We have previously demonstrated that VU0486846, an orally available positive allosteric modulator (PAM) targeting M1 muscarinic acetylcholine receptors, enhances cognitive function and reduces Aβ pathology in female APPswe/PSEN1ΔE9 (APP/PS1) mice. However, it remained unclear whether these improvements were linked to a decrease in neuroglial activation. To investigate, we treated nine-month-old APP/PS1 and wildtype mice with VU0486846 for 8 weeks and analyzed brain slices for markers of microglial activation (ionized calcium binding adaptor molecule 1, Iba1) and astrocyte activation (Glial fibrillary acidic protein, GFAP). We find that VU0486846 reduces the presence of Iba1-positive microglia and GFAP-positive astrocytes in the hippocampus of female APP/PS1 mice and limits the recruitment of these cells to remaining Aβ plaques. This study sheds light on an additional mechanism through which novel M1 mAChR PAMs exhibit disease-modifying effects by reducing neuroglial activation and underscore the potential of these ligands for the treatment of AD, especially in females.

Gut microbiota deficiency aggravates arsenic-induced toxicity by affecting bioaccumulation and biotransformation in C57BL/6J mice

Gut microbiome can influence the arsenic metabolism in mammals. Confusingly, gut microbiome was found to both mitigate and exacerbate arsenic toxicity. In this study, the role of gut microbiota in arsenic bioaccumulation, biotransformation, and organ toxicity in C57BL/6J mice was investigated. Gut microbiota deficiency model was established by antibiotics (Ab) cocktail AVNM. Conventional and gut microbiota deficiency mice were exposed to NaAsO2 for 4 weeks. Comparing with Ab-treated mice, the total arsenic (tAs) in the tissues was significantly reduced in conventional mice, which was opposed to the results of those in feces. Interestingly, dimethyl arsenite (DMA) was the most abundant metabolite in the feces of Ab-treated mice, while arsenic acid (AsV) had the highest proportion in the feces of conventional mice with approximately 16-fold than that in Ab-treated mice, indicating the critical role of gut microbiota in metabolizing arsenious acid (AsIII) to AsV. Additionally, the liver and kidney in Ab-treated mice showed more severe pathological changes and apoptosis. The significant increased level of ionized calcium-binding adapter molecule 1 (IBA-1) was also found in the brains of Ab-treated mice. Our results indicated that gut microbiota protected the host from arsenic-induced toxicity in liver, kidney, and brain by reducing the arsenic accumulation.

Microglial reactivity in brainstem chemosensory nuclei in response to hypercapnia.

Microglia, the resident immune cells of the CNS, surveil, detect, and respond to various extracellular signals. Depending on the nature of these signals, an integrative microglial response can be triggered, resulting in a phenotypic transformation. Here, we evaluate whether hypercapnia modifies microglia phenotype in brainstem respiratory-related nuclei. Adult C57BL/6 inbred mice were exposed to 10% CO2 enriched air (hypercapnia), or pure air (control), for 10 or 30min and immediately processed for immunohistochemistry to detect the ubiquitous microglia marker, ionized calcium binding adaptor molecule 1 (Iba1). Hypercapnia for thirty, but not 10min reduced the Iba1 labeling percent coverage in the ventral respiratory column (VRC), raphe nucleus (RN), and nucleus tractus solitarius (NTS) and the number of primary branches in VRC. The morphological changes persisted, at least, for 60min breathing air after the hypercapnic challenge. No significant changes were observed in Iba1+ cells in the spinal trigeminal nucleus (Sp5) and the hippocampus. In CF-1 outbred mice, 10% CO2 followed by 60min of breathing air, resulted in the reduction of Iba1 labeling percent coverage and the number and length of primary branches in VRC, RN, and NTS. No morphological change was observed in Iba1+ cells in Sp5 and hippocampus. Double immunofluorescence revealed that prolonged hypercapnia increased the expression of CD86, an inflammatory marker for reactive state microglia, in Iba1+ cells in VRC, RN, and NTS, but not in Sp5 and hippocampus in CF-1 mice. By contrast, the expression of CD206, a marker of regulatory state microglia, persisted unmodified. In brainstem, but not in hippocampal microglia cultures, hypercapnia increased the level of IL1β, but not that of TGFβ measured by ELISA. Our results show that microglia from respiratory-related chemosensory nuclei, are reactive to prolonged hypercapnia acquiring an inflammatory-like phenotype.

Telmisartan Protects Mitochondrial Function, Gait, and Neuronal Apoptosis by Activating the Akt/GSK3β/PGC1α Pathway in an MPTP-Induced Mouse Model of Parkinson's Disease.

Mitochondrial dysfunction is one of the major hallmarks of Parkinson's disease (PD). Recently, angiotensin II type 1 and type 2 receptors (AT1R, AT2R) were reported to be present on the mitochondrial membrane. Both are crucial players in the brain renin-angiotensin system (RAS). Current evidence indicates that blockade of brain AT1R protects dopaminergic neurons in PD. Thus, the current study was aimed to explore the effects of Telmisartan (Tel), a selective AT1R blocker, on mitochondrial function and a mouse model by exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [250 mg/kg body weight (10 divided i.p. injections, each 25 mg/kg body weight at 3.5 days interval) + Probenecid 250 mg/kg]. Gait function was assessed by beam walk, and mice were euthanized on the 35th day and their brain tissues isolated for Western blot analysis. Pretreatment with Tel significantly protected motor functions during the beam walk in MPTP-treated mice. Tel attenuated the increased levels of AT1R, α-syn, and inflammatory markers such as inducible nitric oxide synthase (iNOS) and ionized calcium-binding adaptor molecule 1 (IBA1) in MPTP-treated mice. In addition, Tel preserved the expression of AT2R, tyrosine hydroxylase (TH), p-Akt/Akt, and p-GSK3β (Ser-9)/GSK3β, as well as protecting mitofusin protein 1 (MFN1) and Peroxisome proliferator-activated receptor-gamma coactivator-α (PGC1α), a critical activator of mitochondrial biogenesis. These results indicate that Tel protects mitochondrial function and gait in a mouse model of PD by modulating the Akt/GSK3β/PGC1α pathway.

Luteolin amends neural neurotransmitters, antioxidants, and inflammatory markers in the cerebral cortex of Adderall exposed rats

BackgroundAdderall is a central nervous system stimulant while luteolin has neuroprotective activity. This study aimed to determine whether luteolin can amend neural neurotransmitters, antioxidants, and inflammatory markers in the cerebral cortex of Adderall exposed rats. MethodsThirty-six male albino rats were divided into 6 equal groups, Control, Luteolin (1 g/kg)-treated, and Luteolin (2 g/kg)-treated groups: normal rats were orally administrated once a day with 2 ml distilled water, luteolin (1 g/kg), and luteolin (2 g/kg), respectively for 4 weeks. Adderall rats, Adderall rats + luteolin (1 g/kg)-treated, and Adderall rats + luteolin (2 g/kg)-treated groups: normal rats were orally administrated once a day with 10 mg/kg of Adderall, 3 days/week for 4 weeks, then these rats orally administrated daily once a day with 2 ml of distilled water, luteolin (1 g/kg), and luteolin (2 g/kg), respectively for another 4 weeks. Results and conclusionAdderall decreased superoxide dismutase, glutathione peroxidase, catalase, NADPH oxidase, interleukin-10, serotonin, dopamine, norepinephrine, γ-aminobutyric acid, and acetylcoline estrase but increased malondialdehyde, conjugated dienes, oxidative index, tumour necrosis factor-α, interleukin-1β, and interleukin-6 levels in the cerebral cortex. Adderall increased the expression of glial fibrillary acidic protein, ionized calcium binding adaptor molecule 1, and anti-calbindin in the cerebral cortex of Adderall-treated rats. In Adderall-treated rats, daily oral administration of luteolin for 4 weeks brought all these parameters back to values that were close to control where higher dose was more effective than lower dose. The importance of this research is to provide natural compound that amends Adderall-related neural disturbances and this natural compound is cheap, avaliable without any side effect and it does not interfer with Adderall efficiency.

Microcurrent therapy as the nonpharmacological new protocol against Alzheimer's disease.

Alzheimer's disease (AD) poses an increasing global health challenge and is marked by gradual cognitive deterioration, memory impairment, and neuroinflammation. Innovative therapeutic approaches as non-pharmacological protocol are urgently needed with side effect risk of drugs. Microcurrent therapy, a non-invasive modality involving low-level electrical currents, has emerged as a potential solution to address AD's complex pathogenesis. This study investigates the optimal application of microcurrent therapy as a clinical protocol for AD, utilizing a comprehensive approach that integrates behavioral assessments and neuroinflammation evaluation in a mouse model of dementia. The results reveal that microcurrent therapy holds promise in ameliorating memory impairment and reducing neuroinflammation in AD. Behavioral assessments, including the Novel Object Recognition Test (NOR) and Radial Arm Maze Test (RAM), demonstrated improved cognitive function following microcurrent therapy. Furthermore, microcurrent therapy inhibited expression of neuroinflammatory proteins, including ionized calcium binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP) in current-treated group. Mechanistic insights suggest that microcurrent therapy may modulate neuroinflammation through the regulation of MAPK signaling pathways. This study emphasizes the prospect of microcurrent therapy as a safe and efficacious non-pharmacological strategy for Alzheimer's disease (AD), providing optimism to the countless individuals impacted by this debilitating ailment. These results contribute to the developments of an innovative clinical protocol for AD and recovery from neurological injury, underscoring the significance of investigating unconventional therapeutic approaches for addressing this complex condition.

Involvement of NOTCH1-mediated Microglia Activation in Neuromodulation of Chronic Prostatitis-related Pain.

This study aimed to investigate the role of NOTCH receptor 1 (NOTCH1)-mediated activation of microglia in the L5-S2 spinal dorsal horn in chronic prostatitis pain. Rats were divided into chronic prostatitis (CP) group and control group. Complete Freund's adjuvant was injected into the prostate, and prostate pathology and pain-related behavior were monitored to assess the successful establishment of the CP-related pain model. The dorsal horn of the L5-S2 spinal cord was collected for the detection of ionized calcium-binding adapter molecule 1 (IBA-1) and NOTCH1 expression by quantitative real time polymerase chain reaction and the detection of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by enzyme-linked immunosorbent assay. Electrical excitability was assessed with whole-cell patch clamp. In addition, NOTCH1 receptor inhibitor or inhibitor of microglial cell activation was injected into the subarachnoid space, and the pro-inflammatory cytokines in the spinal cord were detected. In the CP group, the expression of NOTCH1, IBA-1, TNF-α and IL-1β began to increase at 4 days, peaked at 12 days, and began to decline at 24 days, and it was significantly higher than in the control group (p<0.01). Inhibition of microglia or NOTCH1 receptor markedly reduced the content of TNF-α and IL-1β in the spinal cord (p<0.05). At 4, 12 and 24 days, the amplitude and frequency of neuronal action potential increased and the threshold decreased markedly as compared to the control group (p<0.05), and spontaneous action potential was noted. NOTCH1 mediates the activation of microglia in the L5-S2 spinal cord, leading to the secretion of inflammatory factors and enhanced electrical excitability of neurons, which is related to persistent and refractory chronic prostatitis-related pain.

Grape seed-derived procyanidins decreases neuropathic pain and nerve regeneration by suppression of toll-like receptor 4-myeloid differentiation factor-88 signaling.

Background: Recent studies have shown that peripheral nerve regeneration process is closely related to neuropathic pain. Toll-like receptor 4 (TLR4) signaling was involved in different types of pain and nerve regeneration. TLR4 induced the recruitment of myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB-depended transcriptional process in sensory neurons and glial cells, which produced multiple cytokines and promoted the induction and persistence of pain. Our study aimed to investigate procyanidins's effect on pain and nerve regeneration via TLR4-Myd88 signaling. Methods: Spinal nerve ligation (SNL) model was established to measure the analgesic effect of procyanidins. Anatomical measurement of peripheral nerve regeneration was measured by microscopy and growth associated protein 43 (GAP43) staining. Western blotting and/or immunofluorescent staining were utilized to detect TLR4, myeloid differentiation factor-88 adaptor protein (MyD88), ionized calcium-binding adapter molecule 1 (IBA1) and nuclear factor kappa-B-p65 (NF-κB-p65) expression, as well as the activation of astrocyte and microglia. The antagonist of TLR4 (LPS-RS-Ultra, LRU) were intrathecally administrated to assess the behavioral effects of blocking TLR4 signaling on pain and nerve regeneration. Result: Procyanidins reduced mechanical allodynia, thermal hyperalgesia and significantly suppressed the number of nerve fibers regenerated and the degree of myelination in SNL model. Compared with sham group, TLR4, MyD88, IBA1 and phosphorylation of NF-κB-p65 were upregulated in SNL rats which were reversed by procyanidins administration. Additionally, procyanidins also suppressed activation of spinal astrocytes and glial cells. Conclusion: Suppression of TLR4-MyD88 signaling contributes to the alleviation of neuropathic pain and reduction of nerve regeneration by procyanidins.

Purpurogallin ameliorates sevoflurane-induced cognitive impairment and hippocampal neuroinflammation in neonatal mice

Postoperative cognitive dysfunction contributes to memory impairment, which is involved in neurotoxicity, endoplasmic reticulum stress, calcium overload and hippocampal neuron apoptosis. Purpurogallin can mitigate ischemia-induced neuronal injury and microglial inflammation. However, whether purpurogallin affects cognitive dysfunction remains unclear. To investigate the efficacy of purpurogallin on cognitive dysfunction and explore the potential signal pathways, Sevoflurane (SEV)-induced model was established on mice, and water maze testing was performed to record escape latency, platform residence time, platform crossings and swimming speed analysis. The concentration of Interleukin (IL)-6, IL-1β and Tumor necrosis factor (TNF)-α in hippocampus tissue were evaluated using Enzyme-linked immunosorbent assay (ELISA). The hippocampal neurons were analyzed using Nissl staining. The Iba-1 was evaluated using immunofluorescent assay (IFA), while the cell apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blots were performed to evaluate the expression of Ionized calcium binding adaptor molecule 1 (Iba1), integrin α-M (CD11b), Bax, integrin α-M (Bcl-2), p-65 and IκBα, as well as the phosphorylation of p-65 and IκBα. Purpurogallin at dose of 150 mg/kg significantly reduced escape latency in SEV-induced mice (p &lt; 0.001), without affecting swimming speed. It increased time spent in the target quadrant and restored platform crossings reduced by SEV. Purpurogallin also mitigated the elevated IL-6, IL-1β and Transforming growth factor alpha (TGF-α) levels caused by SEV in the hippocampus, preserved Nissl bodies, and reduced Iba-I and CD11b expression (p &lt; 0.01). It alleviated cell apoptosis by downregulating Bax and upregulating Bcl-2, while inhibiting p-65 phosphorylation and promoting IκBα phosphorylation induced by SEV (p &lt; 0.05). Purpurogallin effectively countered SEV-induced cognitive impairment, neuroinflammation, hippocampal injury, microglial activation and neuronal apoptosis through inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, which facilitate purpurogallin to become a drug candidate for postoperative cognitive dysfunction.

Kilohertz high-frequency electrical stimulation ameliorate hyperalgesia by modulating transient receptor potential vanilloid-1 and N-methyl-D-aspartate receptor-2B signaling pathways in chronic constriction injury of sciatic nerve mice.

The number of patients with neuropathic pain is increasing in recent years, but drug treatments for neuropathic pain have a low success rate and often come with significant side effects. Consequently, the development of innovative therapeutic strategies has become an urgent necessity. Kilohertz High Frequency Electrical Stimulation (KHES) offers pain relief without inducing paresthesia. However, the specific therapeutic effects of KHES on neuropathic pain and its underlying mechanisms remain ambiguous, warranting further investigation. In our previous study, we utilized the Gene Expression Omnibus (GEO) database to identify datasets related to neuropathic pain mice. The majority of the identified pathways were found to be associated with inflammatory responses. From these pathways, we selected the transient receptor potential vanilloid-1 (TRPV1) and N-methyl-D-aspartate receptor-2B (NMDAR2B) pathway for further exploration. Mice were randomly divided into four groups: a Sham group, a Sham/KHES group, a chronic constriction injury of the sciatic nerve (CCI) group, and a CCI/KHES stimulation group. KHES administered 30 min every day for 1week. We evaluated the paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL). The expression of TRPV1 and NMDAR2B in the spinal cord were analyzed using quantitative reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence assay. KHES significantly alleviated the mechanical and thermal allodynia in neuropathic pain mice. KHES effectively suppressed the expression of TRPV1 and NMDAR2B, consequently inhibiting the activation of glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA1) in the spinal cord. The administration of the TRPV1 pathway activator partially reversed the antinociceptive effects of KHES, while the TRPV1 pathway inhibitor achieved analgesic effects similar to KHES. KHES inhibited the activation of spinal dorsal horn glial cells, especially astrocytes and microglia, by inhibiting the activation of the TRPV1/NMDAR2B signaling pathway, ultimately alleviating neuropathic pain.

Electroacupuncture improves allodynia and central sensitization via modulation of microglial activation associated P2X4R and inflammation in a rat model of migraine.

Background: Recent studies have demonstrated that activated microglia were involved in the pathogenesis of central sensitization characterized by cutaneous allodynia in migraine. Activation of microglia is accompanied by increased expression of its receptors and release of inflammatory mediators. Acupuncture and its developed electroacupuncture (EA) have been recommended as an alternative therapy for migraine and are widely used for relieving migraine-associated pain. However, it remains rare studies that show whether EA exerts anti-migraine effects via inhibiting microglial activation related to a release of microglial receptors and the inflammatory pathway. Therefore, this study aimed to investigate EA' ability to ameliorate central sensitization via modulation of microglial activation, microglial receptor, and inflammatory response using a rat model of migraine induced by repeated epidural chemical stimulation. Methods: In the present study, a rat model of migraine was established by epidural repeated inflammatory soup (IS) stimulation and treated with EA at Fengchi (GB20) and Yanglingquan (GB34) and acupuncture at sham-acupoints. Pain hypersensitivity was further determined by measuring the mechanical withdrawal threshold using the von-Frey filament. The changes in c-Fos and ionized calcium binding adaptor molecule 1 (Ibal-1) labeled microglia in the trigeminal nucleus caudalis (TNC) were examined by immunflurescence to assess the central sensitization and whether accompanied with microglia activation. In addition, the expression of Ibal-1, microglial purinoceptor P2X4, and its associated inflammatory signaling pathway mediators, including interleukin (IL)-1β, NOD-like receptor protein 3 (NLRP3), and Caspase-1 in the TNC were investigated by western blot and real-time polymerase chain reaction analysis. Results: Allodynia increased of c-Fos, and activated microglia were observed after repeated IS stimulation. EA alleviated the decrease in mechanical withdrawal thresholds, reduced the activation of c-Fos and microglia labeled with Ibal-1, downregulated the level of microglial purinoceptor P2X4, and limited the inflammatory response (NLRP3/Caspase-1/IL-1β signaling pathway) in the TNC of migraine rat model. Conclusions: Our results indicate that the anti-hyperalgesia effects of EA ameliorate central sensitization in IS-induced migraine by regulating microglial activation related to P2X4R and NLRP3/IL-1β inflammatory pathway.

Eriocalyxin B alleviated ischemic cerebral injury by limiting microglia-mediated excessive neuroinflammation in mice.

Excessive neuroinflammation mediated by microglia has a detrimental effect on the progression of ischemic stroke. Eriocalyxin B (EriB) was found with a neuroprotective effect in mice with Parkinson's disease via the suppression of microglial overactivation. This study aimed to investigate the roles of EriB in permanent middle cerebral artery occlusion (pMCAO) mice. The pMCAO was induced in the internal carotid artery of the mice by the intraluminal filament method, and EriB (10 mg/kg) was administered immediately after surgery by intraperitoneal injection. The behavior score, 2,3,5-triphenyltetrazole chloride staining, Nissl staining, TUNEL, immunohistochemistry, immunofluorescence, PCR, ELISA, and immunoblotting revealed that EriB administration reduced brain infarct and neuron death and ameliorated neuroinflammation and microglia overactivation in pMCAO mice, manifested by alterations of TUNEL-positive cell numbers, ionized calcium binding adaptor molecule 1 (Iba-1)-positive cell numbers, and expression of tumor necrosis factor-α, interleukin 6, IL-1β, inducible nitric oxide synthase, and arginase 1. In addition, EriB suppressed ischemia-induced activation of nuclear factor kappa B (NF-κB) signaling in the brain penumbra, suggesting the involvement of NF-κB in EriB function. In conclusion, EriB exerted anti-inflammatory effects in ischemia stroke by regulating the NF-κB signaling pathway, and this may provide insights into the neuroprotective effect of EriB in the treatment of ischemic stroke.

Protective effects of the salt-induced kinase inhibitor HG-9-91-01 on sepsis-associated cognitive dysfunction in mice and the underlying mechanisms.

Sepsis-associated cognitive dysfunction is a common complication in patients with sepsis and lack of effective treatment. Its pathological mechanisms remain unclear. Salt-induced kinase (SIK) is an important molecule in the regulation of metabolism, immunity, and inflammatory response. It is associated with the development of many neurological diseases. This study aims to investigate the expression of SIK in the hippocampus of septic mice, and to evaluate the role and mechanism of the SIK inhibitor HG-9-91-01 in sepsis-associated cognitive dysfunction. Firstly, C57BL/6 mice were randomly divided into a control group (Con group) and a sepsis model group [lipopolysaccharide (LPS) group]. The model group was injected intraperitoneally with LPS at a dose of 8 mg/kg and the Con group was injected with an equal volume of normal saline. Hippocampal tissues were harvested at 1, 3, and 6 days after injection and the expressions of SIK1, SIK2, and SIK3 were detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting. Secondly, C57BL/6 mice were randomly divided into a Con group, a LPS group, and a SIK inhibitor group (HG group). The LPS and HG groups were injected with LPS to establish a sepsis model; in the HG group, HG-9-91-01 (10 mg/kg) was injected intraperitoneally at 3-6 days after LPS injection, and the LPS group was injected with the same volume of vehicle. Cognitive function was assessed at 7-11 days after LPS injection using the Morris water maze (MWM). Hippocampal tissues were harvested after the behavioral tests, and the mRNA levels of inflammatory factors and microglial markers were assessed by qPCR. The protein levels of inducible nitric oxide synthase (iNOS), CD68, ionized calcium binding adaptor molecule 1 (Iba-1), N-methyl-D-aspartate (NMDA) receptor (NR) subunit, cAMP response element-binding protein (CREB)-regulated transcription coactivator 1 (CRTC1), and insulin-like growth factor 1 (IGF-1) were detected by Western blotting. Immunohistochemistry (IHC) was used to detect the expression of Iba-1 positive cells in the CA1, CA3 and dentate gyrus (DG) of the hippocampus, followed by Sholl analysis. Compared with the Con group, the mRNA and protein levels of SIK1, SIK2, and SIK3 in the hippocampus were increased in the LPS group (all P<0.05). Compared with the Con group, mice in the LPS group had a significantly longer escape latency, a lower percentage of target quadrant dwell time and a reduced locomotor speed (all P<0.05); the HG group had a decreased escape latency and an increased percentage of time spent in the target quadrant in comparison with the LPS group (both P<0.05). The mRNA levels of inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], and the M1-type microglial markers iNOS and CD68 in the hippocampus of the LPS group were increased in comparison with the Con group, while the M2-type microglial markers CD206 and arginase-1 (Arg-1) were decreased. Compared with the LPS group, the mRNA levels of TNF-α, IL-1β, IL-6, and iNOS were downregulated, while the levels of CD206 and Arg-1 were upregulated in the HG group (all P<0.05). The protein levels of iNOS, CD68, and Iba-1 in the hippocampus of the LPS group were increased in comparison with the Con group, but they were downregulated in the HG group in comparison with the LPS group (all P<0.05). The number of Iba-1 positive cells in CA1, CA3, and DG of the hippocampus was increased in the LPS group in comparison with the Con group, but they were decreased in the HG group in comparison with the LPS group (all P<0.05). Sholl analysis showed that the number of intersections at all radii between 8-38 µm from the microglial soma was decreased in the LPS group in comparison with the Con group (all P<0.05). Compared with the LPS group, the number of intersections at all radii between 14-20 µm was significantly increased in the HG group (all P<0.05). The protein levels of NR subunit NR1, NR2A, NR2B, and IGF-1 were downregulated in the hippocampus of the LPS group in comparison with the Con group, while the expression of phosphorylated CRTC1 (p-CRTC1) was increased. Compared with the LPS group, the levels of NR1, NR2A, NR2B, and IGF-1 were upregulated, while p-CRTC1 was downregulated in the HG group (all P<0.05). SIK expression is upregulated in the hippocampus of septic mice. The SIK inhibitor HG-9-91-01 ameliorates sepsis-associated cognitive dysfunction in mice, and the mechanism may involve in the activation of the CRTC1/IGF-1 pathway, inhibition of neuroinflammation, and enhancement of synaptic plasticity.

Memory impairment induced by aluminum nanoparticles is associated with hippocampal IL-1 and IBA-1 upregulation in mice

ABSTRACT Objectives Increasing evidence indicates a link between aluminum (Al) intake and Alzheimer’s disease (AD). The main entry of Al into the human body is through oral route, and in the digestive tract, under the influence of the pH change, Al can be transformed into Al nanoparticles (Al-NP). However, studies related to the effect of Al-NP on the brain are limited and need further investigation. Neuro-inflammation is considered as one of the principal features of AD. Microglial activation and expression of the inflammatory cytokine IL-1β (interleukin-1β) in the brain have been used as hallmarks of brain inflammation. Therefore, in the present study, the hippocampal levels of ionized calcium-binding adaptor molecule 1 (IBA-1), as the marker of microglia activation, and IL-1β were assessed. Methods Adult male NMRI mice were treated with Al-NP (5 or 10 mg/kg) for 5 days. A novel object recognition (NOR) test was used to assess memory. Following cognitive assessments, the hippocampal tissues were isolated to analyze the levels of IL-1β and IBA-1 as well as beta actin proteins using western blot technique. Results Al-NP in both doses of 5 and 10 mg/kg impaired NOR memory in mice. In addition, Al-NP increased IL-1β and IBA-1 in the hippocampus. Discussion These findings indicate that the memory impairing effect of Al-NP coincides with hippocampal inflammation. According to the proposed relationship between AD and Al toxicity, this study can increase the knowledge about the toxic effects of Al-NP and highlight the need to limit the use of this nanoparticle.

Moxibustion influences hippocampal microglia polarization via IL-33/ST2 pathway in Alzheimer's disease mice.

To observe the effect of moxibustion on the polarization of microglia towards M2 direction in Alzheimer's disease (AD) mice through the interleukin-33 (IL-33)/growth stimulating gene 2 protein (ST2) signaling pathway. Five-month-old APP/PS1 male mice were randomly divided into model and moxibustion (Moxi) groups, and C57BL/6J mice of the same age were as the control group, with 9 mice in each group. In the Moxi group, moxibustion was applied at "Baihui" (GV20) and "Yongquan" (KI1) for 30 min, once a day, 5 days a week for 4 weeks. The spatial learning memory ability was observed by the Morris water maze test. The relative expressions of IL-33 and ST2 in hippocampus were detected by Western blot. The positive expression of amyloid-β (Aβ), phosphorylated Tau (p-Tau), IL-33/ionized calcium binding adapter molecule 1(Iba-1), ST2/Iba-1, arginase 1 (Arg1)/Iba-1 and indu-cible nitric oxide synthase (iNOS)/Iba-1 in hippocampal CA1 region were detected by immunofluorescence. Compared with the control group, the escape latency of the mice in the model group was prolonged (P<0.001, P<0.01), the number of times to enter the effective area and the percentage of target quadrant swimming time were reduced (P<0.001), the positive expression of both Aβ and p-Tau, the positive expression of iNOS/Iba-1 in the hippocampal CA1 region was increased (P<0.001), while the expression of IL-33 and ST2 protein in hippocampal tissue, the positive expression levels of IL-33/Iba-1, ST2/Iba-1 and Arg1/Iba-1 in hippocampal CA1 region were all decreased (P<0.05, P<0.001). After treatment, compared with the model group, the escape latency of the mice in the moxibustion group was shortened (P<0.001, P<0.01), the number of entries into the effective area and the percentage of target quadrant swimming time were increased (P<0.001), the positive expression of Aβ and p-Tau in the hippocampal CA1 region, and the positive expression of iNOS/Iba-1 were decreased (P<0.001), while the expression of IL-33 and ST2 protein in the hippocampal tissue, the positive expression of IL-33/Iba-1, ST2/Iba-1 and Arg1/Iba-1 in hippocampal CA1 region were all increased (P<0.05, P<0.01, P<0.001). Moxibustion can improve the spatial learning and memory abilities, reduce the pathological deposition of Aβ and p-Tau in APP/PS1 mice, which may be related to its function in up-regulating the IL-33/ST2 signaling pathway to regulate the polarization of microglia towards M2 direction.