The therapeutic mechanism of Shenyuan Gan in lipopolysaccharide-induced neuroinflammation in BV2 microglial cells

Objective To study the therapeutic effects of Shenyuan Gan(参远苷,SYG)on the inflammat-ory response in BV2 microglial cells induced by lipopolysaccharide(LPS).Methods The cytotoxicity of SYG to BV2 microglial cells was evaluated using a Cell Counting Kit-8(CCK-8)assay,and the effect of SYG concentrations on LPS-induced BV2 microglial cells was studied.The morphological changes were observed using an optical microscope.The nitric oxide(NO)concentration in cell culture supernatant was determined using Griess re-agent.The expression of cytokines and inflammatory mediators were also measured by an en-zyme-linked immunosorbent assay(ELISA).Western blot analysis was used to determine the levels of inducible NO synthase(iNOS),nuclear factor-kappa B(NF-κB)p65,alpha inhibitor of NF-κB(IκB-α),phosphorylation-IκB-α(p-IκB-α),NOD-like receptor 3(NLRP3),and cas-pase-1 expression.Moreover,the expression of iNOS,NLRP3,and ionized calcium binding adapter molecule 1(Iba1)was also observed using immunofluorescent staining.Results SYG had a low cytotoxic effect on BV2 microglial cells and could significantly decr-ease LPS-induced morphological changes of BV2 microglial cells(P<0.05).ELISA results showed that SYG significantly inhibited the LPS-induced increase in interleukin(IL)-1βand IL-6 in BV2 microglia cells(P<0.05),and Western blot analysis showed that the phosphoryla-tion levels of iNOS,NF-κB p65,and IκB-αas well as NLRP3 and caspase-1 expression were also significantly decreased,and IκB-αexpression was increased after SYG treatment(P<0.05,compared with the LPS-treated group).The immunofluorescence results were consist-ent with the Western blot results,and Iba1 staining indicated that the cell morphology tended to be resting.These results indicate that SYG has a certain inhibitory effect on LPS-induced inflammation in BV2 microglial cells.Conclusion SYG can inhibit LPS-induced release of inflammatory factors in BV2 microglial cells by affecting the phosphorylation levels of NF-κB p65 and IκB-α.SYG is a valuable candid-ate for treating neuroinflammation-related diseases.

Targeting the P2X7 receptor in microglial cells to prevent brain inflammation

Microglial cells are the key innate immune cells in the brain and they are crucial in maintaining brain parenchyma homeostasis.Under physiological conditions,microglial cells assume a ramified morphology with a small cell body and an extensive network of fine processes,which secrete neurotrophic factors and patrol the surroundings in search for pathogens and eliminate cellular debris via phagocytosis.Microglial cells express a repertoire of pattern recognition receptors(PRRs)that enable them to detect diverse danger-associated molecular patterns(DAMPs)released from damaged cells or cells under stress,or pathogen-associated molecular patterns generated by pathogens during infection.

Valerian (Valeriana officinalis) extract inhibits TNF-α and iNOS gene expression in mouse LPS-activated microglial cells

Background:Inflammation and damage to neurons and other cells in the nervous system can cause disorders of the central nervous system.Microglial cells are activated by pathogen infection and injury to release nitric oxide.Valerian(Valeriana officinalis)has been used as a sedative for the treatment of neurological diseases.This study evaluated inflammation of microglial cells and tumor necrosis factorαand induced nitric oxide synthetase gene expression influenced by valerian extract.Methods:Microglial cells were isolated from mice.Lipopolysaccharide(1 ng/mL)was used to induce inflammation and nitric oxide production in cells for an hour.The inflamed cells were then treated with different concentrations(0.1,0.5,2.5,20,and 50μL/mL)of valerian alcoholic extract for 1 and 24 h.nitric oxide production and tumor necrosis factorαand induced nitric oxide synthetase gene expression were determine by Griess assay and real-time polymerase chain reaction,respectively.Results:The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed no toxicity in several concentrations of the valerian extract.In addition,concentrations from 0.1 to 2.5μL/mL significantly reduced inflammation and nitric oxide production in mouse microglial cells to levels observed in control samples.Furthermore,tumor necrosis factorαand induced nitric oxide synthetase gene expression decreased when 2.5μL/mL extract was used.Conclusion:Based on these results,it can be concluded that 2.5μL/mL valerian alcoholic extract is effective as a candidate alternative medicine for reducing inflammation and nitric oxide production and consequently,the inflammatory symptoms of neurodegeneration.

Pectolinarin inhibited LPS-stimulated inflammation in microglial BV_(2) cells via NF-κB signaling pathway

Background:Neuro-inflammation is regarded as one of the critical pathogenesis in neurodegenerative diseases,which is characterized by the activated microglial cells.Pectolinarin(Pec),a natural flavonoid that exists in many Chinese herbal medicines,has been reported to have various biological activities.However,the effects and mechanisms on neuro-inflammation are not clear.Methods:In this study,the inhibitory effects and mechanisms of Pec on neuro-inflammation were investigated in the LPS-stimulated microglial BV_(2) cells.BV_(2) microglial cells were treated with Pec or vehicle,followed by LPS.Enzyme-linked immunosorbent assay,real-time quantitative PCR,nitric oxide and reactive oxygen species assay,and western blot were performed to examine the effects of Pec on neuro-inflammatory responses.Results:We showed that Pec significantly inhibited the expression of tumor necrosis factorαand interleukin 6 in mRNA and protein levels induced by LPS.Moreover,the production of nitric oxide,iNOS,reactive oxygen species,and COX-2 were suppressed by Pec in LPS-stimulated microglial BV_(2) cells.In addition,Pec inhibited LPS-induced inflammation via nuclear factor kappa B signaling pathway,as evidenced by the reduction of the phosphorylation of inhibitor of nuclear factor kappa-B kinase,the degradation of IκBα,and the nuclear translocation of p65.Conclusion:Taken together,Pec exhibited anti-inflammatory effects in LPS-stimulated microglial BV_(2) cells via nuclear factor kappa B signaling pathway,which might provide therapeutic potential for neuro-inflammation and neurodegenerative diseases.

Inhibition of the cGAS–STING pathway:contributing to the treatment of cerebral ischemia-reperfusion injury

The cGAS–STING pathway plays an important role in ischemia-reperfusion injury in the heart,liver,brain,and kidney,but its role and mechanisms in cerebral ischemia-reperfusion injury have not been systematically reviewed.Here,we outline the components of the cGAS–STING pathway and then analyze its role in autophagy,ferroptosis,cellular pyroptosis,disequilibrium of calcium homeostasis,inflammatory responses,disruption of the blood–brain barrier,microglia transformation,and complement system activation following cerebral ischemia-reperfusion injury.We further analyze the value of cGAS–STING pathway inhibitors in the treatment of cerebral ischemia-reperfusion injury and conclude that the pathway can regulate cerebral ischemia-reperfusion injury through multiple mechanisms.Inhibition of the cGAS–STING pathway may be helpful in the treatment of cerebral ischemia-reperfusion injury.

Treadmill exercise improves hippocampal neural plasticity and relieves cognitive deficits in a mouse model of epilepsy

Epilepsy frequently leads to cognitive dysfunction and approaches to treatment remain limited.Although regular exercise effectively improves learning and memory functions across multiple neurological diseases,its application in patients with epilepsy remains controversial.Here,we adopted a 14-day treadmill-exercise paradigm in a pilocarpine injection-induced mouse model of epilepsy.Cognitive assays confirmed the improvement of object and spatial memory after endurance training,and electrophysiological studies revealed the maintenance of hippocampal plasticity as a result of physical exercise.Investigations of the mechanisms underlying this effect revealed that exercise protected parvalbumin interneurons,probably via the suppression of neuroinflammation and improved integrity of blood-brain barrier.In summary,this work identified a previously unknown mechanism through which exercise improves cognitive rehabilitation in epilepsy.

Advantages of nanocarriers for basic research in the field of traumatic brain injury

A major challenge for the efficient treatment of traumatic brain injury is the need for therapeutic molecules to cross the blood-brain barrier to enter and accumulate in brain tissue.To overcome this problem,researchers have begun to focus on nanocarriers and other brain-targeting drug delivery systems.In this review,we summarize the epidemiology,basic pathophysiology,current clinical treatment,the establishment of models,and the evaluation indicators that are commonly used for traumatic brain injury.We also report the current status of traumatic brain injury when treated with nanocarriers such as liposomes and vesicles.Nanocarriers can overcome a variety of key biological barriers,improve drug bioavailability,increase intracellular penetration and retention time,achieve drug enrichment,control drug release,and achieve brain-targeting drug delivery.However,the application of nanocarriers remains in the basic research stage and has yet to be fully translated to the clinic.

Automatic counting of microglial cell activation and its applications

Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal ganglion cells.This disease results in vision loss and blindness.Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma; however an early detection and treatment,could offer neuronal protection and avoid later serious damages to the visual function.A full understanding of the etiology of the disease will still require the contribution of many scientific efforts.Glial activation has been observed in glaucoma,being microglial proliferation a hallmark in this neurodegenerative disease.A typical project studying these cellular changes involved in glaucoma often needs thousands of images- from several animals- covering different layers and regions of the retina.The gold standard to evaluate them is the manual count.This method requires a large amount of time from specialized personnel.It is a tedious process and prone to human error.We present here a new method to count microglial cells by using a computer algorithm.It counts in one hour the same number of images that a researcher counts in four weeks,with no loss of reliability.

Brain and spinal cord trauma:what we know about the therapeutic potential of insulin growth factor 1 gene therapy

Although little attention has been paid to cognitive and emotional dysfunctions observed in patients after spinal co rd injury,several reports have described impairments in cognitive abilities.Our group also has contributed significantly to the study of cognitive impairments in a rat model of spinal co rd injury.These findings are very significant because they demonstrate that cognitive and mood deficits are not induced by lifestyle changes,drugs of abuse,and combined medication.They are related to changes in brain structures involved in cognition and emotion,such as the hippocampus.Chronic spinal cord injury decreases neurogenesis,enhances glial reactivity leading to hippocampal neuroinflammation,and trigge rs cognitive deficits.These brain distal abnormalities are recently called te rtiary damage.Given that there is no treatment for Tertiary Damage,insulin growth factor 1 gene therapy emerges as a good candidate.Insulin growth factor 1 gene thera py recove rs neurogenesis and induces the polarization from pro-inflammato ry towards anti-inflammatory microglial phenotypes,which represents a potential strategy to treat the neuroinflammation that supports te rtiary damage.Insulin growth factor 1 gene therapy can be extended to other central nervous system pathologies such as traumatic brain injury where the neuroinflammatory component is crucial.Insulin growth factor 1 gene therapy could emerge as a new therapeutic strategy for treating traumatic brain injury and spinal cord injury.

Stress injuries and autophagy in mouse hippocampus after chronic cold exposure

Cold exposure is an external stress factor that causes skin frostbite as well as a variety of diseases.Estrogen might participate in neuroprotection after cold exposure,but its precise mechanism remains unclear.In this study,mice were exposed to 10°C for 7 days and 0–4°C for 30 days to induce a model of chronic cold exposure.Results showed that oxidative stress-related c-fos and cyclooxygenase 2 expressions,MAP1LC3-labeled autophagic cells,Iba1-labeled activated microglia,and interleukin-1β-positive pyramidal cells were increased in the hippocampal CA1 area.Chronic cold exposure markedly elevated the levels of estrogen in the blood and the estrogen receptor,G protein-coupled receptor 30.These results indicate that neuroimmunoreactivity is involved in chronic cold exposure-induced pathological alterations,including oxidative stress,neuronal autophagy,and neuroimmunoreactivity.Moreover,estrogen exerts a neuroprotective effect on cold exposure.

Expression of NG2 and platelet-derived growth factor receptor alpha in the developing neonatal rat brain

Platelet-derived growth factor receptor alpha （PDGFRct） is a marker of oligodendrocyte precursor cells in the central nervous system. NG2 is also considered a marker of oligodendrocyte precursor cells. However, whether there are differences in the distribution and morphol- ogy of oligodendrocyte precursor cells labeled by NG2 or PDGFRa in the developing neonatal rat brain remains unclear. In this study, by immunohistochemical staining, NG2 positive （NG2＋） cells were ubiquitous in the molecular layer, external pyramidal layer, internal pyramidal layer, and polymorphic layer of the cerebral cortex, and corpus callosum, external capsule, piriform cortex, and medial septal nucleus. NG2~ cells were stellate or fusiform in shape with long processes that were progressively decreased and shortened over the course of brain development. The distribution and morphology of PDGFRct positive （PDGFRa＋） cells were coincident with NG2＋ cells. The co- localization of NG2 and PDGFRu in the cell bodies and processes of some cells was confirmed by double immunofluorescence labeling. Moreover, cells double-labeled for NG2 and PDGFRa were predominantly in the early postnatal stage of development. The numbers of NG2＋/PDGFRa＋ cells and PDGFRa＋ cells decreased, but the number of NG2＋ cells increased from postnatal days 3 to 14 in the developing brain. In addition, amoeboid microglial cells of the corpus callosum, newborn brain macrophages in the normal developing brain, did not express NG2 or PDGFRu, but NG2 expression was detected in amoeboid microglia after hypoxia. The present results suggest that NG2 and PDGFRct are specific markers of oligodendrocyte precursor cells at different stages during early development. Additionally, the NG2 protein is involved in inflammatory and pathological processes of amoeboid microglial cells.

Molecular Analysis of Clerodendrum formicarum Effects in Painful Diabetic Neuropathy in Rat

The pathophysiology of diabetic neuropathic pain is due to primarily metabolic and vascular factors. There is an increase in sorbitol and fructose, glycated end products, reactive oxygen species and activation of protein kinase C in the diabetic state. All these factors lead to direct damage to the nerves. Taking effective clinical management of neuropathic pain is based on a pharmacological treatment that has shown their limits and many side effects. The hypothesis of central sensitization inhibited by Clerodendrum formicarum, an African pharmacopoeia plant used to treat headaches, arthritis, epilepsy and chronic pain could act on astrocytes and microglial cells. The objective of this work is to study the effect of Clerodendrum formicarum (100, 150 and 200 mg/kg body weight) on astrocytes and microglial cells in a model of diabetic neuropathic pain induced by alloxan monohydrate (150 mg/kg). We noted a suppression of mechanical allodynia and mechanical hyperalgesia respectively by the Von Frey filaments test and the pressure test on the paw by the Clerodendrum formicarumextracts (ECF) at different doses from 2 h at the first injection of the ECF. After 5 days of treatment, we expressed by Western Blot bands of different proteins and by quantitative RT-PCR, we determined inhibition of the expression of GFAP, CD11b and isoforms 1 and 2 of cyclooxygenase. These results suggest that ECF inhibits the activation of astrocytes, microglial cells and cyclooxygenase signaling pathway.

Mitochondrial dysfunction,oxidative stress and apoptotic induction in microglial BV-2 cells treated with sodium arsenate

The treatment of microglial BV-2 cells with sodium arsenate（As（V）：0.1-400 μmol/L — 48 hr）induces a dose-dependent response.The neurotoxic effects of high concentrations of As（V）（100,200 and 400 μmol/L） are characterized by increased levels of mitochondrial complexesⅠ,Ⅱ,and Ⅳ followed by increased superoxide anion generation.Moreover,As（V） triggers an apoptotic mode of cell death,demonstrated by an apoptotic SubG1 peak,associated with an alteration of plasma membrane integrity.There is also a decrease in transmembrane mitochondrial potential and mitochondrial adenosine triphosphate ATP.It is therefore tempting to speculate that As（V） triggers mitochondrial dysfunction,which may lead to defective oxidative phosphorylation subsequently causing mitochondrial oxidative damage,which in turn induces an apoptotic mode of cell death.

Evolving Models and Tools for Microglial Studies in the Central Nervous System

Microglia play multiple roles in such processes as brain development,homeostasis,and pathology.Due to their diverse mechanisms of functions,the complex sub-classifications,and the large differences between different species,especially compared with humans,very different or even opposite conclusions can be drawn from studies with different research models.The choice of appropriate research models and the associated tools are thus key ingredients of studies on microglia.Mice are the most commonly used animal models.In this review,we summarize in vitro and in vivo models of mouse and human-derived microglial research models,including microglial cell lines,primary microglia,induced microglia-like cells,transgenic mice,human-mouse chimeric models,and microglial replacement models.We also summarize recent developments in novel single-cell and in vivo imaging technologies.We hope our review can serve as an efficient reference for the future study of microglia.