PrP 106-126 Altered PrP mRNA Gene Expression in Mouse Microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases.The pathogenic agent is an abnormal prion protein aggregate.Microglial activation in the centre nervous system is a characteristic feature of prion disease.In this study,we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR.PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126,with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly.Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126,and indicate that microglial cells might play a critical role in prion pathogenesis.

Tamoxifen Induces Apoptosis of Mouse Microglia Cell Line BV-2 Cells via both Mitochondrial and Death Receptor Pathways

Little is known about whether tamoxifen (TAM) can affect resting state microglia apoptosis and about the cellular mechanism that may account for this. To explore this question, we incubated the microglia cell line BV-2 cells with TAM at different concentrations. Cell viability was assessed by the MTT assay, and flow cytometric analysis was performed to detect the cell apoptosis rate. Furthermore, mitochondrial membrane potential (Δψm) was tested by flow cytometry, and Bax, Bcl-2, Fas, and Fas-L expression was detected by Western blot. The results demonstrated that TAM decreased cell viability and induced apoptosis of BV-2 cells in a concentration- and time-dependent manner. In addition, disruption of Δψm was followed by up-regulated expression of pro-apoptotic Bax, Fas and Fas-L, and down-regulated expression of anti-apoptotic Bcl-2. These results indicate that TAM may induce apoptosis of BV-2 cells through both mitochondria- and death receptor-mediated pathways.

Conditioned medium from human dental pulp stem cells treats spinal cord injury by inhibiting microglial pyroptosis

Human dental pulp stem cell transplantation has been shown to be an effective therapeutic strategy for spinal cord injury.However,whether the human dental pulp stem cell secretome can contribute to functional recovery after spinal cord injury remains unclear.In the present study,we established a rat model of spinal cord injury based on impact injury from a dropped weight and then intraperitoneally injected the rats with conditioned medium from human dental pulp stem cells.We found that the conditioned medium effectively promoted the recovery of sensory and motor functions in rats with spinal cord injury,decreased expression of the microglial pyroptosis markers NLRP3,GSDMD,caspase-1,and interleukin-1β,promoted axonal and myelin regeneration,and inhibited the formation of glial scars.In addition,in a lipopolysaccharide-induced BV2 microglia model,conditioned medium from human dental pulp stem cells protected cells from pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway.These results indicate that conditioned medium from human dental pulp stem cells can reduce microglial pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway,thereby promoting the recovery of neurological function after spinal cord injury.Therefore,conditioned medium from human dental pulp stem cells may become an alternative therapy for spinal cord injury.

Lipopolysaccharide-activated microglial-induced neuroglial cell differentiation in bone marrow mesenchymal stem cells

BACKGROUND： Microglia are very sensitive to environmental changes, often becoming activated by pathological conditions. Activated microglia can exert a dual role in injury and repair in various diseases of the central nervous system, including cerebral ischemia, Parkinson＇s disease, and Alzheimer＇s disease. OBJECTIVE： An immortal microglial cell line, BV2, was treated with varying concentrations of lipopolysaccharide （LPS） to induce a pathological situation. Supernatant was harvested and incubated with bone marrow mesenchymal stem cells and, concomitantly, bone marrow mesenchymal stem cell differentiation was observed. DESIGN： A controlled observation, in vitro experiment. SETTING： Department of Neurology, First Affiliated Hospital of China Medical University. MATERIALS： Five male 2-3-week-old Sprague Dawley rats were purchased from Animal Laboratory Center of China Medical University and included in this study. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The microglial cell line BV2 was produced by Cell Research Institute of Chinese Academy of Sciences. LPS was produced by Sigma Company, USA. METHODS： This study was performed in the Central Laboratory of China Medical University from September 2006 to March 2007. Rat femoral and tibial bone marrow was collected for separation and primary culture of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cell cultures were divided into 5 groups： control group, non-activated group, as well as low-, medium-, and high-dose LPS groups. In the control group, bone marrow mesenchymal stem cells were cultured with Dulbecco＇s modified Eagle＇s medium （DMEM） supplemented with fetal bovine serum （volume fraction 0. 1）. In the non-activated group, bone marrow mesenchymal stem cells were incubated with non-activated BV2 supernatant. In the low-, medium-, and high-dose LPS groups, bone marrow mesenchymal stem cells were incubated with LPS （0.01,0.1 and 1 μg/L, respectively）-activated BV2 supernatant. MAIN OUTCOME MEASURES： Expression of glial fibrillary acidic protein （GFAP） and neuron-specific enolase （NSE） in bone marrow mesenchymal stem cells was detected by immunofluorescence staining. RESULTS： GFAP-positive cells were detected in each group; however, the greatest number were found in the high-dose LPS group. The number of GFAP-positive cells was significantly greater in the high- and medium-dose groups, compared to the control, non-activated, and low-dose LPS groups （P 〈 0.05）. However there was no significant difference between the medium- and high-dose LPS groups （P 〉 0.05）. NSE-positive cells were also detected in each group. However, there was no significant difference between any two groups （P 〉 0.05）. CONCLUSION： Microglia, when activated to some ertent, could induce neuroglial cell differentiation from bone marrow mesenchymal stem cells; however, they did not exhibit the capacity to markedly promote neuronal cell differentiation. When microglia are activated, the capacity to induce bone marrow mesenchymal stem cell differentiation reaches a peak level, but is not increased with greater activation rates.

Retinal ganglion cell death in a DBA/2J mouse model of glaucoma Microglial activation and intraocular pressure

BACKGROUND： Retinal microglia has been shown to reactivate in a murine model of pigmentary glaucoma. However, the relationship between microglial activation and intraocular pressure （lOP） elevation and retinal ganglion cell （RGC） death is still unclear. OBJECTIVE： To verify that microglial activation and tumor necrosis factor alpha （TNF-α） expression is involved in RGC death with elevated lOP and prolonged time of glaucomatous optic nerve lesion in a DBA/2J mouse model of glaucoma. DESIGN, TIME AND SETTING： This randomized, controlled, animal experiment was performed at the Peking University Third Hospital, Peking University Eye Center, China between December 2006 and May 2008.MATEFIiALS： DBA/2J mice and C57BL/6J mice （Jackson Laboratory, USA）, rat anti-mouse CD11 b monoclonal antibody （Serotec, UK）, and goat anti-TNF-α polyclonal antibody （Sigma, USA） were used in this study.METHODS： A total of 100 female, DBA/2J mice at 3, 6, 9, 12, and 14 months of age （20 mice per age group） were used for the glaucoma model, and 18 C57BL/6J mice at 3, 9, 14 months of age （6 mice per age group） were used as normal controls. The anterior segment of the eye was observed using a slit-lamp biomicroscope, lOP was measured using a microneedle system. Morphology and number of retinal microglia were observed using immunohistochemistry. RGCs were quantified using Nissl staining. Co-localization of TNF-α and microglia was observed using double-labeling immunofluorescence. Excavation of the optic nerve head was observed utilizing hematoxylin-eosin staining. MAIN OUTCOME MEASURES： The following parameters were measured： lOP levels, numbers of RGCs and activated microglia, and TNF-α expression. RESULTS： In 6-month-old DBA/2J mice, dispersed pigment was observed, and some mice developed increased IOP. At 9 months of age, lOP levels reached a peak. In 3-month-old DBA/2J mice, microglia were activated. In 6-month-old DBA/2J mice, the number of activated microglia was significantly increased and migrated to the outer retinal layer. In 9-month-old mice, TNF-a expression was co-localized with microglia. Significant RGC loss occurred in mice aged 9 to 14 months, with the presence of optic nerve fiber loss and optical nerve head excavation, lOP returned to normal levels at 12 months of age, but microglia remained activated, which was consistent with RGC loss. CONCLUSION： Retinal microglial activation was partially attributed to increased lOP. Activated microglia might be mainly responsible for RGC loss. TNF-α expression was evident in the inner retinal layer. However, the relationship between TNF-α and RGC loss remains poorly understood.

美满霉素对脂多糖诱导的BV-2小胶质细胞肿瘤坏死因子-α表达的影响

目的探讨美满霉素(MC)对脂多糖(LPS)诱导的BV-2小胶质细胞肿瘤坏死因子-α(TNF-α)表达的影响。方法采用相应浓度的MC或LPS(100 ng/ml)对MC组及0.1、1、10、100μmol/L MC+LPS组、LPS组、空白对照组BV-2小胶质细胞进行预处理。ELISA法检测BV-2小胶质细胞TNF-α蛋白的表达,逆转录-PCR检测细胞TNF-αmRNA表达。结果与空白对照组比较,0.1、1μmol/L MC+LPS组及LPS组的TNF-α水平显著升高(均P<0.01)。10、100μmol/L MC+LPS组TNF-α水平显著低于LPS组(均P<0.01)。与空白对照组比较,LPS组及10μmol/L MC+LPS组TNF-αmRNA的表达水平显著增高(均P<0.01)。10μmol/L MC+LPS组TNF-αmRNA表达水平显著低于LPS组(P<0.01)。结论 MC预处理(≥10μmol/L)可显著抑制LPS诱导的小胶质细胞TNF-α的表达。

鱼藤酮对小鼠小胶质细胞 BV-2存活率及一氧化氮含量的影响

目的：研究鱼藤酮对小鼠小胶质细胞BV-2存活率及一氧化氮含量的影响。方法 BV-2细胞以5×10^7/L密度接种到细胞培养板中，分别加入鱼藤酮0、1×10^-11、1×10^-10、1×10^-9、1×10^-8、1×10^-7、1×10^-6、1×10^-5 mol/L后0、6、12、24、48、72 h等测定细胞存活率。另以1×10^-8 mol/L鱼藤酮干预BV-2细胞48 h，测定上清液中超氧化物歧化酶、过氧化物酶、总巯基、超氧阴离子和NO含量并与未给予鱼藤酮干预的对照组比较。结果5×10^7/L BV-2细胞于0、6、12、24、48、72 h细胞增殖活力分别为0．035±0．001、0．132±0．006、0．334±0．017、1．073±0．044、2．272±0．172、0．776±0．032，可见48 h达到细胞生长曲线的峰值。鱼藤酮（1×10^-6 mol/L ）干预 BV-2细胞24、48、72 h 细胞存活率分别降低至（63．4±10．1）％、（51．7±12．2）％、（33．9±11．2）％；鱼藤酮（1×10^-7 mol/L）干预BV-2细胞72 h细胞存活率降低至（50．8±2．9）％。1×10^-8 mol/L 鱼藤酮干预48 h 后 BV-2细胞上清液一氧化氮水平达（27．6±6．2）μmol/L，明显高于对照组的（13．3±2．5）μmol/L （t＝-2．135， P＝0．044）。结论鱼藤酮在一定浓度范围内可激活小胶质细胞，但是超出此浓度范围时则可能导致小胶质细胞存活率降低。

EP2受体拮抗剂AH6809对BV-2小胶质细胞凋亡的影响

目的:观察不同浓度前列腺素E_2受体2(EP2)抑制剂AH6809对BV-2细胞的致凋亡作用,探索AH6809使用浓度与BV-2细胞凋亡的关系。方法:将不同浓度(10、20、30、40、80、160μmol/L)的AH6809依次加入BV-2细胞培养液中,运用倒置显微镜、免疫荧光双标、TUNEL检测、流式细胞术和Western Blot技术,对AH6809干预后的BV-2细胞的形态和凋亡情况进行观察。结果:AH6809在40μmol/L以上时,观察到BV-2细胞开始出现凋亡迹象,并且随着AH6809浓度的递增,发生凋亡的BV-2细胞呈现出逐渐增多的趋势,表现出浓度依赖性特征,差异具有统计学意义(P <0. 05)。结论:AH6809对BV-2细胞有致凋亡作用,使用时需要考虑浓度对细胞凋亡的影响。

甲酰肽受体-2促进LPS诱导的BV-2细胞炎症反应

目的考察脂多糖(lipopolysaccharide,LPS)激活的BV-2细胞中甲酰肽受体-2(formyl peptide receptor-2,FPR2)的表达及其对细胞炎症反应的作用。方法 1 mg·L^(-1)的LPS刺激BV-2细胞12 h,建立体外小胶质细胞炎症模型。Western blot法检测FPR2的表达;分别用FPR2特异性激动剂MMK^(-1)和拮抗剂Boc-2孵育LPS-BV-2细胞后,ELISA法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白介素1β(interleukin^(-1)β,IL^(-1)β)的分泌情况;Western blot法检测下游信号分子NF-κB的磷酸化;Transwell小室法考察LPS-BV-2细胞的迁移情况。结果 LPS可使BV-2细胞上的FPR2表达上调,并激活NF-κB。与LPS组比较,激动剂MMK^(-1)可促进LPS-BV-2细胞的TNF-α、IL^(-1)β的分泌和趋化,拮抗剂Boc-2可抑制这些反应。结论 LPS可诱导BV-2细胞膜表面FPR2表达上调,FPR2可使LPS诱发的炎症反应增强。

Rutin pretreatment promotes microglial M1 to M2 phenotype polarization

Microglial cells are important resident innate immune components in the central nervous system that are often activated during neuroinflammation.Activated microglia can display one of two phenotypes,M1 or M2,which each play distinct roles in neuroinflammation.Rutin,a dietary flavonoid,exhibits protective effects against neuroinflammation.However,whether rutin is able to influence the M1/M2 polarization of microglia remains unclear.In this study,in vitro BV-2 cell models of neuroinflammation were established using 100 ng/mL lipopolysaccharide to investigate the effects of 1-hour rutin pretreatment on microglial polarization.The results revealed that rutin pretreatment reduced the expression of the proinflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6 and increased the secretion of interleukin-10.Rutin pretreatment also downregulated the expression of the M1 microglial markers CD86 and inducible nitric oxide synthase and upregulated the expression of the M2 microglial markers arginase 1 and CD206.Rutin pretreatment inhibited the expression of Toll-like receptor 4 and myeloid differentiation factor 88 and blocked the phosphorylation of I kappa B kinase and nuclear factor-kappa B.These results showed that rutin pretreatment may promote the phenotypic switch of microglia M1 to M2 by inhibiting the Toll-like receptor 4/nuclear factor-kappa B signaling pathway to alleviate lipopolysaccharide-induced neuroinflammation.

Exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation after traumatic brain injury

Exosomes derived from bone marrow mesenchymal stem cells can inhibit neuroinflammation through regulating microglial phenotypes and promoting nerve injury repair.However,the underlying molecular mechanism remains unclear.In this study,we investigated the mechanism by which exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation.Our in vitro co-culture experiments showed that bone marrow mesenchymal stem cells and their exosomes promoted the polarization of activated BV2 microglia to their anti-inflammatory phenotype,inhibited the expression of proinflammatory cytokines,and increased the expression of anti-inflammatory cytokines.Our in vivo experiments showed that tail vein injection of exosomes reduced cell apoptosis in cortical tissue of mouse models of traumatic brain injury,inhibited neuroinflammation,and promoted the transformation of microglia to the anti-inflammatory phenotype.We screened some microRNAs related to neuroinflammation using microRNA sequencing and found that microRNA-181b seemed to be actively involved in the process.Finally,we regulated the expression of miR181b in the brain tissue of mouse models of traumatic brain injury using lentiviral transfection.We found that miR181b overexpression effectively reduced apoptosis and neuroinflamatory response after traumatic brain injury and promoted the transformation of microglia to the anti-inflammatory phenotype.The interleukin 10/STAT3 pathway was activated during this process.These findings suggest that the inhibitory effects of exosomes derived from bone marrow mesenchymal stem cells on neuroinflamation after traumatic brain injury may be realized by the action of miR181b on the interleukin 10/STAT3 pathway.

髓系细胞触发受体2在高糖处理的小胶质细胞中的表达及作用

目的 探索高糖条件下小胶质细胞中髓系细胞触发受体2(triggering receptor expressed on myeloid cells 2, TREM2)的表达情况,以及TREM2在高糖条件下小胶质细胞增殖、迁移和吞噬中的作用。方法 小胶质细胞分为对照组、高糖处理组(67.5 mmol/L葡萄糖,24 h),检测小胶质细胞数量、Iba1和TREM2的表达水平;转染TREM2的siRNA,检测小胶质细胞增殖和迁移能力的变化;加入带有荧光标签的淀粉样蛋白β(Aβ),观察小胶质细胞对Aβ吞噬能力的影响。结果 与正常小胶质细胞相比,高糖处理后小胶质细胞的数量明显下降(P<0.001),而TREM2和Iba1表达显著升高(P<0.001)。高糖和TREM2均不影响小胶质细胞的增殖能力。与正常组相比,高糖处理后小胶质细胞迁移能力下降(P<0.05),而TREM2对高糖小胶质细胞的迁移能力无显著影响。与正常小胶质细胞相比,高糖处理组小胶质细胞对Aβ的吞噬能力显著下降(P<0.001),TREM2 siRNA敲减后高糖小胶质细胞对Aβ的吞噬能力进一步下降(P<0.001)。结论 高糖处理后小胶质细胞TREM2表达明显升高,其主要影响小胶质细胞对Aβ的吞噬能力。

Mitochonic acid 5 regulates mitofusin 2 to protect microglia

Microglial apoptosis is associated with neuroinflammation and no effective strategies are currently available to protect microglia against inflammation-induced apoptosis. Mouse microglial BV-2 cells(5 × 10^6) were incubated with 10 μg/mL lipopolysaccharides for 12 hours to mimic an inflammatory environment. Then the cells were co-cultured with mitochonic acid 5(MA-5) for another 12 hours. MA-5 improved the survival of lipopolysaccharide-exposed cells. MA-5 decreased the activity of caspase-3, which is associated with apoptosis. MA-5 reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells, and increased adenosine triphosphate levels in cells. MA-5 decreased the open state of the mitochondrial permeability transition pore and reduced calcium overload and diffusion of second mitochondria-derived activator of caspase(Smac). MA-5 decreased the expression of apoptosis-related proteins(mitochondrial Smac, cytoplasmic Smac, pro-caspase-3, cleaved-caspase-3, and caspase-9), and increased the levels of anti-apoptotic proteins(Bcl2 and X-linked inhibitor of apoptosis protein), mitochondria-related proteins(mitochondrial fusion protein 2, mitochondrial microtubule-associated proteins 1 A/1 B light chain 3 B II), and autophagy-related proteins(Beclin1, p62 and autophagy related 5). However, MA-5 did not promote mitochondrial homeostasis or decrease microglial apoptosis when Mitofusin 2 expression was silenced. This shows that MA-5 increased Mitofusin 2-related mitophagy, reversed cellular energy production and maintained energy metabolism in BV-2 cells in response to lipopolysaccharide-induced inflammation. These findings indicate that MA-5 may promote the survival of microglial cells via Mitofusin 2-related mitophagy in response to lipopolysaccharide-induced inflammation.

醛酮还原酶1成员B1(AKR1B1)激活NF-κB通路诱导小鼠BV-2小胶质细胞活化抑制视网膜神经节细胞活性

目的探讨醛酮还原酶1成员B1(AKR1B1)在调控小胶质细胞极化中的机制及其对视网膜神经节细胞(RGC)活性的影响。方法利用脂多糖(LPS)诱导BV-2小胶质细胞极化,分别利用AKR1B1小干涉RNA(siRNA)和抑制剂泊那司他(ponalrestat/Statil)作用BV-2细胞;实时定量PCR检测BV-2小胶质细胞AKR1B1、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、环加氧酶2(COX2)、诱导型一氧化氮合酶(iNOS)的mRNA表达,ELISA检测BV-2小胶质细胞培养上清液TNF-α、IL-1β含量。采用BV-2条件培养基处理原代RGC,免疫荧光细胞化学染色检测RGC脑特异性同源盒蛋白3a(Brn-3a)的表达以确定RGC活性,TUNEL染色检测RGC凋亡;Western blot法检测RGC核因子κBp65(NF-κBp65)、核因子κB抑制物激酶(IKK)蛋白磷酸化情况。结果LPS诱导BV-2细胞中TNF-α、IL-1β、COX2和iNOS的mRNA表达增加,培养液上清中TNF-α、IL-1β含量增加。BV-2细胞条件培养基导致RGC活力降低、凋亡增加;敲低AKR1B1后,可阻断BV-2细胞M1型极化,恢复RGC活力;敲低AKR1B1可阻断LPS诱导的IKK磷酸化和NF-κBp65的入核。结论AKR1B1可通过激活NF-κB通路诱导小胶质细胞的活化,进而降低RGC的活力并促进其凋亡。

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.

温肾通督方促进小鼠脊髓损伤的修复

背景:前期研究证实,温肾通督方可通过抑制脾脏B细胞焦亡、促进微血管内皮细胞吞噬髓鞘碎片、影响小胶质细胞迁移和浸润、促进受损神经元恢复、降低脊髓损伤后神经元凋亡等促进脊髓损伤恢复,但其机制尚不明确。目的:探讨温肾通督方对脊髓损伤小鼠髓系细胞触发受体2(triggering receptor expressed on myeloid cells 2,TREM2)及PI3K/Akt信号通路的影响。方法:取36只C57BL/6小鼠,采用随机数字表法分为假手术组、模型组、温肾通督方组,每组12只。模型组与温肾通督方组采用改良Allen’s法制备小鼠T10脊髓损伤模型,造模后第1天,温肾通督方组灌胃给予温肾通督方,假手术组、模型组灌胃给予生理盐水,每天1次,连续给药28 d。给药期间,通过BMS评分和斜板实验评价各组小鼠运动功能;造模后第7,28天,采用苏木精-伊红染色观察各组小鼠脊髓组织病理变化,免疫荧光染色双标法检测脊髓组织离子化钙结合适配分子1(ionized calcium binding adaptor molecule 1,IBA1)、TREM2蛋白表达,Western blot检测脊髓组织TREM2、PI3K、p-PI3K、Akt、p-Akt、Bcl2、Bax、Caspase3蛋白表达。结果与结论:①BMS评分和斜板实验结果表明,脊髓损伤造模后小鼠后肢运动功能下降,而经过温肾通督方治疗后,脊髓损伤小鼠后肢运动功能明显提升。②苏木精-伊红染色结果显示,与模型组比较,温肾通督方小鼠脊髓组织病理结构明显改善,表现为背侧白质和神经元萎缩程度、细胞质空泡化降低及炎性细胞浸润减少等。③免疫荧光染色结果显示,造模后第7天,模型组IBA1、TREM2蛋白表达均低于假手术组(P<0.05),温肾通督方组IBA1、TREM2蛋白表达均高于模型组(P<0.05);造模后第28天,模型组TREM2蛋白表达低于假手术组(P<0.05),温肾通督方组小鼠脊髓组织中的TREM2蛋白表达高于模型组(P<0.05)。④Western blot检测结果表明,造模后第7天,与假手术组相比,模型组TREM2、Akt蛋白表达及Bcl2/Bax比值降低(P<0.05),p-Akt、Bax蛋白表达及p-Akt/Akt比值升高(P<0.05);与模型组相比,温肾通督方组TREM2、PI3K、p-PI3K、Akt、p-Akt、Bcl2蛋白表达及p-PI3K/PI3K、p-Akt/Ak、Bcl2/Bax比值升高(P<0.05),Bax、Caspase3蛋白表达降低(P<0.05)。造模后第28天,与假手术组相比,模型组TREM2、PI3K、p-PI3K、Akt、p-Akt、Bcl2蛋白表达及Bcl2/Bax比值降低(P<0.05),Bax蛋白表达升高(P<0.05);与模型组相比,温肾通督方组TREM2、PI3K、Akt、p-Akt、Bcl2蛋白表达及Bcl2/Bax比值升高(P<0.05),Bax蛋白表达降低(P<0.05)。⑤结果表明,温肾通督方可能通过上调小胶质细胞TREM2激活PI3K/Akt信号通路,抑制神经元凋亡发挥神经保护作用,进而促进脊髓损伤的修复。

他莫昔芬介导小胶质细胞LRRK-2表达对炎症刺激PC12细胞的影响

目的探讨他莫昔芬对小胶质细胞的富含亮氨酸重复序列激酶2(LRRK2)基因及相关炎症因子表达的影响。方法取原代小胶质细胞分为CON组、脂多糖(LPS)组、LPS+他莫昔芬组、LPS+LRRK2组;以LPS刺激激活小胶质细胞,用他莫昔芬调控小胶质细胞激活;将各组小胶质细胞与大鼠肾上腺嗜铬瘤细胞(PC12)细胞共培养24 h;Western Blot法检测小胶质细胞的LRRK2、一氧化氮合成酶(iNOS)表达,酶联免疫吸附法(ELISA)测定TNF-α、IL-1β水平;Hochest染核法检测PC12细胞凋亡/存活比。结果他莫昔芬抑制LPS刺激后小胶质细胞的LRRK2表达,通过抑制LRRK2下调iNOS、TNF-α及IL-1β释放,进而降低小胶质细胞与PC12细胞共培养体系炎症表达及PC12细胞凋亡/存活比。结论 LRRK2是小胶质细胞炎症因子iNOS释放的上游调控靶点,他莫昔芬可通过调控LRRK2基因抑制小胶质细胞激活,从而减轻炎症相关多巴胺能神经元损伤。

Interleukin-4 promotes microglial polarization toward a neuroprotective phenotype after retinal ischemia/reperfusion injury

Glaucoma results from irreversible loss of retinal ganglion cells(RGCs)through an unclear mechanism.Microglial polarization and neuroinflammation play an important role in retinal degeneration.Our study aimed to explore the function of microglial polarization during glaucoma progression and identify a strategy to alleviate retinal neuroinflammation.Retinal ischemia/reperfusion injury was induced in C57BL/6 mice.In a separate cohort of animals,interleukin(IL)-4(50 ng/mL,2μL per injection)or vehicle was intravitreally injected after retinal ischemia/reperfusion injury.RGC loss was assessed by counting cells that were positive for the RGC marker RNA binding protein,mRNA processing factor in retinal flat mounts.The expression of classically activated(M1)and alternatively activated(M2)microglial markers were assessed by quantitative reverse transcription-polymerase chain reaction,immunofluorescence,and western blotting.The results showed that progressive RGC loss was accompanied by a continuous decrease in M2 microglia during the late phase of the 28-day period after retinal ischemia/reperfusion injury.IL-4 was undetectable in the retina at all time points,and intravitreal IL-4 administration markedly improved M2 microglial marker expression and ameliorated RGC loss in the late phase post-retinal ischemia/reperfusion injury.In summary,we observed that IL-4 treatment maintained a high number of M2 microglia after RIR and promoted RGC survival.

髓样细胞触发受体2在神经退行性疾病中的作用

髓样细胞触发受体(triggering receptor expressed on myeloid cell,TREM)家族是由Bouchon等⑴于2000年在骨髓细胞表面发现的.TREM2是TREM家族的重要成员之一,被认为是参与调节免疫反应和小胶质细胞吞噬作用的关键信号因子。大多数研究证明TREM2具有抗炎活性,而炎性反应是多数神经退行性疾病的共同特征。近年来有关TREM2与神经退行性疾病之间的关系越来越受到重视,已有研究表明.TREM2在阿尔茨海默病(alzheimer's disease,AD)[2]、帕金森病(parkinson's disease,PD)[3]肌萎缩脊髓侧索硬化症(amyotrophic lateral sclerosis,ALS)⑷、Nasu-Hakola病(nasuhakola disease,NHD)[5]等神经退行性疾病的发病机制中发挥着重要作用。

TREM2与脑内小胶质细胞作用对中枢神经系统的影响

小胶质细胞是中枢神经系统中的固有免疫细胞。在生理条件下,小胶质细胞起免疫监视作用,参与突触修饰、清除凋亡细胞。在病理条件下,小胶质细胞由静息状态转化为激活态,向病变部位迁移、增殖,吞噬清除异常物质、分泌细胞因子,参与氧化应激和神经修复。小胶质细胞在众多中枢神经系统疾病中被称为“双刃剑”,以阿尔茨海默病(Alzheimer disease,AD)为例,一方面小胶质细胞可吞噬、清除异常蛋白聚集产物,另一方面,过度活化的小胶质细胞损伤突触及神经元。全基因组关联研究中确定的与AD相关的大多数基因都在小胶质细胞中高度表达或仅表达于小胶质细胞[1].