Background: Microglia-mediated neuroinflammation is one of the most prominent characteristics of multiple sclerosis (MS), a chronic demyelination disease. As one of the main active ingredients in Astragali radix, total flavonoids of Astragalus (TFA) has multiple pharmacological effects such as immunomodulation, anti-inflammation and and anti-tumor. However, little is known about whether TFA could inhibit microglia-mediated neuroinflammation in MS. Purpose: This study was aimed to elucidate whether TFA could inhibit microglia-mediated neuroinflammation in MS. Study design: In the present study, we explored the protective effect of TFA on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice for the first time, and discussed its mechanism from the aspect of anti-microglia-mediated neuroinflammation. Methods: The mice received oral administration of TFA (25 and 50 mg/kg) daily from two days before immunization and continued until day 21 post-immunization. The effect of TFA on EAE in mice and its mechanism were investigated by ELISA, Western blot, real-time PCR, luciferase reporter assay, histopathology and immunohistochemistry. Results: TFA were shown to alleviate the severity of EAE in mice. It inhibited the excessive activation of microglia both in spinal cords of EAE mice and in LPS-stimulated BV-2 cells, evidenced by weakening the production of inflammatory mediators such as NO, TNF-alpha, IL-6, and IL-1 beta markedly at either protein or mRNA level. Further study demonstrated that TFA repressed the phosphorylation, nuclear translocation and transcriptional activity of NF kappa B, and inhibited the activation of AKT and JNK signaling in BV-2 cells induced by LPS. The agonists of AKT and JNK, anisomycin and SC79, could partly abolish the inhibitory effect of TFA on the production of inflammatory mediators in BV-2 cells induced by LPS. Conclusions: Taken together, our results clarified that TFA inhibited microglia-mediated inflammation in EAE mice probably through deactivating JNK/AKT/NF kappa B signaling pathways. The novel findings may lay a theoretical foundation for the clinical application of TFA in the treatment of MS.

AIMS:Dendritic cells (DCs) actively participate in the pathogenesis of multiple sclerosis (MS), an autoimmune disease. Astragaloside IV (ASI), an active monomer isolated from the Chinese medicine Astragalus membranaceus, has a wide range of pharmacological effects. We aimed to elucidate the effects of ASI on the development of DCs in the early stage of MS/EAE.MAIN METHODS:The mice were administered with ASI (20 mg/kg) daily 3 days in advance of EAE induction and continuously until day 7 post-immunization. The effect of ASI on CD11c+ DC cells from bone marrow (BMDCs) or the spleen of EAE mice at day 7 post-immunization were investigated respectively by flow cytometry, ELISA, western blot, real-time PCR and immunofluorescence.KEY FINDINGS:ASI administration in the early stage of EAE was demonstrated to delay the onset and alleviate the severity of the disease. ASI inhibited the maturation and the antigen presentation of DCs in spleen of EAE mice and LPS-stimulated BMDCs, as evidenced by decreased expressions of CD11c, CD86, CD40 and MHC II. Accordingly, DCs treated by ASI secreted less IL-6 and IL-12, and prevented the differentiation of CD4+ T cells into Th1 and Th17 cells, which was probably through inhibiting the activation of NFκB and MAPKs signaling pathways.SIGNIFICANCE:Our results implicated the alleviative effect of early ASI administration on EAE might be mediated by suppressing the maturation and function of DCs. The novel findings may add to our knowledge of ASI in the potentially clinical treatment of MS.

BACKGROUND AND PURPOSE:Sleep deprivation compromises learning and memory in both humans and animals, and can be reversed by administration of modafinil, a drug promoting wakefulness. Dysfunctional autophagy increases activation of apoptotic cascades, ultimately leading to increased neuronal death, which can be alleviated by autophagy inhibitors. This study aimed to investigate the alleviative effect and mechanism of modafinil on the excessive autophagy occurring in the hippocampus of mice with deficiency of learning and memory induced by sleep deprivation.EXPERIMENTAL APPROACH:The Morris water maze was used to assess the effects of modafinil on male C57BL/6Slac mice after 48-hr sleep deprivation. The HT-22 hippocampal neuronal cell line was also used. Nissl staining, transmission electron microscope, immunofluorescence, Western blot, transient transfection, and autophagy inducer were used to study the effect and mechanism of modafinil on hippocampal neurons with excessive autophagy and apoptosis.KEY RESULTS:Modafinil improved learning and memory in sleep-deprived mice, associated with the inhibition of excessive autophage and apoptosis and an enhanced activation of the PI3K/Akt/mTOR/P70S6K signalling pathway in hippocampal neurons. These effects of modafinil were abolished by rapamycin. In addition, modafinil suppressed the aberrant autophagy and apoptosis induced by rapamycin and reactivated PI3K/Akt/mTOR/P70S6K signals in HT-22 cells.CONCLUSIONS AND IMPLICATIONS:These results suggested that modafinil alleviated impaired learning and memory of sleep-deprived mice potentially by suppressing excessive autophagy and apoptosis of hippocampal neurons. This novel mechanism may add to our knowledge of modafinil in the clinical treatment of impaired memory caused by sleep loss.

目的 探讨黄芪提取物(RAE)对小鼠实验性自身免疫性脑脊髓炎(EAE)的作用及机制.方法 采用髓鞘少突胶质糖蛋白(MOG35-55)诱导雌性C57BL/6小鼠建立EAE小鼠模型.将小鼠随机分为正常对照组、模型组及模型+RAE 125,250和500 mg·kg-1组.模型+RAE组于造模前2 dig给予RAE,每天1次,共23 d.给药的同时采用双盲法记录神经功能评分及体质量变化;HE和劳克坚劳蓝(LFB)髓鞘染色检测脊髓组织炎症浸润和脱髓鞘情况;流式细胞分析技术检测脾CD4,CD11b和CD11c阳性细胞百分比;Western印迹法检测脊髓组织NF-κB、磷酸化NF-κB(p-NF-κB)、NF-κB抑制蛋白(IκBα)、磷酸化NF-κB抑制蛋白(p-IκBα)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、Bax、Bcl2、活化胱天蛋白酶3及钙离子结合衔接分子1(Iba1)等蛋白表达.结果 与正常对照组相比,模型组神经功能评分增高(P<0.01),体质量显著降低(P<0.05,P<0.01);在给药第3周,模型组相比,模型+RAE 125和500 mg·kg-1组EAE小鼠的发病症状明显改善,体质量降低减轻(P<0.01).与正常对照组相比,模型组小鼠脊髓组织炎症浸润、脱髓鞘现象明显(P<0.01),模型+RAE组脊髓组织炎症浸润及脱髓鞘明显改善(P<0.01);模型组脾CD4,CD11b和CD11c阳性细胞百分比显著增高(P<0.01),模型+RAE组其增高得到逆转(P<0.01).与正常对照组相比,模型组脊髓NF-κB通路蛋白磷酸化及Iba1,Bax和活化胱天蛋白酶3等蛋白表达均增加,PI3K/Akt通路蛋白磷酸化及Bcl2/Bax蛋白表达比值降低(P<0.01);模型+RAE 125 mg·kg-1组上述蛋白表达的变化明显被逆转(P<0.05,P<0.01).结论 RAE能有效缓解EAE小鼠的发病症状,减少中枢神经系统的炎症浸润和脱髓鞘,其作用机制可能与降低周边炎症细胞激活、抑制中枢神经系统神经炎症以及减少神经细胞的凋亡相关.

目的 研究鹅去氧胆酸(CDCA)抗脂多糖(LPS)诱导的BV2小胶质细胞炎症反应的作用及其机制.方法 BV2细胞与CDCA 25～100μmol·L-1预孵育2 h,加LPS 200 mg·L-1继续培养22 h,采用Griess试剂法检测一氧化氮(NO)含量;Western印迹法检测细胞内环氧合酶2(COX-2)和诱导型一氧化氮合酶(iNOS)蛋白表达水平;RT-PCR法检测细胞肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β和G蛋白偶联受体5(TGR5)mRNA表达水平.BV2细胞与CDCA 25～100μmol·L-1预孵育2 h,加入LPS 200 mg·L-1继续培养1 h,Western印迹法检测NF-κB、NF-κB抑制蛋白α(IκBα)和丝氨酸/苏氨酸蛋白激酶(Akt)磷酸化水平;细胞免疫荧光法观察NF-κB入核情况.结果 与正常对照组相比,模型组培养基中NO含量显著增加(P<0.01);COX-2和iNOS蛋白表达水平升高(P<0.05,P<0.01);TNF-α,IL-6和IL-1βmRNA表达显著上调(P<0.01);TGR5 mRNA表达显著下调(P<0.01);并且NF-κB,IκBα和AKT磷酸化水平显著增加(P<0.05,P<0.01),NF-κB入核增多.与模型组相比,CDCA显著减少培养基中NO含量(P<0.01),降低COX-2和iNOS蛋白表达水平(P<0.05,P<0.01);显著下调TNF-α,IL-6和IL-1βmRNA表达(P<0.01);显著上调TGR5 mRNA表达(P<0.01).与模型组相比,CDCA显著降低NF-κB,IκBα和AKT磷酸化水平(P<0.05,P<0.01),并观察到NF-κB核转位减少现象.结论 CDCA可显著抑制LPS诱导BV2细胞炎症反应,其作用机制可能与激活TGR5、抑制Akt/NF-κB信号通路相关.

CD4+ T cells, especially T-helper (Th) cells (Th1, Th2 and Th17) and regulatory T cells (Treg) play pivotal role in the pathogenesis of multiple sclerosis (MS), a demyelinating autoimmune disease occurring in central nervous system (CNS). Astragaloside IV (ASI, CAS: 84687-43-4) is one of the saponins isolated from Astragalus membranceus, a traditional Chinese medicine with immunomodulatory effect. So far, whether ASI has curative effect on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and how it affects the subsets of CD4+ T cells, as well as the underlying mechanism have not been clearly elucidated. In the present study, ASI was found to ameliorate the progression and hamper the recurrence of EAE effectively in the treatment regimens. It significantly reduced the demyelination and inflammatory infiltration of CNS in EAE mice by suppressing the percentage of Th1 and Th17 cells, which was closely associated with the inhibition of JAK/STAT and NF-κB signaling pathways. ASI also increased the percentage of Treg cells in spleen and CNS, which was accompanied by elevated Foxp3. However, in vitro experiments disclosed that ASI could regulate the differentiation of Th17 and Treg cells but not Th1 cells. In addition, it induced the apoptosis of MOG-stimulated CD4+ T cells probably through modulating STAT3/Bcl-2/Bax signaling pathways. Together, our findings suggested that ASI can modulate the differentiation of autoreactive CD4+ T cells and is a potential prodrug or drug for the treatment of MS and other similar autoimmune diseases.

Gypenoside IX (GP IX) is a pure compound isolated from Panax notoginseng . Gypenosides have been implicated to benefit the recovery of enormous neurological disorders. By suppressing the activation of astrocytes, gypenosides can improve the cognitive impairment. However, so far, little is known about whether GP IX could restrain the inflammatory responses in astrocytes or reactive astrogliosis. In present study, the anti-inflammatory effects of GP IX were investigated in reactive astrocytes induced by proinflammatory mediators both in vitro and in vivo . GP IX significantly reduced the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) at either protein or mRNA level in glial cell line C6 cells stimulated by lipopolysaccharide (LPS)/TNF-α combination. It also alleviated the astrogliosis and decreased the production of inflammatory mediators in brain cortex of LPS-treated mice. Further study disclosed that GP IX inhibited nuclear translocation of nuclear factor kappa B (NFκB) and reduced its transcriptional activity. Meanwhile, GP IX significantly attenuated the phosphorylation of NFκB, inhibitor of kappa B (IκB), Akt, and p38 mitogen-activated protein kinase (MAPK) under inflammatory conditions both in vitro and in vivo . These findings indicated that GP IX might suppress reactive astrogliosis by suppressing Akt/p38 MAPK/NFκB signaling pathways. And GP IX might be a promising drug candidate or prodrug for the therapy of neuroinflammatory disorders characterized with reactive astrogliosis.