Bu Shen Yi Sui Capsule Promotes Myelin Repair by Modulating the Transformation of A1/A2 Reactive Astrocytes In Vivo and In Vitro.

Background/Aims. Multiple sclerosis (MS) is an autoimmune disorder that affects the central nervous system (CNS) primarily hallmarked by neuroinflammation and demyelination. The activation of astrocytes exerts double-edged sword effects, which perform an integral function in demyelination and remyelination. In this research, we examined the therapeutic effects of the Bu Shen Yi Sui capsule (BSYS), a traditional Chinese medicine prescription, in a cuprizone- (CPZ-) triggered demyelination model of MS (CPZ mice). This research intended to evaluate if BSYS might promote remyelination by shifting A1 astrocytes to A2 astrocytes. Methods. The effects of BSYS on astrocyte polarization and the potential mechanisms were explored in vitro and in vivo utilizing real-time quantitative reverse transcription PCR, immunofluorescence, and Western blotting. Histopathology, expression of inflammatory cytokines (IL-10, IL-1ß, and IL-6), growth factors (TGF-ß, BDNF), and motor coordination were assessed to verify the effects of BSYS (3.02 g/kg/d) on CPZ mice. In vitro, A1 astrocytes were induced by TNF-α (30 ng/mL), IL-1α (3 ng/mL), and C1q (400 ng/mL), following which the effect of BSYS-containing serum (concentration of 15%) on the transformation of A1/A2 reactive astrocytes was also evaluated. Results and Conclusions. BSYS treatment improved motor function in CPZ mice as assessed by rotarod tests. Intragastric administration of BSYS considerably lowered the proportion of A1 astrocytes, but the number of A2 astrocytes, MOG+, PLP+, CNPase+, and MBP+ cells was upregulated. Meanwhile, dysregulation of glutathione peroxidase, malondialdehyde, and superoxide dismutase was reversed in CPZ mice after treatment with BSYS. In addition, the lesion area and expression of proinflammatory cytokines were decreased and neuronal protection factors and anti-inflammatory cytokines were increased. In vitro, BSYS-containing serum suppressed the A1 astrocytic markers' expression and elevated the expression levels of A2 markers in primary astrocytes triggered by C1q, TNF-α, and IL-1α. Importantly, the miR-155/SOCS1 signaling pathway was involved in the modulation of the A1/A2 phenotype shift. Overall, this study demonstrated that BSYS has neuroprotective effects in myelin repair by modulating astrocyte polarization via the miR-155/SOCS1 pathway.

Alcohol Increases Exosome Release from Microglia to Promote Complement C1q-Induced Cellular Death of Proopiomelanocortin Neurons in the Hypothalamus in a Rat Model of Fetal Alcohol Spectrum Disorders.

Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing ß-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the ß-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the ß-endorphin neuronal killing activity of microglial exosomes both in vivo and in vitro Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in ß-endorphin neuronal cells in both in vitro and in vivo systems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of ß-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced ß-endorphin neuronal death.SIGNIFICANCE STATEMENT Neurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.

Complimentary action: C1q increases ganglion cell survival in an in vitro model of retinal degeneration.

Using a previously described retinal explant culture system as an acute injury model, we here explore the role of C1q, the initiator of the classical complement pathway, in neuronal cell survival and retinal homeostasis. Full-thickness adult rat retinal explants were divided into four groups, receiving the following supplementation: C1q (50nM), C1-inhibitor (C1-inh; Berinert; 500mg/l), C1q+C1-inh, and no supplementation (culture controls). Explants were kept for 12h or 2days after which they were examined morphologically and with a panel of immunohistochemical markers. C1q supplementation protects ganglion cells from degeneration within the explant in vitro system. This effect is correlated to an attenuated endogenous production of C1q, and a quiesced gliotic response.