Mesencephalic astrocyte-derived neurotrophic factor (MANF) prevents the neuroinflammation induced dopaminergic neurodegeneration.

BACKGROUND: The excessive activation of the microglia leads to the release of inflammatory factors that contribute to neuronal cell loss and neurodegeneration in Parkinson's Disease (PD). Mesencephalic astrocyte-derived neurotrophic factor (MANF) that belongs to a newly found neurotrophic factors (NTFs) family has been reported to promote neuronal survival in the PD models. However, the effects of the MANF on neuroinflammation in PD remain unclear. METHODS: AAV8-MANF virus was constructed to determine whether the high expression of MANF can protect the neuroinflammation-induced dopaminergic neurodegeneration in rats with 6-OHDA-induced PD. Rotarod performance test, immunofluorescent staining and western bolt were employed to evaluate the behavioral dysfunction, dopaminergic neurodegeneration, microglia activation, and signal activation. 6-OHDA treated SH-SY5Y cells and LPS treated BV-2 cells were used as the in vitro model for MANF neuroprotective and neuroinflammation mechanisms. Cell vitality and apoptosis were evaluated with MTT, CCK-8 and flow cytometric analysis. The AKT/GSK3ß-Nrf2 signaling and the TNF-α/IL6 expression were measured by Western Blot. RESULTS: Our findings indicated that the elevated MANF expression by the AAV8-MANF administration ameliorated the motor dysfunction and protected the dopaminergic neurons in the 6-OHDA treated rats. The upregulated CD11b in the rat SN caused by the 6-OHDA administration was significantly attenuated by the pretreatment of the AAV8-MANF. Furthermore, the levels of p-AKT, p-GSK3ß, BCL-2, and Nrf-2 were upregulated by the high expression of the MANF. Under the oxidative stress of the 6-OHDA, the MANF significantly reduced the apoptotic effect of the TNF-α on the SH-SY5Y cells. In the LPS treated BV-2 cells, the MANF reduced the production of the TNF-α and IL-6, via enhancing the Nrf-2, p-Akt, p-GSK3ß, and p-NF-κß level. CONCLUSIONS: These results suggested that the MANF prevented the dopaminergic neurodegeneration caused by the microglia activation in PD via activation of the AKT/GSK3ß-Nrf-2 signaling axis.

Selective mGluR1 Negative Allosteric Modulator Reduces Blood-Brain Barrier Permeability and Cerebral Edema After Experimental Subarachnoid Hemorrhage.

The blood-brain barrier (BBB) disruption leads to the vasogenic brain edema and contributes to the early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the mechanisms underlying the BBB damage following SAH are poorly understood. Here we reported that the neurotransmitter glutamate of cerebrospinal fluid (CSF) was dramatically increased in SAH patients with symptoms of cerebral edema. Using the rat SAH model, we found that SAH caused the increase of CSF glutamate level and BBB permeability in EBI, intracerebroventricular injection of exogenous glutamate deteriorated BBB damage and cerebral edema, while intraperitoneally injection of metabotropic glutamate receptor 1(mGluR1) negative allosteric modulator JNJ16259685 significantly attenuated SAH-induced BBB damage and cerebral edema. In an in vitro BBB model, we showed that glutamate increased monolayer permeability of human brain microvascular endothelial cells (HBMEC), whereas JNJ16259685 preserved glutamate-damaged BBB integrity in HBMEC. Mechanically, glutamate downregulated the level and phosphorylation of vasodilator-stimulated phosphoprotein (VASP), decreased the tight junction protein occludin, and increased AQP4 expression at 72 h after SAH. However, JNJ16259685 significantly increased VASP, p-VASP, and occludin, and reduced AQP level at 72 h after SAH. Altogether, our results suggest an important role of glutamate in disruption of BBB function and inhibition of mGluR1 with JNJ16259685 reduced BBB damage and cerebral edema after SAH.

Enhanced Therapeutic Potential of Nano-Curcumin Against Subarachnoid Hemorrhage-Induced Blood-Brain Barrier Disruption Through Inhibition of Inflammatory Response and Oxidative Stress.

Curcumin and nano-curcumin both exhibit neuroprotective effects in early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH). However, the mechanism that whether curcumin and its nanoparticles affect the blood-brain barrier (BBB) following SAH remains unclear. This study investigated the effect of curcumin and the poly(lactide-co-glycolide) (PLGA)-encapsulated curcumin nanoparticles (Cur-NPs) on BBB disruption and evaluated the possible mechanism underlying BBB dysfunction in EBI using the endovascular perforation rat SAH model. The results indicated that Cur-NPs showed enhanced therapeutic effects than that of curcumin in improving neurological function, reducing brain water content, and Evans blue dye extravasation after SAH. Mechanically, Cur-NPs attenuated BBB dysfunction after SAH by preventing the disruption of tight junction protein (ZO-1, occludin, and claudin-5). Cur-NPs also up-regulated glutamate transporter-1 and attenuated glutamate concentration of cerebrospinal fluid following SAH. Moreover, inhibition of inflammatory response and microglia activation both contributed to Cur-NPs' protective effects. Additionally, Cur-NPs markedly suppressed SAH-mediated oxidative stress and eventually reversed SAH-induced cell apoptosis in rats. Our findings revealed that the strategy of using Cur-NPs could be a promising way in improving neurological function in EBI after experimental rat SAH.

[Preparation and characterization of monoclonal antibodies specific for CjaA protein of Campylobacter jejuni].

AIM: Expression, purification of Campylobacter jejuni CjaA protein and development of monoclonal antibodies (mAbs) against this protein. METHODS: The C. jejuni cjaA gene was amplified and inserted into the expression plasmids, pGEX-6p-1 and pET30a (+). The purified rGST-CjaA protein was used as an immunogen in 8-week-old BALB/c mice, and injected subcutaneously. The purified rHis-CjaA protein used as a detecting antigen for screening mAbs against CjaA was prepared. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays. RESULTS: The recombinant expression plasmids, pGEX-6p-1-cjaA and pET30a(+)-cjaA were obtained. The sizes of the recombinant proteins, rGST-CjaA and rHis-CjaA, were consistent with their predicted size. Specific reaction was found between CjaA positive serum and expressed protein by Western blot assay, confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines, designated 2B6, 3C2 and 4F11, secreting mAbs against CjaA were obtained. Their immunoglobulin subclasses were all IgG1. The ELISA titers of the ascites fluid were 1:1×10(5);, 1:2×10(5); and 1:4×10(5);, respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-CjaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with CjaA and not the tags for the expression vectors. CONCLUSION: The successful preparation of three mAbs specific for the CjaA protein lays the foundation for further study regarding the biological characteristics of CjaA and the pathogenesis of C. jejuni.

Intranasal immunization with chitosan/pCAGGS-flaA nanoparticles inhibits Campylobacter jejuni in a White Leghorn model.

Campylobacter jejuni is the most common zoonotic bacterium associated with human diarrhea, and chickens are considered to be one of the most important sources for human infection, with no effective prophylactic treatment available. We describe here a prophylactic strategy using chitosan-DNA intranasal immunization to induce specific immune responses. The chitosan used for intranasal administration is a natural mucus absorption enhancer, which results in transgenic DNA expression in chicken nasopharynx. Chickens immunized with chitosan-DNA nanoparticles, which carried a gene for the major structural protein FlaA, produced significantly increased levels of serum anti-Campylobacter jejuni IgG and intestinal mucosal antibody (IgA), compared to those treated with chitosan-DNA (pCAGGS). Chitosan-pCAGGS-flaA intranasal immunization induced reductions of bacterial expellation by 2-3 log(10) and 2 log(10) in large intestine and cecum of chickens, respectively, when administered with the isolated C. jejuni strain. This study demonstrated that intranasal delivery of chitosan-DNA vaccine successfully induced effective immune response and might be a promising vaccine candidate against C. jejuni infection.