Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells.

Japanese encephalitis virus (JEV) remains the predominant cause of viral encephalitis worldwide. It reaches the central nervous system upon crossing the blood-brain barrier through pathogenic mechanisms that are not completely understood. Here, using a high-throughput siRNA screening assay combined with verification experiments, we found that JEV enters the primary human brain microvascular endothelial cells (HBMEC) through a caveolae-mediated endocytic pathway. The role of ezrin, an essential host factor for JEV entry based on our screening, in caveolae-mediated JEV internalization was investigated. We observed that JEV internalization in HBMEC is largely dependent on ezrin-mediated actin cytoskeleton polymerization. Moreover, Src, a protein predicted by a STRING database search, was found to be required in JEV entry. By a variety of pharmacological inhibition and immunoprecipitation assays, we found that Src, ezrin, and caveolin-1 were sequentially activated and formed a complex during JEV infection. A combination of in vitro kinase assay and subcellular analysis demonstrated that ezrin is essential for Src-caveolin-1 interactions. In vivo, both Src and ezrin inhibitors protected ICR suckling mice against JEV-induced mortality and diminished mouse brain viral load. Therefore, JEV entry into HBMEC requires the activation of the Src-ezrin-caveolin-1 signalling axis, which provides potential targets for restricting JEV infection.

Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells.

Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell line, does not involve clathrin- and caveolae-dependent endocytic pathways or macropinocytosis but requires GTP-binding protein dynamin 2 and cytoskeleton remodelling. The use of siRNAs targeting endophilin family members revealed that endophlin-A2 is essential for the uptake of EV71 particles by Caco-2 cells. Subcellular analysis revealed that internalized EV71 virions largely colocalized with endophilin-A2 at cytomembrane ruffles and in the perinuclear area. Combined with viral entry kinetics, these data suggest that EV71 enters Caco-2 cells mainly via an endophilin-A2-mediated endocytic (EME) pathway. Finally, we showed that internalized EV71 virions were transported to endosomal sorting complex required for transport (ESCRT)-related multivesicular bodies (MVBs). These data provide attractive therapeutic targets to block EV71 infection.

Dexmedetomidine Protects Against Multi-Organ Dysfunction Induced by Heatstroke via Sustaining The Intestinal Integrity.

Previous studies have indicated that gut-derived endotoxin played a pivotal role for aggravating systemic inflammatory response to multi-organ dysfunction under heatstroke. Dexmedetomidine (DEX) could protect against inflammation and multi-organ injury in various scenarios. The aim of this study was to explore the protective effect of DEX on heatstroke and the mechanism involved. Male C57BL/6 mice were placed in a controlled climate chamber (40 ±â€Š1°C) until the maximum core temperature (Tc, Max) of 42.7°C, the received criterion of heatstroke, was attained, DEX (25 µg/kg) or 0.9% saline was injected intraperitoneally immediately. The results showed that DEX could significantly attenuate multi-organ injury induced by heatstroke, simultaneously decrease levels of serum inflammatory cytokines through inhibiting the intestinal nuclear factor-κB activation. Furthermore, to assess the effects of DEX on intestine mucosal barrier under heatstroke, the levels of plasma endotoxin, FD4, and D-lactate were detected and the expression of tight junction proteins occludin and ZO-1 was analyzed by western blot and immunohistochemistry. Meanwhile, transmission electron microscopy was employed to confirm the ultrastructure of intestine. Interestingly, we found that DEX decreased the intestinal permeability and sustained the integrity of intestinal barrier. Finally, to evaluate the anti-apoptosis effect of DEX, the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were analyzed by western blot, and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) staining was conducted. The results showed that DEX decreased TUNEL-positive cells induced by heatstroke in a Bax/Bcl-2-related manner. Taken together, our results indicate that DEX could protect against inflammation and multi-organ injury induced by heatstroke via sustaining the intestinal integrity.

NLRP3 inflammasome activation mediates radiation-induced pyroptosis in bone marrow-derived macrophages.

A limit to the clinical benefit of radiotherapy is not an incapacity to eliminate tumor cells but rather a limit on its capacity to do so without destroying normal tissue and inducing inflammation. Recent evidence reveals that the inflammasome is essential for mediating radiation-induced cell and tissue damage. In this study, using primary cultured bone marrow-derived macrophages (BMDM) and a mouse radiation model, we explored the role of NLRP3 inflammasome activation and the secondary pyroptosis underlying radiation-induced immune cell death. We observed an increasing proportion of pyroptosis and elevating Caspase-1 activation in 10 and 20 Gy radiation groups. Nlrp3 knock out significantly diminished the quantity of cleaved-Caspase-1 (p10) and IL-1ß as well as the proportion of pyroptosis. Additionally, in vivo research shows that 9.5 Gy of radiation promotes Caspase-1 activation in marginal zone cells and induces death in mice, both of which can be significantly inhibited by knocking out Nlrp3. Thus, based on these findings, we conclude that the NLRP3 inflammasome activation mediates radiation-induced pyroptosis in BMDMs. Targeting NLRP3 inflammasome and pyroptosis may serve as effective strategies to diminish injury caused by radiation.

Activation of the NLRP3 inflammasome in lipopolysaccharide-induced mouse fatigue and its relevance to chronic fatigue syndrome.

BACKGROUND: The NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3) is an intracellular protein complex that plays an important role in innate immune sensing. Its activation leads to the maturation of caspase-1 and regulates the cleavage of interleukin (IL)-1ß and IL-18. Various studies have shown that activation of the immune system plays a pivotal role in the development of fatigue. However, the mechanisms underlying the association between immune activation and fatigue remained elusive, and few reports have described the involvement of NLRP3 inflammasome activation in fatigue. METHODS: We established a mouse fatigue model with lipopolysaccharide (LPS, 3 mg/kg) challenge combined with swim stress. Both behavioural and biochemical parameters were measured to illustrate the characteristics of this model. We also assessed NLRP3 inflammasome activation in the mouse diencephalon, which is the brain region that has been suggested to be responsible for fatigue sensation. To further identify the role of NLRP3 inflammasome activation in the pathogenesis of chronic fatigue syndrome (CFS), NLRP3 KO mice were also subjected to LPS treatment and swim stress, and the same parameters were evaluated. RESULTS: Mice challenged with LPS and subjected to the swim stress test showed decreased locomotor activity, decreased fall-off time in a rota-rod test and increased serum levels of IL-1ß and IL-6 compared with untreated mice. Serum levels of lactic acid and malondialdehyde (MDA) were not significantly altered in the treated mice. We demonstrated increased NLRP3 expression, IL-1ß production and caspase-1 activation in the diencephalons of the treated mice. In NLRP3 KO mice, we found remarkably increased locomotor activity with longer fall-off times and decreased serum IL-1ß levels compared with those of wild-type (WT) mice after LPS challenge and the swim stress test. IL-1ß levels in the diencephalon were also significantly decreased in the NLRP3 KO mice. By contrast, IL-6 levels were not significantly altered. CONCLUSIONS: These findings suggest that LPS-induced fatigue is an IL-1ß-dependent process and that the NLRP3/caspase-1 pathway is involved in the mechanisms of LPS-induced fatigue behaviours. NLRP3/caspase-1 inhibition may be a promising therapy for fatigue treatment.

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3ß, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease.

[Multi-central randomized controlled observation on clinical therapeutic effect of the new Bian-stone therapy on scapulohumeral periarthritis].

OBJECTIVE: To study the clinical curative effect of the new Bian-stone therapy. METHODS: According to clinical trail principle of multi-centers, randomized grouping, parallel control and single-blind, the observation group (n=120) were treated by the new Bian-stone therapy, with multi-functional Bian-stone respectively dredging qi and blood of channels on the neck and shoulder, the shoulder and back, the limbs, and the control group (n=120) by electroacupuncture with Jianyu (LI 15), Jianliao (SJ 14), Jianzhen (SI 9), and other points selected. Their therapeutic effects were compared. RESULTS: The two therapies had effects of analgesia, improving functional activity and transient analgesia. The total effective rate of the analgesic effect was 86.8% in the observation group and 72.5% in the control group with a significant difference between the two groups (P < 0.05), the observation group being better than the control group; the total effective rate of shoulder functional activity was 86.8% in the observation group and 79.8% in the control group with no significant difference between the two groups (P > 0.05). CONCLUSION: The new Bian-stone therapy has obvious therapeutic effect on scapulohumeral periarthritis, with effects of analgesia, improving functional activity, and transient analgesia, and good long-term therapeutic effect.

[Randomized controlled trials of acupuncture at Tiaokou (ST 38) for treatment of periarthritis of shoulder].

OBJECTIVE: To study the basic therapeutic function of Tiaokou (ST 38). METHODS: According to clinically multi-central randomized controlled and single-blind test principle, 257 cases of periarthritis of shoulder were divided into two groups, a test group (n = 124) treated with oral anti-inflammatory analgesic medicine combined with acupuncture at Tiaokou (ST 38), and a control group (n = 133) treated with oral anti-inflammatory analgesic medicine. Their therapeutic effects were compared. RESULTS: The total effective rate for stopping pain was 96.0% in the test group and 91.7% in the control group with a very significant difference between the two groups (P< 0.01). And the total effective rate for improvement of shoulder activity was 86.3% in the test group and 59.4% in the control group with a very significant difference between the two groups (P<0.01). CONCLUSION: Oral anti-inflammatory analgesic medicine combined with acupuncture has obvious therapeutic effect on periarthritis of shoulder, which is better than that of simple oral anti-inflammatory analgesic medicine.