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Objective To predict the potential mechanism of Punicalagin in the treatment of Inflammatory bowel disease by network pharmacology.Methods The intersection genes of Punicalagin and IBD were obtained from the database,and PPI,GO and KEGG pathways were enriched and analyzed.Punicalagin and the target were verified by molecular docking.C57BL/6J mice were drunk dextran sulfate sodium to establish inflammatory enteritis model,and were given Punicalagin for 7 d of intervention.During the administration,signs of mice in each group were observed,daily disease activity index was calculated;Intestinal permeability test after administration;The colon tissue was stained with hematoxylin eosin to observe the pathological changes and calculate the histological damage score;Detection of tumor necrosis factor(TNF-α),Interleukin-10(IL-10),myeloperoxidase(MPO),chemokine 1(CXCL1)and other cytokines in colon tissue of mice by ELISA.Detection of TNF-α,IL-6,MPO and CXCL1 level in mouse serum by ELISA.CCK8 method was used to determine the effect of Punicalagin on the proliferation activity of caco-2 cells.The levels of cytokines released by caco-2 cells induced by lipopolysaccharide(LPS)were detected by ELISA.Results 14 common targets of Punicalagin and IBD were obtained,including tumor necrosis factor(TNF),arachidonic acid-5-lipoxygenase(ALOX5)and vascular endothelial growth factor A(VEGFA).KEGG enrichment analysis predicted that the treatment of IBD by Punicalagin mainly acted on arachidonic acid signaling pathway,age-rage signaling pathway,VEGR signaling pathway and Ras signaling pathway.Molecular docking showed that Punicalagin had good docking activity with TNF receptor.Compared with the model group,the decreasing range of body mass in Punicalagin group abated(P<0.01);the disease activity index of Punicalagin group decreased significantly(P<0.01);The congestion and edema of colonic mucosa were significantly reduced,and the histological injury score was significantly reduced(P<0.01);The level of TNF-α,IL-1β,MPO,CXCL1,IL-6,IL-18,IFN-γ in colon tissue was significantly decreased(P<0.01);20-300 μmol·L-1 Punicalagin promoted caco-2 cell proliferation and inhibited TNF-α secretion induced by LPS,up-regulation of IL-10 levels.Conclusion Punicalagin inhibits the secretion of TNF-α and other proinflammatory factors,up-regulation of the level of anti-inflammatory factor IL-10,and improvement of colonic inflammatory response in IBD mice.
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Antibody-dependent enhancement (ADE) has been reported in several virus infections including dengue fever virus, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronavirus infection. To study whether ADE is involved in COVID-19 infections, in vitro pseudotyped SARS-CoV-2 entry into Raji cells, K562 cells, and primary B cells mediated by plasma from recovered COVID-19 patients were employed as models. The enhancement of SARS-CoV-2 entry into cells was more commonly detected in plasma from severely-affected elderly patients with high titers of SARS-CoV-2 spike protein-specific antibodies. Cellular entry was mediated via the engagement of Fc{gamma}RII receptor through virus-cell membrane fusion, but not by endocytosis. Peptide array scanning analyses showed that antibodies which promote SARS-CoV-2 infection targeted the variable regions of the RBD domain. To further characterize the association between the spike-specific antibody and ADE, an RBD-specific monoclonal antibody (7F3) was isolated from a recovered patient, which potently inhibited SARS-Cov-2 infection of ACE-2 expressing cells and also mediated ADE in Raji cells. Site-directed mutagenesis the spike RBD domain reduced the neutralization activity of 7F3, but did not abolish its binding to the RBD domain. Structural analysis using cryo-electron microscopy (Cryo-EM) revealed that 7F3 binds to spike proteins at a shift-angled pattern with one "up" and two "down" RBDs, resulting in partial overlapping with the receptor binding motif (RBM), while a neutralizing monoclonal antibody that lacked ADE activity binds to spike proteins with three "up" RBDs, resulting in complete overlapping with RBM. Our results revealed that ADE mediated by SARS-CoV-2 spike-specific antibodies could result from binding to the receptor in slightly different pattern from antibodies mediating neutralizations. Studies on ADE using antibodies from recovered patients via cell biology and structural biology technology could be of use for developing novel therapeutic and preventive measures for control of COVID-19 infection.