ABSTRACT
Aim To investigate the mechanism of the effect of a specific Kvl. 3 blocker PAP-1 on DSS-in-duced colitis. Methods C57BL/6 mice were divided into normal control group, PAP-1 control group, DSS model group and DSS + PAP-1 group. DSS-induced colitis model was constructed by adding DSS (final concentration of 5% ) to the drinking water of mice. PAP-1 (3 jxg • g~1, 3 times a day for 7 days) was adminis tered intraperitoneally, while an equal volume of solvent was intraperitoneally injected to normal control group and model group. The changes of body weight of mice and the inflammatory activity index (DAI) were recorded every day. After the experiment, the peritoneal macrophages, spleen and colon tissues of mice were collected. Some colon tissues were used for colonic HE staining , and the tissue homogenate was used to detect MPO activities and inflammatory factors. Immunofluorescence confocal microscopy was used to observe the exudation of colonic macrophages. Transmission e-lectron microscopy was used to observe the autophagy-related structures of macrophages, qPCR and Western blot were used to detect the expression of iNOS, IL-1 £ and autophagy-related markers LC3-II , p62, and Bec-lin-1 in mouse colon, peritoneal macrophages and spleen macrophages. Results The DSS-induced mouse colitis model was successfully constructed. PAP-1 reduced weight loss of DSS-induced colitis mice (P <0. 05) and decreased DAI score (P <0. 05) and colon HI score ( P < 0. 05). The MPO activity and the levels of IL-1, IL-6 and TNF-ot decreased in colon tissue of DSS + PAP-1 group compared to DSS model group (P <0. 05). The expression of F4/80 in DSS + PAP-1 group was significantly lower than that in DSS model group based on immunofluorescence microscopic observation. Under the electron microscope, the autophagic bubbles in peritoneal macrophages and spleen macrophages in DSS + PAP-1 group increased compared with those in DSS model group. qPCR and West-em blot showed that in DSS + PAP-1 group, compared with DSS model group, the expression ofiNOS, IL-lfJ and p62 significantly decreased, while the expression of LC3-H and Beclin-1 markedly increased in colon, peritoneal macrophages and spleen macrophages ( P < 0. 05). Conclusions PAP-1 reduces the impaired autophagy of macrophages in mice with colitis, further decreases inflammatory cytokine production, and ultimately ameliorates the immunoinflammatory damage of colitis.
ABSTRACT
To investigate the changes in activation and proliferation of Tregs after targeted RNA inter-ference of Kvl. 3 channel genes and in vitro administration of eplerenone (EPL). Methods After lenti-virus vector was transfected to regulatory T cells (Tregs) of rats, qPCR and whole-cell patch-clamp methods were used to detect gene knockout efficiency, and EOSA method was used to detect cytokine secretion IL-10 and TGF-p levels of Tregs group,Tregs + EPL group,RNAi-Tregs group,and RNAi-Tregs + EPL group. Results Lentivirus vector was successfully transfected into Tregs cells, and the mRNA level and current density of Kvl. 3 channel was 78% and 71.3% respectively; compared with Tregs group, extracellular and intracellular TGF-p levels in RNAi-Tregs group were significantly reduced (P < 0. 01), and extracellular and intracellular TGF-(3 levels in Tregs + EPL group were also reduced (P < 0. 05 ) ; compared with RNAi-Tregs group, extracellular and in-tracellular TGF-fJ levels in RNAi-Tregs + EPL group showed no change. However, IL-10 levels in Tregs group,Tregs + EPL group, RNAi-Tregs group, RNAi-Tregs + EPL group showed no significant change. Conclusions Kvl.3 channel mediates the activation and proliferation of Tregs cells, while EPL can reduce the activation and proliferation of Tregs cells by directly inhibiting Kvl.3 channel and reducing the secretion of TGF-fi levels, further indicating that EPL is a specific blocker of Kvl. 3 channel.