Effect Of Kv1.3 Blocker On Autophagy Of Macrophages In Experimental Colitis

Background:Autophagy is considered as a cellular stress response that plays important roles in many physiological processes,including innate immunity.Macrophages play an important role in the innate immune inflammatory response and have been thought to be a primary type of innate immune cell that is usually activated in DSS-induced colitis.Dysfunctional autophagy is recognized as a contributing factor in IBD with promoting proinflammatory cytokine production by macrophages.Kv1.3 potassium channel,is present in macrophages,where it controls activation and proliferation processes together with Kv1.5.PAP-1 is a specific Kv1.3 channel blocker,which has been applied in many animal exprements.However,there is rare report concerning whether PAP-1 may reduce colitis by regulating macrophage autophagy.Therefore,to study the effect of PAP-1 on colitis of IBD and the underlying mechanisms is of great theoretical and practical significance.Objectives:To investigate the mechanism of a specific Kv1.3 blocker PAP-1 on DSS-induced colitis.Methods: C57BL/6 mice were divided into normal control group,normal control + PAP-1 group,DSS model group and DSS + PAP-1 group.DSS-induced colitis model was constructed by adding DSS(final concentration of 5%)to the mouse drinking water.PAP-1(3μg·g-1,3 times a day for 7 days)was administered intraperitoneally.The normal control group and the model group were intraperitoneally injected with an equal volume of solvent.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 for colonic HE staining,and the tissue homogenate was used to detect MPO activity and inflammatory factor IL-1,IL-6,IL-10 and TNF-α.Immunofluorescence confocal microscopy was used to observe the exudation of colonic F4/80+ macrophages.Transmission electron microscopy was used to observe the autophagy-related structures of peritoneal macrophages and spleen macrophages;RT-q PCR and Western blotting were used to detect the expression of i NOS,IL-1β and autophagy-related markers LC3-II,p62,and Beclin1 in mouse colon,peritoneal macrophages and spleen macrophages.Results: The DSS-induced mice colitis model was successfully constructed.PAP-1 reduced the weight loss of DSS-induced colitis mice(P<0.05),and decreased DAI score(P< 0.05)and colon HI score(P< 0.05),compared with the DSS model group.The MPO activity and the levels of IL-1,IL-6 and TNF-α were decreased(P<0.05),and the level of IL-10 was increased(P< 0.05),in the colon tissue of DSS+PAP-1 group comparing to the DSS model group.The expression of F4/80 in the DSS+PAP-1 group was significantly lower than that in the DSS model group by immunofluorescence microscopy observed.Under the electron microscope,the autophagic bubble in the peritoneal macrophages and spleen macrophages in the DSS+PAP-1 group were increased compared with the DSS model group.RT-q PCR and western blotting showed that in the DSS+PAP-1 group,compared with the DSS model group,the expression of i NOS,IL-1β and p62 were significantly decreased,and the expression of LC3-II and Beclin1 was significantly increased on colon,peritoneal macrophages and spleen macrophages(P<0.05).Conclusions:PAP-1 could reduce the impaired autophagy of macrophages with colitis mice,further reduce inflammatory cytokine production,and ultimate reduce the immunoinflammatory damage of colitis.