The Effect And Mechanism Of Palmatine On DSS-induced Ulcerative Colitis

ObjectivePalmatine is an herbal derived isoquinoline alkaloid with antibacterial,anti-inflammatory and anti-cancer activities,which clinical applications inlcludes enteritis,pelvic inflammation and gastritis.The study aims to investigate the protective effect of palmatine on mice experimental colitis induced by DSS and explore the underlying mechanisms.Methods1.Effect of palmatine on DSS-induced colitis in mice.Male Balb/C mice were randomly divided into six groups:control group,DSS group,Palmatine groups and SASP group.Mice in control group was given distilled water,while the other mice were given 3%(w/v)DSS in drinking water for continuous 7 days to establish ulcerative colitis.At the same time,mice in palmatine and sulfasalazine groups were intragastricly administrated with palmatine(10,40,100 mg/lkg)and sulfasalazine(200 mg/kg)for consecutive 7 days respectively,whereas,the control group and DSS group were given the same volume of distilled water.The bodyweight and the presence of gross blood as well as stool consistency were recorded daily from day 1 to day 8.On day 8,all animals were euthanaized.The length of colon was measured,the histopathological score and MPO activity of colon tissue were detected,and macrophage activation marker F4/80 was examined by immunofluorescence.Moveover,the levels of cytokines including IL-1β and TNF-αwere measured using ELISA and the protein expression of NLRP3,caspase-1 and IL-1β were analyzed by Western blot.2.Potential mechanism of palmatine.(1)Effect of palmatine on the NLRP3 inflammasome activation in THP-Ms.THP-1 cells were stimulated with 300 nM PM A for 24 hours to differentiate into macrophages.After stimulated with 1μg/mL LPS for 3 hours and 5mM ATP for 1 hour to establish model of NLRP3 inflammasome activation,macrophages were treated with palmatine(50,100,200 μM)for 3 hours.The expression of NLRP3,caspase-1 and IL-1β in THP-Ms were measured using Western blot,and the expression and distribution of caspase-1 and ASC in THP-Ms was detected by immunofluorescence.In addition,the secretion of cytokine IL-1β in supernatant was determined using ELISA.(2)Effect of palmatine on mitophagy in THP-Ms.The level of mitochondrial ROS in THP-Ms was analyzed by using flow cytometry.The mitophagy-related proteins including P62,LC3,PINK1 and Parkin were determined by using immunofluorescence and western blotting.In addition,we used mitotracker and lysotracker to label mitochondria and lysosome respectively to identify mitophagy.(3)The influence of mitophagy inhibitor on palmatine’s effect on THP-Ms.THP-Ms were stimulated with 1μg/mL LPS for 3 hours and 5 mM ATP for 1 hour,and then treated with palmatine(200 μM)and CsA(5 nM)for 3 hours.The protein expression of caspase-1 and PINK1 was detected by Western blot,and the secretion of IL-1β was determined using ELISA.(4)The influence of PINK1-siRNA on palmatine’s effect on THP-Ms.The expression of PINK1 in THP-Ms was silenced by PINK1-siRNA.THP-Ms were stimulated with 1μg/mL LPS for 3 hours and 5mM ATP for 1 hour,and then treated with palmatine(200 μM)for 3 hours.The protein expression of caspase-1 and PINK1 was detected by Western blot,and the secretion of IL-1β was determined using ELISA.3.The influence of mitophagy inhibitor on the in vivo effect of palmatine.Male Balb/C mice were randomly divided into five groups:control group,DSS group,Palmatine group,Palmatine+CsA group,CsA group.Mice in control group was given distilled water,while the other mice were given 3%(w/v)DSS in drinking water for continuous 7 days to establish ulcerative colitis.At the same time,Palmatine(100 mg/kg)was given orally,while cyclosporine A(20 mg/kg),a mitophagy inhibitor,was administrated intraperitoneally for continuous 7 days.The bodyweight and the presence of gross blood as well as stool consistency were recorded daily from day 1 to day 8.On day 8,animals were euthanasized and colons were separated.The clonic levels of cytokines IL-1β and TNF-α was determined by ELISA and the protein expression of caspase-1,IL-1β,LC3,PINK1 and Parkin were detected using Western blot.Results1.The results showed that DSS caused weight loss,hematochezia,colon shortening and severe colon injury in mice.It also activated macrophages in mice colon,increased colonic MPO activity and elevated the levies of IL-1β and TNF-α.Furthermore,the DSS mice exhibited high levels of NLRP3,cleaved-caspase-1 and cleaved-IL-1β.However,palmatine(40 and 100 mg/kg)significantly ameliorated the symptoms of DSS-induced colitis in mice,decreased MPO activity,and inhibited secretion of IL-1β and TNF-α.In addition,palmatine down-regulated the activation of macrophages and inhibited the protein expression of NLRP3,cleaved-caspase-1 and cleaved-IL-1β.2.In vitro experiments showed that LPS and ATP stimulated THP-Ms,increased the protein expression ofNLRP3,cleaved-caspase-1 and cleaved-IL-1β,and enhanced the secretion of IL-1β and the production of mitochondrial ROS in THP-Ms.However,palmatine inhibited the protein expression of NLRP3,cleaved-caspase-1 and cleaved-IL-1β and the secretion of IL-1β in supernatant,and decreased mitochondrial ROS.Simultaneously,palmatine induced the fusion of mitochondria and lysosome,facilitated the conversion of LC3-Ⅰ to LC3-Ⅱ and reduced the expression of P62.Moreover,palmatine elevated the expressions of PINK1 and Parkin,and promoted the mitochondrial recruitment of PINK1 and Parkin in THP-Ms.3.In vitro experiments showed that either inhibiting mitophagy by CsA or silencing PINK1 expression by PINK-siRNA evidently blocked the inhibitory effects of palmatine on NLRP3 inflammasome.4.In vivo experiment revealed that under the intervention of CsA,the protective effect of palmatine on DSS mice was significantly impaired,and inhibitory effects of palmatin on colonic cytokines and mitophagy proteins was weakened.ConclusionPalmatine reduced the accumulation of damaged mitochondria and the production of excessive mitochondrial ROS by enhancing PINK1/Parkin-mediated mitophagy.By this way,Palmatine may inhibit NLRP3 inflammasome activation and decreas secretion of proinflammatory cytokines in macrophages,and therefore alleviate DSS-induced ulcerative colitis in mice.