| ObjectiveUlcerative colitis is an inflammatory bowel disease that can cause continuous mucosal inflammation from the rectum to the proximal colon to varying degrees.The immune disorder caused by the disorder of intestinal flora and the destruction of the intestinal barrier defense mechanism are important pathogenesis.Studies have shown that activating mitophagy can inhibit the activation of NLRP3 inflammasome.thereby alleviating inflammation and restoring immune balance.At the same time,as the previous literature research reported,the live form of Lactobacillus acidophilus can repair the intestinal barrier damage caused by UC.Postbiotics is a good substitute for probiotics,however,there are few studies focusing on the influence of different administration forms of Lactobacillus acidophilus on UC.and there is no report on its pharmacological mechanism involving NLRP3 inflammasome or mitophagy.Moreover,as an important executor of enterobacteria host interaction,whether the co metabolites of bacteria host participate in different forms of administration of Lactobacillus acidophilus in the treatment of UC remains unknown.In summary,this study aims to investigate the therapeutic effects of live,heat-killed and culture supernatant of Lactobacillus acidophilus in DSS induced UC model rats.The content of SCFAs and the abundance of SCFAs-producing bacteria in feces of UC model rats were determined by gas chromatography and RT-qPCR,respectively.The activation of NLRP3 inflammasome and level of autophagy in rat colon tissue were analyzed.Secondly,we evaluated the repair effect of SCFAs regulated by Lactobacillus acidophilus on LPS induced Caco-2 cell damage model,and then probed into its possible targets and its correlation with NLRP3 inflammasome and mitophagy by using small molecule inhibitors and agonists.thirdly,combined with mitophagy inhibitors,we assessed the effect of SCFAs regulated by Lactobacillus acidophilus on the mitophagy/NLRP3 inflammasome pathway in DSS induced UC mice.Finally,the upstream mechanism of SCFA regulated by Lactobacillus acidophilus regulating mitophagy/inflammasome signaling pathway was investigated.In conclusion,this study aims to evaluate the efficacy of different forms of administration of Lactobacillus acidophilus in UC model and clarify its mechanism of treatment of UC,so as to provide theoretical reference for its clinical application in the prevention and treatment of inflammatory bowel disease.Methods1.Therapeutic effects of various administration forms of Lactobacillus acidophilus on UC model ratsFirst,the pharmacological effects of different administration forms of Lactobacillus acidophilus(live Lactobacillus acidophilus,heat-killed Lactobacillus acidophilus and Lactobacillus acidophilus culture supernatant)were evaluated on a DSS-induced UC rat model.The body weight,food intake and water intake,fecal traits and blood in the stool of UC rats were recorded,and then analyzed the DAI score.HE staining was used to observe and score the pathological changes of colon tissue.Serum MPO content was detected by myeloperoxidase kit.The protein and mRNA levels of TNF-α,IL-6,MCP-1 and IL-10 were measured by Elisa and RT-qPCR method.AB-PAS method was used to determine the number of goblet cells in colon tissue and the secretion of total mucin.Immunohistochemical method was used to measure the expression levels of tight junction proteins such as Occludin and Muc2.Meanwhile,RT-qPCR method was used to detect the mRNA levels of colonic intestinal barrier related factors such as Tff3,ZO-1 and Occludin.The ultramicro intestinal barrier structure of colonic epithelial cells was observed by transmission electron microscope.Subsequently,the effects of different administration forms of Lactobacillus acidophilus on SCFAs and SCFAs-producing bacteria in UC model rats were investigated.Gas chromatography was used to determine the content of SCFAs in the feces of rats,and the RTqPCR method was used to determine the abundance of SCFAs-producing bacteria in the feces.Finally,the effects of different administration forms of Lactobacillus acidophilus on NLRP3 inflammasome and autophagy in UC model rats were evaluated.The levels of CAT,GSHPX,SOD and MDA were determined by oxidative stress kit,the mRNA levels of NLRP3,ASC,Caspase 1,IL-1β and IL-18 in colon tissue were determined by RT-qPCR,and the levels of NLRP3 inflammasome proteins and autophagy-related proteins were determined by western blotting.The organelles and autophagosomes of colonic epithelial cells were observed by transmission electron microscope.2.Protective effect and mechanism of SCFAs regulated by Lactobacillus acidophilus on intestinal epithelial injuryFirst,it was verified whether SCFAs regulated by Lactobacillus acidophilus could protect intestinal epithelial damage by regulating NLRP3 inflammasome.Caco-2 cells was stimulated with different concentrations of LPS to establish a viable intestinal epithelial cell damage model.The cell viability and apoptosis was determined by CCK8 and Annexin VFITC/PI methods.The protein expression levels of Occludin and NLRP3 inflammasome was determined by western blotting method.Subsequently,10 μg/mL of LPS was used to stimulate Caco-2 cells,and intervened with SCFAs regulated by Lactobacillus acidophilus.The cell viability and apoptosis were measured.The relative transmittance of FITC-Dextran was measured.The protein expression levels of Occludin and NLRP3 inflammasome was determined.LPS and Nigericin(NLRP3 inflammasome agonist)were used to stimulate Caco-2 cells,which were intervened with SCFAs regulated by Lactobacillus acidophilus.Then,the protein expression of Occludin and IL-1β was measured.To verify whether SCFAs regulated by Lactobacillus acidophilus can protect intestinal epithelial damage by regulating mitophagy/NLRP3 inflammasome pathway at the cellular level and animal level respectively,we use 10μg/mL of LPS to stimulate Caco-2 cells and intervene with SCFAs regulated by Lactobacillus acidophilus.Afterwards,the reactive oxygen species kit was used to measure the level of ROS,JC-1 method was used to measure mitochondrial membrane potential.Later,10 μg/mL of LPS was used to stimulated Caco-2 cells and intervened with mitophagy inhibitor Mdivi-1,mitophagy agonist CCCP,and SCFAs regulated by Lactobacillus acidophilus.The expression level of NLRP3 inflammasome-related proteins(NLRP3,Caspase 1,and IL-1β)and mitophagy-related proteins(LC3 Ⅱ/Ⅰ,Tom20,Hsp60,P62,Parkin and Pink 1)were determined.Subsequently,a DSS-induced UC mice model was established and gavaged SCFAs regulated by Lactobacillus acidophilus and/or injected mitophagy inhibitor CsA every day.The appearance signs and DAI score of UC mice was recorded.HE staining was used to observe and score the pathological changes of colon tissue.The mRNA level of TNF-α,IL-6,IL-1β and IL-10 in colon tissue was detected.AB-PAS method is used to determine the number of goblet cells in colon tissue and the secretion of total mucin.Immunohistochemical method was used to determine the expression levels of tight junction protein including Occludin and Muc2.The ultramicroscopic observation on intestinal barrier structure,mitochondrial damage and autophagy of colonic epithelial cells with transmission electron microscope was implemented.The expression levels of Occludin protein,mitophagy-related proteins and NLRP3 inflammasome-related proteins were determined.3.Study on the mechanism of SCFAs regulated by Lactobacillus acidophilus in regulating mitophagy/NLRP3 inflammasome pathway10 μg/mL of LPS was used to stimulate Caco-2 cells and intervened with SCFAs regulated by Lactobacillus acidoph ilus;the protein expression and mRNA levels of HDAC1 and HDAC2 were measured;the mRNA levels of GPR41,GPR43 and GPR109 were measured.A DSS-induced UC mice model was established,and the UC mice drank daily SCFAs regulated by Lactobacillus acidophilus and/or injected CsA,an inhibitor of mitophagy.Then,the mRNA levels of GPR41,GPR43 and GPR109 in colon tissue were measured.Results1.Therapeutic effects of various administration forms of Lactobacillus acidophilus on UC model ratsThe live and heat-killed forms of Lactobacillus acidophilus can significantly alleviate the weight loss,blood in the stool and pathological damage of colon tissue in UC model rats,reduce the DAI score and serum MPO levels,increase the length of the colon,inhibit the levels of inflammatory factors such as TNF-α,IL-6,IFN-γ and MCP-1,increase the levels of anti-inflammatory factor IL-10.Thus,the number of goblet cells,the expression levels of Tff3,ZO-1,Occludin and Muc2,amd the ultramicro intestinal barrier structure UC model rats was improved.It should be pointed out that in most of the indicators,the improvement effect of the live form of Lactobacillus acidophilus was better than that of the heat-killed form,and compared with the blank culture supernatant of Lactobacillus acidophilus,the culture supernatant of Lactobacillus acidophiluis did not show a significant treatment effect on UC.In addition,the live and inactivated forms of Lactobacillus acidophilus can respectively increase the content of different kinds of SCFAs in the feces of UC model rats,and the live form can also increase the abundance of SCFAs-producing bacteria.Meanwhile,the live form of Lactobacillus acidophilus can increase the level of antioxidant enzymes(GSH-PX and CAT),reduce the level of oxidation product(MDA),decrease the levels of NLRP3 inflammasome-related proteins(NLRP3,Caspase 1,and IL-1β),and increase the protein level of the specific marker protein LC 3Ⅱ/Ⅰ on the autophagosome membrane,reduce the protein expression level of P62,and increase the number of autophagosomes in colonic epithelial cells.However,compared with the live form of Lactobacillus acidophilus,neither the heat-killed form nor the culture supernatant of Lactobacillus acidophilus showed similar effects on the above indicators.2.Protective effect and mechanism of SCFAs regulated by Lactobacillus acidophilus on intestinal epithelial injuryWith the increasing concentration of LPS,the viability of Caco-2 cells was decreased and the apoptosis was more obvious.The occludin protein expression of Caco-2 cells was gradiently decreased,while the NLRP3 inflammasome related protein gradiently increased.It was found that 10 μg/ml of LPS had the most stable and obvious modeling effect.SCFAs regulated by Lactobacillus acidoph ilus could significantly inhibit the decrease of viability and apoptosis of Caco-2 cells induced by LPS,significantly increase the expression of occludin protein,reduce the expression of NLRP3 inflammasome-related protein,and reduce the relative transmittance of FITC-Dextran of damaged monolayer Caco-2 cells.Moreover.Nigericin could down regulate the expression of occludin protein and significantly increase the expression of IL-1β.And the improvement effect of SCFAs regulated by Lactobacillus acidophilus on occludin protein and IL-1β protein was attenuated by Nigericin.SCFAs regulated by Lactobacillus acidophilus can reduce the level of ROS in Caco-2 cells damaged by LPS and lower the mitochondrial membrane potential.In addition,in contrast to Mdivi-1,CCCP and SCFAs regulated by Lactobacillus acidophilus can activate mitophagy,which was manifested by increasing the expression levels of the mitophagyrelated proteins such as LC3 Ⅱ/Ⅰ,Parkin and Pink 1 in Caco-2 cells and reducing that of the mitophagy-related proteins such as Tom20,Hsp60 and P62,and thereby inhibiting the activation of NLRP3 inflammasome which was manifested by reducing the expression levels of NLRP3 inflammasome-related proteins such as NLRP3,Caspase 1,and IL-1β.However,when Mdivi-1 and SCFAs regulated by Lactobacillus acidophilus co-stimulated Caco-2 cells,the regulation of SCFAs regulated by Lactobacillus acidophilus on the mitophagy/NLRP3 inflammasome pathway was significantly weakened.SCFAs regulated by Lactobacillus acidophilus significantly improved the appearance of UC model mice,the number of goblet cells,the expression of tight junction proteins,and ultramicro on intestinal barrier structure of the colonic epithelial cells.Meanwhile,they significantly reduced the expression levels of mitophagy-related proteins such as Tom20,Hsp60 and P62 in colon tissue,increased that of mitophagy-related proteins such as LC3 Ⅱ/Ⅰ,Parkin and Pink 1,and increases the number of autophagosomes in colon tissue.Thus,the expression levels of NLRP3 inflammasome-related proteins such as NLRP3,Caspase 1,and IL-1β were decreased.However,CsA,an inhibitor of mitophagy,can block the aforementioned effects of SCFAs regulated by Lactobacillus acidophilus.3.Study on the mechanism of SCFAs regulated by Lactobacillus acidophilus in regulating mitophagy/NLRP3 inflammasome pathwaySCFAs regulated by Lactobacillus acidophilus have no significant effect on the expression of HD AC in Caco-2 cells,but they can reverse the decline in the Mrna levels of GPR41,GPR43 and GPR109A in Caco-2 cells and the colon tissue of UC model mice.However,after treatment with the mitophagy inhibitor CsA,the activation of G proteincoupled receptors by SCFAs regulated by Lactobacillus acidophilus was significantly weakened.Conclusion1.Compared with the heat-killed Lactobacillus acidophilus and the culture supernatant of Lactobacillus acidophilus,the live form of Lactobacillus acidophilus has the optimal therapeutic effect on UC model rats.This effect may be associated with significantly increasing the content of SCFAs in feces,inhibiting the activation of NLRP3 inflammasome in colon tissues,and increasing the level of autophagy.2.SCFAs regulated by Lactobacillus acidophilus can reduce the levels of proinflammatory cytokines in Caco-2 cells and UC model mice,and then improve intestinal barrier dysfunction,which was inextricably related to the promotion of mitophagy/NLRP3 inflammasome signaling pathway in colon.3.SCFAs regulated by Lactobacillus acidophilus may regulate the mitophagy/NLRP3 inflammasome signal pathway by activating G protein-coupled receptors independently of HD AC activity. |
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