Study On The Role And Mechanism Of Mitochondrial Dysfunction In Elderly Patients With Ulcerative Coliti

Part 1 Comparison of clinical characteristics and prognostic outcomes in adult and elderly patients with ulcerative colitisBackground and Aim:The aim was to compare therapeutic responses and prognosis between adult and elderly UC patients with moderate to severe activity.Methods:148 moderately to severely active UC patients hospitalized between 2000 to 2019 were enrolled consecutively,including 74 patients with age of diagnosis over 60 years and 74 patients diagnosed less than 60 years.Patients were matched by gender,duration(±15%),disease activity,and admission time(±1 year).They were followed up until the latest medical record or December 2019.The primary outcome was UC-related colectomy or death.Results:148 patients were followed over median 37.5 months.For steroid use,76.8%of elderly UC patients were responsive,lower than that in adult group(85.7%).Decreased level of clinical activity index[2.0(-1.5-4.00)vs.6.0(3.0-8.0),P<0.001],reduction of CRP[23.9(3.5-65.5)vs.27.8(9.7-58.1)mg/L,P=0.61]and ESR[9.0(-1.3-30.5)vs.15.5(3.8-36.5)mm/h,P=0.16]at 4 weeks after steroid induction was less obvious in the elderly.More elderly patients manifested steroid dependence and resistance.28.4%of elderly UC patients took colectomy,remarkably more than adult patients(12.2%),which also occurred earlier[8.0(0.5-44.75)vs.39.5(12-57.38)months,P=0.001].Aging(HR 2.868,95%CI 1.290-6.375,P=0.01),male,steroid resistance,and occurrence of complications were independently related with colectomy.The rate of serious infections was significantly higher in the elderly(55.4%vs.35.1%,P=0.013),mainly including cytomegalovirus infection,bacterial infection and extraintestinal infection.Aging(OR 2.774,95%CI 1.355-5.675,P=0.015),extensive colonic involvement,steroid resistance and biologics usage were independently associated with high-risk of concomitant infections in UC patients.Conclusion:Elderly patients with moderate to severe UC undergone more treatment failure and increased risk of UC-related colectomy,mortality and severe infections,predicting demand for more strict and individualized management.Part 2 Mitochondrial damage is an important factor in the differences in the pathogenesis of adult-and elderly-onset colitisBackground and Aim:To analyze the differences of mitochondrial dysfunction between adult and elderly colitis and to reveal the role of mitochondrial damage in the pathogenesis of UC in the elderly.Methods:(1)Colonic mucosal specimens from adult and elderly moderately or severely active UC patients were collected,and performed RNA sequencing analysis.And the expression profile data of young and elderly UC colonic mucosa from the Gene Expression Omnibus(GEO)database was obtained.The mitochondrial related differential genes were got according to MitoMiner and Aging Atlas databases and were then conducted GO and KEGG pathway enrichment analysis.The protein interaction network was built with the STRING database and imported in the Cytoscape software to screen out the mitochondrial related hub genes,following which the gene expression was validated in clinical and animal model samples.(2)Dextran sulfate sodium(DSS)induced acute colitis model was built in young(6-8 weeks)and aged(≥ 16 months)C57BL/6 male mice.Mitochondrial morphological changes were observed by transmission electron microscopy.The ATP levels were determined by colonic homogenate.The total reactive oxygen species(ROS)and mitochondrial ROS in colonic epithelial cells were evaluated by DCFHDA and MitoSOX probes incubation.Quantitative real-time PCR(qRT-PCR)was used to determine the mRNA expression of mitochondria-related molecules in colon and to validate the differential gene expression of RNA sequencing and bioinformatic analysis.Results:(1)The GSE87466 dataset includes 49 young UC patients(<45 years old)and 12 elderly UC patients(≥60 years old)with moderate to severe activity.Finally,101 mitochondrial related differential genes were screened out including mitochondrial electron transport chain subunits genes NDUFAF5,NDUFAF6,ATP regulatory gene ATP 13A3,mitochondrial fission-related genes MFF,MTFR1,etc.GO and KEGG enrichment analysis indicated that their encoding proteins were mainly related to mitochondrial respiratory chain enzyme complexes,composition or function of mitochondrial membrane and mitochondrial transport,etc.The RNA sequencing analysis on colonic mucosa from young and elderly moderately or severely active UC showed there were 713 differentially expressed genes(P<0.05 and|log2FC|>1)between the young and elderly,in which 30 differential genes related to mitochondria were identified according to MitoMiner and Aging Atlas databases.These 30 differential encoding proteins were mainly related to mitochondrial redox regulation,mitochondrial membrane composition and function,oxidative phosphorylation,and peroxisome proliferators-activated receptors(PPAR)signaling pathway.Protein interactive analysis suggested that aldehyde dehydrogenase 1 family member L1(ALDH1L1)and succinyl-CoA:glutarate-CoA transferase(SUGCT)were both downregulated in the elderly colitis group with a potential co-expression relationship.qPCR results showed that the relative expressions of ALDH1L1 mRNA in the colon tissue of young healthy controls,young UC patients,old healthy controls and old UC patients were 1.0±0.13,0.43 ±0.15,0.39±0.04,0.17±0.05 respectively(Young UC vs.Old UC:P=0.06),the relative expressions of SUGCT mRNA were 1.0±0.13,0.77±0.06,0.72±0.24,0.18±0.01 respectively(Young UC vs.Old UC:P<0.05).(2)The DSS-induced acute colitis in the aged mice displayed more obvious weight loss,higher DAI and more severe histological inflammation.Through transmission electron microscope,damaged mitochondrial structure was more severe in the aged colitis group,including the swollen mitochondrion with shallowing matrix and reduced or disappeared cristae.The ATP levels in the aged inflammatory colon tissue were lower than those in the young inflammatory colon.Flow cytometry showed that the positive ratio and fluorescence intensity of DCFHDA and MitoSOX in colonic epithelial cells in aged DSS group were more increased than those in young DSS mice.And qPCR revealed that some mitochondrial related molecules were differentially expressed in young and aged colitis mice,for instance,ME2,Prkaa2,SDHA,PGC1α,DACT2,SUGCT,ALDH1L1 and ECHDC3 mRNA decreased more significantly in the aged colitis group(Young-DSS vs.Old-DSS mRNA relative expression,ME2:0.85±0.18 vs.0.44±0.21,P=0.05;ALDH1L1:0.55±0.32 vs.0.19±0.11,P=0.03;the others P>0.05).Conclusion:1.The colonic mucosa of aged DSS acute colitis mice has more obvious mitochondrial morphological damage,decreased ATP level,and higher ROS levels.2.There are differences in the expression of mitochondrial related molecules in the colon of young and elderly colitis,which are closely associated to mitochondrial membrane,redox regulation,oxidative phosphorylation,ATP regulation and mitochondrial fission.Among them,both ALDH1L1 and SUGCT are downregulated significantly in aged colitis colon which may be a co-expression relationship.Part 3 Preliminary mechanistic study on mitochondrial damage involved in the regulation of aging-inflammatory progressionBackground and Aim:To explore the roles of ALDH1L1 and SUGCT in aginginflammatory progression and mitochondrial damage and to provide evidence for the potential therapeutic value of ALDH1L1 and SUGCT in elderly-onset UC.Methods:(1)The distribution,mRNA and protein expression levels of ALDH1L1 and SUGCT in colon tissue were determined by immunohistochemical staining(IHC),qPCR and Western blot(WB)respectively;(2)HCT116 cells were transfected with small interfering RNA(siRNA)and overexpression plasmids to interfere with the expression of ALDH1L1 or SUGCT,and were given exogenous TNFa stimulation.DCFHDA and MitoSOX probes were used to measure the changes of total cellular ROS and mitochondrial ROS.Mitochondrial stress test was performed by seahorse XFe24 to measure cellular oxygen consumption rate(OCR),and to evaluate basal respiration,maximal respiration capacity,spare respiration capacity,and ATP production and nonmitochondrial respiration.WB detected the changes of NLRP3,IL-1β proteins.The ALDH1L1 or SUGCT-overexpressing cells were pretreated with the ROS scavenger Nacetyl-L-cysteine(NAC),and then stimulated with TNFα,and the inflammatory related factors NLRP3,IL-6,and IL-1β were tested by WB.Results:(1)In clinical and mouse model samples,the mRNA and protein expressions of ALDH1L1 and SUGCT decreased in the colitis group,more significantly in the elderly group.IHC staining showed that ALDH1L1 and SUGCT were mainly distributed in the cytoplasm of colonic epithelial cells.(2)After TNFa intervention,HCT116 cells treated with ALDH1L1 or SUGCT siRNA showed that the increase of total ROS was more obvious than those in the negative control+TNFα group(siALDH1L1:P>0.05;siSUGCT:P<0.05).Compares with negative control,the level of mitochondrial ROS rose less significantly in ALDH1L1-or SUGCT-interfered HCT116 cells(P<0.05).In terms of inflammatory phenotype,the protein levels of NLRP3 and IL-1β in ALDH1L1-or SUGCT-interfered HCT116 cells increased less obviously than those in negative control after TNFa induction(relative expression,siALDH1L1+TNFα vs.NC+TNFα:NLRP3:0.90±0.09 vs.1.38±0.04,P＜0.05;IL-1β:0.77±0.02 vs.1.35±0.31,P<0.05;siSUGCT+TNFα vs.NC+TNFα:NLRP3:0.90±0.06 vs.1.38±0.04,P<0.05;IL-1β:0.77±0.12 vs.1.35±0.31,P<0.05).Through transient transfection of plasmids,flow cytometry showed that HCT116 cells overexpressed ALDH1L1 or SUGCT had more significant increase of total ROS than negative control after stimulation with TNFα(vector-ALDH1L1:P<0.05;vectorSUGCT:P>0.05).The level of mitochondrial ROS also rose more significantly in the overexpression+TNFα group compared with the negative control+TNFα group(P<0.05).In terms of inflammation,the protein levels of NLRP3 and IL-1β in ALDH1L1 and SUGCT overexpressing cells increased more significantly than those in negative control after TNFα stimulation(relative expression,vector-ALDH1L1+TNFα vs.NC+TNFα:NLRP3:1.24±0.07 vs.0.98±0.11,P<0.05;IL-1β:2.40±1.41 vs.1.49±0.31,P>0.05;vector-SUGCT+TNFα vs.NC+TNFα:NLRP3:1.0±0.27 vs.0.98±0.11,P>0.05;IL-1β:1.74 ± 0.46 vs.1.49±0.31,P>0.05).Moreover,NLRP3 activation regulated by ALDH1L1 or SUGCT was partially inhibited in the overexpression cells pretreated with NAC,and the expression of IL-1β,IL-6 reduced to different degrees.Conclusion:1.In the inflammatory condition,the expressions of ALDH1L1 and SUGCT in colon tissue from UC patients or DSS colitis mice were obviously decreased at the mRNA and protein levels,more significantly in the elderly group.2.The expression of ALDH1L1 or SUGCT can affect mitochondrial respiratory function and ATP production,change cellular ROS levels,and regulate oxidative stress injury and inflammatory response probably by affecting the NLRP3 inflammasome signaling pathway.