Preliminary Study On The Difference In Pathogenesis Of Inflammatory Bowel Disease Between The Elderly And The Young

Part 1Mechanisms of cell autophagy in elderly and young inflammatory bowel diseaseObjective:This study aims to compare autophagy activity in elderly and young ulcerative colitis(UC)patients and the healthy population,and to explore whether aging increases the severity of colitis in mice and investigate its possible mechanisms.Methods:1.Colonic biopsies were collected from young and old UC patients as well as young and old healthy subjects(n=10 per group).2.Young(6-8 weeks)and aged(56 weeks)C57BL/6 mice were divided into control and experimental groups(n=6-10 per group).3.Aged(56 weeks)C57BL/6 mice were divided into control,experimental and 3-methyladenine(3-MA)treatment group(n=5-7 per group).4.Dextran sodium sulfate(DSS)was used to induce an acute colitis mouse model in the experimental group.3-MA(10 mg/kg/d)was administered intraperitoneally for 7 days.The autophagosomes of colon tissue were observed by transmission electron microscopy(TEM).Protein expressions of LC3B-II,p62,ATG5,Beclinl and F4/80 in colon tissue of mice were detected by Western blot and immunohistochemistry in colon.mRNA expressions of TNF-a,IL-6,CCL4,CXCL12 and CD86 in colon were measured by qRT-PCR.Electrophoretic mobility shift assay(EMSA)was used to measure the transcriptional activity of NF-?B.Results:1.Clinical samples:The autophagosomes of intestinal epithelial cells(IECs)in the elderly and young UC patients were observed by TEM,but none was observed in the elderly and young control groups.Compared with the control group,the ATG5 immunostaining in IECs was intenser,the protein expression of LC3B-II was higher,while the protein expression of p62 was lower in the elderly and young UC patients.Compared with the young UC patients,the protein expression of LC3B-II was higher,while the protein expression of p62 was lower in the elderly UC patients.2.Animal experiment-DSS-induced colitis in mice2.1 The aged DSS-induced mice showed higher weight loss,disease activity indexes(DAIs),histological scores than in the young DSS-induced mice.The F4/80 immunostaining was intenser and the levels of IL-6,CCL4,CXCL12 were higher in the aged DSS-induced mice than in the young DSS-induced mice.2.2 The autophagosomes of IECs in the aged and young DSS-induced mice were observed by TEM,but none was observed in the aged and young control groups.Compared with the control group,the Beclinl and ATG5 immunostaining in IECs were intenser,the protein expression of LC3B-II was higher,while the protein expression of p62 was lower in the aged and young DSS-induced mice.Compared with the young DSS-induced mice,the protein expression of LC3B-II was higher in the aged DSS-induced mice.3.Animal experiment-3-MA treatment in mice3.1 Compared with the experimental group,the positive cells of Beclinl and ATG5 immunostaining were less,the protein expression of LC3B-? was lower in the 3-MA treatment group.The mice treated with 3-MA showed lower weight loss,DAIs and histological scores than in the experimental group.3.2 Compared with the experimental group,the levels of CCL4,CXCL12,TNF-? and IL-6 were lower,the positive cells of F4/80 immunostaining were less and the level of CD86 was lower in the 3-MA treatment group.The transcriptional activity of NF-?B was lower in the 3-MA treatment group.Conclusions:1.Autophagy is induced in IECs of the elderly and young UC patients.The autophagy activity is higher in the elderly UC patients than that in the young UC patients.2.Aging increases the severity of DSS-induced colitis in mice.The autophagic ability enhances in the aged and young DSS-induced mice.The autophagy activity is higher in the aged DSS-induced mice,which may be speculated to be due to the severer colitis in the aged DSS-induced mice.3.The aged DSS-induced mice treated with autophagy inhibitor 3-MA showed reduced autophagy activity in IECs and less sever colitis.4.3-MA alleviates the DSS-induced colitis in mice.The possible machnism is that 3-MA may reduce the expression of chemotactic factors,reduce macrophage infiltration,inhibit the polarization of M1 macrophage,reduce the levels of TNF-a and IL-6,inhibit the NF-?B signial ways and ultimately relieve the inflammation.Part 2Changes and Mechanisms of gut barrier and gut microbiota in elderly and young inflammatory bowel diseaseObjective:This study aims to compare intestinal mucosal barrier function in elderly and young ulcerative colitis(UC)patients and the healthy population,and to explore the possible mechanisms that aging increases the severity of colitis in mice.Methods:1.Colonic biopsies were collected from young and old UC patients as well as young and old healthy subjects(n=10 per group).2.Young(6-8 weeks)and aged(56 weeks)C57BL/6 mice were divided into control and experimental groups(n=6-10 per group).Dextran sodium sulfate(DSS)was used to induce an acute colitis mouse model in the experimental group.3.The ultrastructure of intestinal epithelial cells(IECs)was examined by transmission electron microscopy(TEM).Protein expression of E-cadherin and occludin were detected by Western blot and immunohistochemistry.Gene sequencing of 16S rRNA was used to profile the composition of fecal and mucosal microbiota in young and aged mice before and after DSS administration.Results:1.Clinical samples:The tight junction(TJ)structure was blurred and swollen in the elderly and young UC patients.The old healthy people showed discontinued TJ strand.The E-cadherin(t=11.06,P<0.001)and occludin(t=1.519,P=0.203)protein expressions in the colonic tissue of the old healthy subjects were lower than in the young healthy people.The protein expressions of E-cadherin(t=1.809,P=0.14)and occludin(t=2.393,P=0.07)were lower in the old UC patients than in the young UC patients.2.Animal experiment-DSS induced colitis in mice2.1 In the young and aged DSS-induced mice,the TJ strands were destroyed.The aged untreated mice showed discontinued TJ strands.The protein expressions of E-cadherin(t=4.614,P=0.004)and occludin(t=3.918,P=0.008)were lower in the aged untreated mice than in the young untreated mice.Compared with the results observed in the young DSS-induced mice,the protein expressions of E-cadherin(t=0.932,P=0.387)and occludin(t=0.775,P=0.468)were lower in the aged DSS-induced mice.2.2.Significant differences were observed in the composition of the gut microbiota between the young and aged mice.Before the DSS treatment,in the aged mice,the fraction of beneficial bacteria(Lactobacillus)was lower,while the fraction of the harmful bacteria(Turicibacter,Parasutterella)was higher than in the young mice.After the DSS treatment,in the aged mice,the fraction of beneficial bacteria(Odoribacter and Alistipes)was lower,while the fraction of harmful bacteria(Turicibacter)was higher than in the young mice.Conclusions:Aging of the human colon is characterized by impairment of the intestinal barrier.Age-related deterioration of gastrointestinal barrier function and gut microbial dysbiosis may be involved in the pathogenesis of severer colitis in the aged mice.