The Role And Mechanism Of Low-methoxyl Pectin In The Prevention Of Type 1 Diabetes

Type 1 diabetes（T1D）is an autoimmune disease,which is characterized by pancreaticβ-cells damage.As a worldwide medical problem,none of effective treatments has been found for several decades.Accumulating evidence showed that intestinal homeostasis imbalance is one of the important factors of T1D onset.Meanwhile,it has been reported that low-methoxyl pectin（LMP）shows its effect of maintaining intestinal homeostasis.However,there are almost no reports on LMP and T1D.Therefore,in order to explore the effect of LMP dietary intervention on the pathogenesis of T1D and its potential mechanism.Firstly,4-week-old female non-obese diabetic（NOD）mice were weaned onto either control or 5%LMP supplemented diets for 40 weeks.The effects of LMP supplement on the incidence of T1D and intestinal homeostasis were monitored,and the mechanisms of LMP protecting NOD mice from diabetes was studied preliminary.Next,the inflammatory environment was simulated in vitro on intestinal organoids to screen the target of LMP and confirming that LMP maintains intestinal homeostasis depends on histone demethylase KDM5a initially.Intestinal homeostasis imbalance can induce inflammatory bowel disease（IBD）.KDM5a expression was inhibited and overexpressed in colitis mice to confirm the effects of KDM5a expression on intestinal homeostasis and to elucidate the potential mechanism.Finally,the effects of KDM5a expression on diabetes incidence and immune regulation were studied,and the potential mechanism was clarified.The main results of this study are as follows:After 40 weeks of dietary intervention,53.8%of the LMP-fed mice vs.all mice in the control group had developed overt diabetes.Lower levels of insulitis,inflammatory response in pancreas and caecum were observed in LMP-treated mice.The intestinal barrier was strengthened after LMP treatment.The frequency of regulatory T cell（Treg）increased significantly in pancreas,pancreatic lymph node（PLN）and mesenteric lymph node（MLN）in LMP-treated mice compared with NOD mice.Finally,we found that microbiota is required when LMP exerts its protective effects by antibiotics treatment experiments combined with fecal transfer experiments.To further clarify the mechanism of LMP protecting NOD mice,the inflammatory environment was simulated in vitro on intestinal organoids.The results showed that the expression of KDM5a increased after LPS stimulation.The inflammatory levels of intestinal organoids increased significantly.The expression of tight junction proteins and the mRNA levels of antimicrobial peptides decreased sharply after LPS stimulation.The inflammatory levels of organoid decreased significantly,and the intestinal barrier was significantly strengthened after LMP treatment.The effect of LMP on maintaining intestinal homeostasis disappeared on KDM5a-overexpression organoids.Finally,the microbiota composition was analyzed.We found that LMP-treatment promotes the probiotic proliferation specifically.To further clarify the important role of KDM5a in maintaining intestinal homeostasis,we examined the expression of KDM5a in the colon of mice with colitis,and found that KDM5a expression was significantly increased.Next,we inhibited and overexpressed KDM5a expression by lentiviral and found that reduced severity of colitis and enhanced levels of intestinal homeostasis were observed after inhibiting KDM5a expression.Opposite trends were observed after KDM5a-overexpression treatment.In cnlp^-/-mice,we found that KDM5a inhibition did not protect cnlp^-/-mice from colitis.CRAMP is required when KDM5a inhibition exerts its protective effects.Exogenous CRAMP supplement attenuated colitis by administrating CRAMP-L.plantarum orally.Finally,to explore the effect of KDM5a on T1D incidence,lentiviral particle were used to inhibit or overexpress KDM5a in NOD mice.We found that suppressing the expression of KDM5a reduced the incidence of T1D in NOD mice.At 40th week,the diabetes incidence was 47.62%.Meanwhile,overexpressing KDM5a increased diabetes incidence.All the mice in this group were diabetic at 34th week.The expression of MHC-II and co-stimulatory molecules CD80/86 were decreased in CD11b⁺DC,and the frequency of Treg was increased and the frequency of Th1 was decreased after suppressing KDM5a expression.KDM5a inhibition enhanced the enrichment of H3K4me3 in the proximal socs3 promoter region,thus activating socs3 gene transcription and,therefore,impacting the antigen presenting ability of CD11b⁺DC and cytokine expression.However,overexpressing KDM5a showed the opposite trends.These results showed that LMP dietary intervention reduced the diabetes incidence effectively by maintaining intestinal homeostasis,and histone demethylase,KDM5a,plays a pivotal role in this progress.Inhibiting KDM5a expression reduced the intestinal inflammatory response,intestinal barrier and improves the microbiota composition.These results may provide experimental basis and theoretical support for the LMP as a dietary additive or KDM5a inhibitor in further application.