| BackgroundDiabetes mellitus is a chronic metabolic disease,which is characteristic of chronically elevated glucose concentrations and caused by multiple and complex factors.In the latest data from the International Diabetes Federation,it is estimated that 537million people have diabetes in 2021 and this number is projected to reach 700 million by 2045.“Guidelines for the Prevention and Treatment of type 2 diabetes mellitus(T2DM)in China(2020 Edition)”announced by Diabetes Branch of Chinese Medical Association shows that the incidence of adult diabetes mellitus has been reached to11.2%and approximately 90%are T2DM.T2DM,one of the major public health problems,has become the sixth largest disability factors which not only brings great pain to patients,but also cause a heavy burden to social economy.The development of T2DM is affected by complex factors including genetics,environment,and lifestyle,and the curative measures such as drugs and metabolic surgery have become the routine treatment method for T2DM.However,effective prevented methods are still lacked.To further investigate the pathogenesis of T2DM is needed to seek more effective prevention and treatment measures.Recent researches indicated that dietary factors(such as high energy and high-fat diet)promote the development of T2DM and the intestinal barrier dysfunction palys an important role in the occurrence of T2DM induced by dietary factors.In this concept,the intestinal endotoxemia initiates obesity and diabetes.Intestinal flora disturbance caused by dietary factors tigger the increasing of intestinal tract permeability and lead the translocation of intestinal flora through intestinal barrier.The flux of lipopolysaccharide(LPS)from the intestinal lumen into the blood results in a low-grade chronic tissue inflammation which in turn to cause the development of insulin resistance and obesity.The intestinal immune system,an integral part of the intestinal barrier,which includes Foxp3~+Tregs,dendritic cell,Th17,and innate lymphoid cells(ILCs).ILCs are specific lymphocytes which play an important role in the limitation of pathogens invasion and maintain of intestinal barrier integrity through initial immune responses.ILCs mainly located in intestine and receive signals from other resident cells and intestinal metabolites to secret IL-22 that promote tissue damage recovery,protect intestinal barrier integrity,and resist microbial insults.The gut immune system coordinates with intestinal epithelial cells to promote intestinal barrier maintenance and this cross-talk is dysregulated during HFD-feeding through changing the expression of intestinal immune cells and cytokines.Therefore,further research on the influence of HFD to the intestinal regional immune system is needed to reinforce the scientific viewpoint about intestinal inflammation initiate T2DM and provide theoretical basis to explore new intervention targets for T2DM prevention and treatment.A growing body of research shows that natural phytochemicals have become optimistic candidates for the prevention and treatment of T2DM.Dihydromyricetin(DHM),which is also known as ampelopsin,is one of natural flavonoid primarily extracted from grape family and myricaceae,especially Ampelopsis grossedentata(commonly known as"rattan tea").Our previous study showed that DHM effectively regulated metabolic pathways to ameliorate insulin resistance and maintain glucose homeostasis.Although the ability of DHM to intervene with the prevention and treatment of T2DM is established,the specific mechanism regarding the way by which this compound regulate T2DM remains elusive.Meanwhile,researches showed that DHM regulated liver cell lipid metabolism and maintained redox homeostasis via Silent mating-type information regulation 2 homolog 3(SIRT3).This study indicated that SIRT3 may be a potential target for DHM to exert biological effects in vivo.SIRT3 is an important deacetylase enzyme found in the mitochondria,which acts as a"molecular switch"of mitochondrial energy metabolism and positively regulates mitochondrial electron transfer chain enzyme complex,fatty acidβoxidation,tricarboxylic acid cycle and ketone body generation.The upregulation of the expression levels of proteins associated with lipid oxidation and mitochondrial respiratory function has been found to promote ILC3 activation and proliferation.Moreover,SIRT3 promote Signal transducer and activator of transcription 3(STAT3)phosphorylation via ERK signaling and STAT3signaling activation has been found to be a critical regulator for the production of IL-22in ILC3s.Previous study also indicated that DHM effectively maintained intestinal barrier function by alleviating intestinal flora disturbance.Based on the above background,we hypothesized that DHM could reduce intestinal chronic inflammation,improve insulin resistance,and prevent T2DM through promote IL-22 secretion of ILC3s.The current findings firstly reported that DHM could enhance the immune function of ILC3s in small intestine lamina propria to protect against the intestinal barrier integrity damage,which subsequently improved T2DM development.This study would further reveal the pathogenesis of T2DM from the perspective regarding intestinal regional immune pathway,and provide new intervention strategies for the prevention and treatment of T2DM through the dietary pathway.ObjectiveTo investigate the effect of ILC3 cells in small intestinal lamina propria on the HFD-induced intestinal barrier damage and T2DM,and further illustrate the effect and and mechanism of DHM on ameliorating HFD-indcued T2DM through regulating immune function of ILC3 cells via a SIRT3-dependent mechanism.Our research was aimed to provide a potential nutrition intervention strategy and target for the prevention of T2DM.Methods1.The C57BL/6-WT male mice were fed with control diets or high-fat diets(HFD)for 8 weeks.Bodyweights,food intake,and blood glucose were recorded to evaluate the model building.Intestinal permeability and the expression of intestinal tight junction(TJ)protein were detected to evaluate the effects of HFD on the intestinal epithelial barrier integrity.The number of intestinal Peyer patches,intestine intraepithelial lymphocytes(IELs),and lamina propria lymphocytes(LPLs)in small intestine and colon and lymphocytic immunological phenotype of small intestinal LPLs were calculated to evaluate the effect of HFD on the intestinal regional immunity.Further,the detection of IL-22 expression in small intestinal LPLs and IL-22 intervention experiment were conducted to evaluate the effects of IL-22 on HFD-induced intestinal barrier damage.2.The C57BL/6-WT and Rag1 knockout(adaptive immune deficiency with lack of T and B cells)male mice were fed with HFD for 8 weeks to establish T2DM model.DHM was thoroughly mixed into the diets,and the food pellets and water were provided ad libitum.Bodyweight,food intake,blood glucose,oral glucose tolerance and insulin sensitivity in indicated groups were compared to evaluate the effects of DHM on HFD-induced T2DM.Blood LPS level,intestinal permeability,intestinal epithelial structure and small intestinal epithelial tight junction protein expression were detected to evaluate the effects of DHM on the HFD-induced intestinal barrier destruction.The proliferation and IL-22 secretion of ILC3 cells in small intestinal LPLs were detected to evaluate the effects of DHM on the immune function of ILC3 cells.To further determine the effects of DHM on ILC3 cells,ILC3 cells in small intestinal LP were isolated to conduct transcriptome analysis to analyze the regulatory effect of DHM on ILC3 expression.3.The SIRT3 knockout(SIRT3KO)mice and wild-type(WT)mice were fed with HFD for 16 weeks to establish T2DM model.DHM was thoroughly mixed into the diets,and the food pellets and water were provided ad libitum.Bodyweights,food intake,blood glucose,oral glucose tolerance and insulin sensitivity in indicated groups were compared to evaluate the effects of SIRT3 on the DHM-mediated protected functions on HFD-induce T2DM.The proliferation and IL-22 secretion of ILC3 cells in small intestinal LPLs were detected to evaluate the effects of SIRT3 on the DHM-induced regutory influence on ILC3 cells.Blood LPS level,intestinal permeability,intestinal epithelial structure and small intestinal epithelial tight junction protein expression were monitored to illustrate the effects of SIRT3 on DHM-induced protected functions on HFD-induecd intestinal barrier damage.4.MNK3 cells(in vitro cell line of ILC3 cells)were used to establish in vitro model under saturated fatty acids and DHM intervention.A variety of specific inhibitors,lentivirus transfection,cell metabolism detection,ROS detection by DCFH-DA and Mito SOX probe were employed to explore the molecular mechanism under the effects of DHM on ILC3 cells.The effects of DHM on the expression and activity of SIRT3 in PA-induced MNK3 cell model were detected by q RT-PCR,Western blotting and enzyme activity detection to demonstrate the regulatory effect of DHM on SIRT3 expression of ILC3 cells.The expression of immune-related functional genes and IL-22 in PA-induced MNK3 cell model were detected using SIRT3 inhibitor and lentivirus transfection to clarify the role of SIRT3 in the effects of DHM on ILC3 cells.The oxygen consumption and ROS level of MNK3 cells were further calculated to explore the possible mechanism in the effects of DHM on the IL-22 secretion of ILC3 cells via SIRT3.Then AMPK inhibitor dorsomorphin was employed to detect the expression of AMPK/PGC1α/SIRT3signaling to demonstrate the upstream mechanism of the effects of DHM on ILC3 cells.Finally,the effects of DHM on ERK,CREB,STAT3 phosphorylation and IL-22expression were detected using SIRT3 knockdown virus and STAT3 inhibitor static to further reveal the molecular mechanism of the effects of DHM on the IL-22 secretion of MNK3 cells via SIRT3.Results1.HFD changed the intestinal regional immune homeostasis by reducing the IL-22 expression of ILC3 cells in small intestinal LPLs to destruct in intestinal barrier integrity.(1)Compared with control group,HFD significantly decreased the small intestinal epithelial tight junction protein expression and enhanced the the intestinal permeability.(2)Compared with control group,HFD significantly reduced the length of small intestine and colon tissue,decreased the number of Peyer’s patches,small intestine IELs and LPLs,downregulated the number of CD3~+T cells,CD3~+TCRαβ~+cells,CD3~+TCRγδ~+cells,CD3~+TCRαβ~+CD4~-CD8αβ~-cells,CD3~+TCRαβ~+CD4~+cells,CD3~+TCRαβ~+CD8αβ~+cells,CD3~-CD45~+CD90.2~+cells and CD3~-CD45~+CD90.2~+RORγt~+ILC3 cells significantly.(3)HFD significantly reduced the expression of IL-22 in innate lymphocytes,and decreased the number and IL-22 secretion of CD3~-CD45~+CD90.2~+RORγt~+ILC3 cells in small intestinal LPLs.IL-22 intervention effectively upregulated HFD-induced decreasing expression of small intestinal epithelial tight junction protein expression,and reduced intestinal barrier permeability.2.DHM alleviated HFD-induced T2DM in wild-type and adaptive immune deficient mice through the protection of intestinal barrier integrity via promoting the immune-related function and IL-22 expression of ILC3 cells in small intestinal LPLs.(1)Compared with HFD group,DHM supplementation significantly inhibited bodyweights and blood glucose increasing,enhanced glucose tolerance and insulin sensitivity.DHM alleviated inceased intestinal permeability and blood LPS level and decreased expression of small intestinal epithelial tight junction protein induced by HFD.(2)Compared with the control group,HFD enhanced the intestinal inflammation,decreased the frequencies,proliferation,and IL-22 production of ILC3 cells in small intestinal LPLs.DHM supplementation effectively alleviated the immune-related function damage induced by HFD.(3)The transcriptome sequencing of small intestinal lamina propria ILC3 cells sorted from Rag1 knockout mice showed that DHM significantly changed the differentially expressed genes(DEGs)of ILC3 cells induced by HFD,regulated twenty cell signaling pathways including oxidative phosphorylation,protein digestion and adsorption and Th17 cell differentiation,effectively promoted the gene expreesion associated with RORγt and IL-22 expression regulating,immune system,and TLR signaling pathway and downregulated lipid metabolism related genes.3.DHM promoted STAT3 phosphorylation and IL-22 secretion through increasing SIRT3 expression via enhancing AMPK phosphorylation,which in turn to alleviate HFD-induced intestinal barrier damage and T2DM.(1)PA inhibited cell proliferation,mitochondrial function genes(Pgc1α,Tfam,Nrf1)expreesion,and cell respiration and decreased ROS production in MNK3 cells.DHM effectively promoted the expression of PGC1α,SIRT3,and AMPK phosphorylation,restored mitochondria respiratory capacity,and promoted IL-22 secretion.The effects of DHM on PA-induced MNK3 cell model was blocked by SIRT3 inhibition and the upregulation of PGC1αand SIRT3 expression induced by DHM was attenuated by AMPK inhibitor.(2)Compared with HFD group,the HFD+DHM feeding WT mice reduced bodyweights and blood glucose,enhanced glucose tolerance and insulin sensitivity,increased the number and IL-22 secretion of ILC3 cells in small intestinal LPLs,decreased intestinal permeability,and alleviated intestinal inflammation.In contrast,the effects of DHM on WT mice was restricted in SIRT3 KO mice.(3)DHM effectively upregulated ERK/CREB/STAT3 phosphorylation,and promoted IL-22 expression in MNK3 cells.STAT3 inhibitor significantly inhibited the function of DHM on the IL-22 expression in MNK3 cells.Meanwhile,SIRT3 knockdown cell model shows that SIRT3 deficiency significantly inhibited DHM-induced activation of ERK/CREB/STAT3,thus suppressing the role of DHM on IL-22 expression promoting.ConclusionHFD destructed intestinal barrier integrity through changing the intestinal regional immune homeostasis via downregulating IL-22 espression of ILC3 cells in small intestinal LPLs.DHM promoted SIRT3 expression and activity,enhanced mitochondria respiratory capacity,decreased ROS production,increased cell proliferation and IL-22expression in small intestinal ILC3 cells through AMPK/SIRT3/STAT3 signaling pathway.The effects of DHM on ILC3 cells alleviated intestinal epithelial barrier damage and endotoxemia caused by HFD which in turn to ameliorate insulin resistance and T2DM.This study provides new intervention targets and dietary nutrition intervention strategies and scientific evidence for the prevention and treatment of T2DM. |
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