Blueberry Anthocyanins Inhibit Intestinal Inflammation And Regulate Glucolipid Metabolism Through Toll-like Receptor 4 Signal Pathway

Blueberries are rich in nutrients and micronutrients,as well as various active ingredients,such as anthocyanins.Blueberries can reduce the risk of type 2 diabetes,probably because anthocyanins help restore insulin sensitivity,but the specific mechanism is not yet clear.Anthocyanins,as the natural pigment and functional characteristic components of blueberry,have a variety of physiological functions,such as antioxidant,anti-inflammatory,anti-aging,hypoglycemic and other functions.Our previous study found that blueberry anthocyanins,especially the major constituents malvidin-3-glucoside and malvidin-3-galactoside,have anti-inflammatory and hypoglycemic effects.Toll-like receptor 4（TLR4）has long been regarded as a classic innate immune recognition molecule involved in inflammation.Recent studies have found that TLR4 effectively participates in the regulation of glucose and lipid metabolism and is related to the regulation of insulin resistance.We speculate that there is a close relationship between the anti-inflammatory effect and hypoglycemic effect of blueberry anthocyanins,and intestinal TLR4mediated signaling pathway plays a role of central link in regulating glucose and lipid metabolism.In this study,blueberry anthocyanin extracts,and their major constituents malvidin-3-glucoside,malvidin-3-galactoside,and malvidin-3-arabinoside were investigated on the mechanisms of inhibiting intestinal inflammation and regulating glucolipid metabolism.The main research contents and results are as follows:（1）In this study,C57BL/6J（wild）and C57BL/10Sc NJGpt(TLR4^-/-)mice were fed with high fat and high sugar for 5 months,and then streptozotocin was injected to construct a diabetic mouse model.Blueberry anthocyanin extracts（BAE）were administered to diabetic mic via gavage continuously for 5 weeks.The blood glucose and body weights were measured weekly,and glucose tolerance test was performed in the fifth week.The results showed that the wild mice more easily reach the diabete model standard than TLR4^-/-mice,and the changes of body weight and blood glucose in wild mice were more significant than those in TLR4^-/-mice.Intragastric administration of low and high doses of BAE in C57BL/6 mice increased body weight and decreased blood glucose in diabetic mice,but this effect was not found in TLR4^-/-mice.Enzyme-linked immunosorbent assay（ELISA）and real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）were used to investigate whether blueberry anthocyanins acted on TLR4 to inhibit inflammation and regulate glucose and lipid metabolism.The results showed that blueberry anthocyanins could inhibit the inflammatory signaling pathway nuclear factor-Kappa B（NF-κB）and inflammatory factors,such as monocyte chemotactic protein-1（MCP-1）,vascular cell adhesion molecule-1（VCAM-1）in C57BL/6J diabetic mice both in m RNA and protein levels.The mechanism of BAE on inhibiting intestinal inflammation might involve the down-regulation of TLR4 and its downstream signaling pathways,including myeloid differentiation factor 88（My D88）and tumor necrosis factor receptor-related factor 6（TRAF6）.Blueberry anthocyanins could also regulate the m RNA and protein levels of glucose metabolizers,such as glucose-6-phosphatase（G6Pase）,glucose transporter 2（GLUT2）,forkhead transcription factor 1（Fox O-1）,as well as lipid metabolizers,such as acetyl-Co A carboxylase（ACC）,3-hydroxy-3-methyl-valeryl Co A reductase（HMGR）,hormone-sensitive triglyceride lipase（HSL）.The mechanism of BAE on regulating glucolipid metabolism might also involve the down-regulation of TLR4 signaling pathways,which up-regulated intracellular phosphoinositide-3 kinase（PI3K）-endothelial nitric oxide（e NOS）signaling pathway to activate adenosine 5’-monophosphate-activated protein kinase（AMPK）signaling pathway.（2）In this study,normal Caco-2 cells,constructed plasmid vectors,lentivirus infection overexpression and short hairpin RNA（sh RNA）to silence TLR4 gene were used in Caco-2 cells.Western blotting was used to screen and verify the effect of overexpression and knockdown of TLR4 genes in cells.Interleukin-1βwas used to stimulate five kinds of Caco-2 cells to induce inflammation.Enzymed-linked immunosorbent assay（ELISA）and RT-q PCR were used to investigate whether blueberry anthocyanins acted on TLR4 to inhibit cellular inflammation.Blueberry anthocyanins and and their major constituents malvidin-3-glucoside,malvidin-3-galactoside,and malvidin-3-arabinoside could inhibit NF-κB signaling pathway and inflammatory factors（MCP-1 and VCAM-1）both in m RNA and protein levels by down-regulating the TLR4signaling pathways,including TLR4,My D88,and TRAF6,which was consistant with the results of diabetic mice in vivo.However,in Caco-2 cells with TLR4 gene silenced by sh RNA,the inflammation inhibitory effect of blueberry anthocyanins was not significant,while,in overexpressed TLR4 Caco-2 cells,the inflammation inhibitory effect of blueberry anthocyanins was more significant than that in normal Caco-2 cells.（3）In this study,Hep G2 cells with normal Hepg2 cells,constructed plasmid vectors,lentivirus infection overexpression and short hairpin RNA（sh RNA）to silence TLR4 gene were used in Hep G2 cells.The overexpressed and knockout effects of TLR4 cells were screened and verified by western blotting.Five kinds of Hep G2 cells were stimulated with glucose and oleic acid to induce inflammation and disorder of glycolipid metabolism.Whether blueberry anthocyanins act on TLR4 to regulate glucolipid metabolism was investigated by analyzing the m RNA transcription and protein expression using RT-q PCR and ELISA.Blueberry anthocyanins and and their major constituents malvidin-3-glucoside,malvidin-3-galactoside,and malvidin-3-arabinoside could down-regulate TLR4,G6Pase,and ACC.Their mechanism might be that blueberry anthocyanins regulated glucolipid metabolism by down-regulating TLR4 and up-regulating PI3K-e NOS signaling pathway to activate AMPK signaling pathway,which was consistant with the results of diabetic mice in vivo.However,in Hep G2 cells with TLR4 gene silenced by sh RNA,the regulation effect of blueberry anthocyanins on glycolipid metabolism was not significant,while,in overexpressed TLR4 Hep G2 cells,the regulation effect of blueberry anthocyanins on glycolipid metabolism was more significant than that in normal Hep G2 cells,which confirmed the key role of TLR4 for blueberry anthocyanins in the regulation of glycolipid metabolismIn summary,this study used TLR4 gene silencing or overexpression cell model and TLR4gene knockout mouse model to clarify the mechanism of TLR4 mediated blueberry anthocyanin inhibiting intestinal inflammation and regulating glucose and lipid metabolism at the molecular,cellular and animal overall levels.We combined in vitro and in vivo experiments to confirm the conclusion,which would provide the theoretical basis for blueberry anthocyanins to develop new functional food ingredien...