In View Of Intestinal Mucosal Barrier And Immune System To Explore The Effects Of Berberine On Glucose And Lipid Metabolism Disorders

Section 1 In view of intestinal mucosal barrier and immune-endocrine system to explore the effects of berberine on glucose and lipid metabolism disorders in type 2 diabetes mellitus ratsObjective The rats with type 2 diabetes(T2DM)were treated with berberine at low,medium and high doses.The effective mechanisms of berberine on glucose and lipid metabolism disorders were explored from intestinal mucosal barrier,immune system and gastrointestine-deriving hormones.Methods The male Wistar rat model of T2 DM was established by high glucose and fat diet feeding and intravenous injection of streptozocin.The rats were then randomly divided into low,medium or high dose group of berberine,metformin group or diabetic model group.Rats fed with normal diet were set as normal controls.After 9 weeks of medicine treatment,the differences of blood glucose,blood lipid,fasting insulin and intestinal permeability of ileum were compared between various groups.Intestine sections were stained using hematoxylin and eosin(H&E)to assess the histological morphology.Transmission electron microscope was used to evaluate the cell junction changes between intestinal epithelial cells.Flow cytometry was applied for the detection of T cell,NK cells,dendritic cells and macrophages percentages in mesenteric lymph nodes.ELISA was used to detect the changes of gastrointestinal hormones,such as GLP-1,GIP,Ghrelin and Amylin in plasma of diabetic rats.RT-PCR was employed to detect the expressions of cytokines such as IL-6,TNF?,MIP,IL-10 and TGF-? in intestinal tissues of rats in each group.Occludin,ZO-1,Claudin-1,TLR4,My D88 and phosphorylated IKK? in intestinal tissue were detected by Western Blot.The effects of berberine on the permeability of intestinal tissue in rats were observed by ileal perfusion with FITC-dextran.The distribution of NF?B in cytoplasm and nucleus of intestinal epithelial cells was detected by immunofluorescence.Results Berberine decreased blood glucose concentration at different time points of OGTT and triglyceride level in diabetic rats in a concentration-dependent manner.For intestinal permeability,transmission electron microscopy showed berberine prevented the injuries of intestinal epithelial juncitons in diabetic rats.After FITC-dextran was affused into the ligated 10cm-long intestinal cavity,permeability for large molec ules was increased in diabetic rats;however,berberine and metformin co uld inhibit increased intestinal permeability.The beneficial effects of berberine on t he intestinal mucosa integrity were related to the intestinal tight junction proteins Occludin and ZO-1.Flow cytometry showed the proportion of macrophages increased and Tregs cells decreased in mesenteric lymph nodes of diabetic rats.Berberine and metformin could decrease the percentage of macrophages and increase ratio of Treg cells in the intestinal immune system.No significant changes were found about the percentages of CD4+ or CD8+ T cells and dendritic cells labeled by OX-62.For immune factor levels in the intestinal mucosa detected by RT-PCR,IL-1?,TNF-? and MIF m RNA expressions were elevated compared with normal group,while IL-4 and IL-10 m RNA levels were reduced in intestinal tissue of diabetic rats.However,BBR,especially at high dose,showed significant inhibitory effects on the m RNA levels of IL-1?,MIF and TNF-?.Moreover,BBR treatment also increased IL-4 and IL-10 m RN A expressions in intestinal tissue of diabetic rats.The res ults of ELISA showed that GLP-1 and GIP secretions reduced,and islet ? cells-derived Amylin increased in plasma of diabetic rats.Berberine,especially at high dose,could reverse these changes.In the intestinal tissues of diabetic rats,the expression of TLR4,My D88,p-IKK? protein,as well as the transcription o f LBP and C D14 increased,while berberine inhibited the up-regulation of pro-inflammatory signal transduction pathway.Conclusions Berberine can improve glucose and lipid metabolism disorders of diabetic rats in a dose-dependent manner,and the active targets are located in the intestine.Berberine improves the intestinal immune system,intestinal mucosal barrier and gastrointestinal hormones.These effects are related to the regulation of TLR4/ My D88/p-IKK?/NF?B signaling pathway.Section 2 Mechanisms of berberine in proinflammatory M1 polarization of macrophages based on TLR4/My D88/NF?B signaling pathwayObjective The effects and mechanisms of berberine on the inflammatory polarization of Raw264.7 cells and primary peritoneal macrophages induced by LPS were explored.Methods The intervention concentrations of BBR and metformin were determined by MTT assays.The macrophage inflammatory polarization model was established with LPS exposure at different concentrations for 3,6,12 or 24 hours.The changes of cell morphology during polarization were observed by electron microscope.ELISA assay was used to detect the secretion of inflammatory factor TNF? after intervention with berberine.RT-PCR was used to detect expression of M1 inflammatory factors IL-6,i NOS2,IL-1? and MIP.The transcriptions of TLR1,TLR2,TLR4,TLR5,TLR6 and TNFR were also evaluated by RT-PCR.The expressions of p-AMPK and TLR4 were detected by Western Blot.The cytoplasmic and nuclear distribution of NF?B was observed by confocal microscope.The binding of TLR4 to My D88 was tested by Co-IP,and the affinity of berberine and TLR4 was assessed by molecular docking.Primary peritoneal macrophages were extracted from female Kunming mice.F4/80+ peritoneal macrophages ratio was determined by flow cytometry.The effect of BBR on the morphological changes of peritoneal macrophages exposed to LPS was observed.After treatment with berberine,TNF? secretion changes of peritoneal macrophages were detected by ELISA.M1 macrophage ratio was measured by flow cytometry.Results Exposed to LPS,the secretion of TNF? and the transcriptions of inflammatory factors IL-6,IL-1?,MIP and i NOS2 increased in primary peritoneal macrophages and Raw264.7 macrophages.Besides,cell morphology changed and protrusions appeared gradually during the course of inflammatory polarization.BBR could partially inhibit the secretion of TNF? at different time points,and it also attenuated the transcriptions of inflammatory markers induced by LPS.LPS increased proportion of F4/80+C D11c+ M1 polarized macrophages,and the change was inhibited by BBR pretreatment.LPS increased the nuclear distribution of NF?B at 3h;however,BBR significantly decreased the changes of NF?B distribution.Western Blot showed the expressions of TLR4 and p-AMPK did not change significantly at 3h after LPS intervention.Meanwhile,Co IP results showed that the combination between TLR4 and My D88 increased,and BBR could inhibit the binding of TLR4 to My D88.Molecular docking suggested BBR co uld bind to several chains of TLR4,and the affinity of BBR to the binding site of A chain was the highest.Conclusions Inflammatory changes were induced after LPS stimulation for 3 hours in Raw264.7 cells,and BBR pretreatment co uld depress the inflammatory polarization of Raw264.7 macrophages and primary peritoneal macrophages.After LPS stimulation for 6h,the expression changes of TLR4 and p-AMPK occurred.BBR might combine with TLR4 and disturb the signal transduction of TLR4/My D88/NF?B signaling pathway,which could be the mechanism of BBR on attenuating inflammation in the early phase.Section 3 A preliminary study of the inhibitory effects of berberine on the inflammatory changes of macrophages induced by palmitic acidObjective The impact of berberine on palmitic acid-induced inflammation in Raw264.7 cells was observed.Methods The effects of palmitic acid with or without BSA on macrophages were observed.Exposed to palmitic acid for 3 hour,6 hours and 24 hours,the TNF? levels in the cell supernatant were detected,and the intervention concentration of palmitic acid for Raw264.7 cells was ascertained by MTT and ELISA assay.The expressions of IL-6,MIP-1 and i NOS2 stimulated by palmitic acid or berberine were detected by RT-PCR.Triglyceride kit was used for the determination of intracellular triglyceride accumulation after the stimulation of palmitic acid for different time.F ree fatty acid kit was used to detect fatty acid concentration in cells and supernatant after palmitic acid intervention for 6 hours and 24 hours,to observe the uptake of palmitic acid and lipid storage in Raw264.7 cells.Results BSA could induce inflammation in Raw264.7 cells,and palmitic acid without BSA was used for intervening cells.Berberine inhibited the inflammatory response induced by palmitic acid at 6 hour and 24 hour.There was no significant difference in intracellular triglyceride levels after the exposure to palmitic acid for 6 hour and 24 hours.Free fatty acid contents were changed after palmitic acid stimulation for 6 hours.Conclusions Berberine depressed palmitic acid-induced inflammation in Raw264.7 cells.But it seemed that inflammation induced by palmitic acid in macrophages was independent of the accumulation of triglycerides in the cells.The mechanism identification of berberine in improving palmitic acid-induced inflammation of macrophage needs further study.