| Objective:Obesity is one of the world’s biggest public health crises,BMI of 650million adults more than 30kg/m~2.The existence of chronic low-level inflammation in obese individuals promote the occurrence and development of insulin resistance,metabolic syndrome,type 2 diabetes,hypertension,non-alcoholic fatty liver,coronary atherosclerosis,cancer and other diseases,which seriously affects the quality and longevity of people and causes a huge economic burden.In obese human and rodents,the occurrence of chronic low-level inflammation is closely related to the pyroptosis of adipocytes.White adipose tissue is main fat reservoir and the largest endocrine organ in the body,secreting adipokines and cytokines.Adipokines are involved in a variety of metabolic and physiological signaling,including the regulation of insulin signaling,glucose uptake,fatty acid oxidation,and other energy production and metabolic processes.Cytokines are involved in the regulation of inflammation.Pyroptosis is a newly discovered mode of programmed cell death,which is accompanied by inflammatory response.When pyroptosis occurs,numerous pores will form in the cell membrane,resulting in the loss of the integrity of the cell membrane and the ability to regulate transport of substances,which eventually leads to the dissolution of the cell membrane and the release of the cell contents and inflammatory cytokines,recruit large numbers of immune cells to further induce inflammatory responses.One of the main causes of pyroptosis in adipocytes is metabolic endotoxemia.The abundance of gut microbiota in obese individuals contain lipopolysaccharide(LPS)is significantly increased.LPS can damage the intestinal barrier and increase intestinal permeability,lead to LPS from the intestinal tract into the blood circulation,which is the metabolic endotoxemia.Mildly elevated LPS in the blood can induce pyroptosis of adipocytes through non-classical pathway.The intestinal barrier system relies on interaction between various barrier components,including mucus gel layers,antimicrobial peptides,immunoglobulin A,epithelial cells,and tight junctions(TJ)between cells.Among these components,TJ is the Important member of the intestinal physical barrier.Multiple studies have demonstrated that in obese individuals,TJ are disrupted,allowing toxins such as LPS to enter the bloodstream from the gut.Orexin is a neuropeptide expressed in the lateral hypothalamus,which is involved in many functions,including eating,sleep/wakefulness,energy balance.It has been reported injection of orexin A can alleviate intestinal damage and increase the expression of TJ in ischemia-reperfusion and ulcerative colitis.In obese individuals,can orexin A regulate intestinal epithelial TJ to improve intestinal permeability,reduce LPS release into blood,which will reduce pyroptosis of adipocytes and secretion of inflammatory cytokine?The PI3K/AKT signaling pathway is widely found in mammalian tissues and organs,which plays an important role in various cell biological processes such as cell proliferation,differentiation,apoptosis,formation and metabolism of proteins.The PI3K/AKT pathway is involved in the regulation of TJ,but the conclusions are not consistent.Some studies demonstrated that the activation of PI3K/AKT pathway can protect TJ,while other studies with opposite view that the activation of PI3K/AKT pathway is involved in the occurrence and development of intestinal mucosal barrier dysfunction in some pathological cases.However,there are few reports on the role of this pathway in TJ injury caused by obesity.Therefore,in this study,male SD rats,intestinal epithelioid cell line 6(IEC-6)from rat and 3T3-L1 preadipocyte were used to investigate whether orexin A could up-regulate intestinal epithelial tight junction through PI3K/AKT pathway to reduce LPS entry into the blood,and then reduce the pyroptosis of adipocytes and secretion of inflammatory factors,relieve chronic low-level inflammation,so as to improve the prognosis of obesity.Methods:Section 1:The effect of orexin A on tight junctions of intestinal epithelium,pyroptosis of adipocytes and secretion of inflammatory cytokines in obese ratsSD male rats were randomly divided into normal diet group(NC group)and high fat diet group(HF group).After 12 weeks,the body weight of rats in normal diet group and high fat diet group was compared to determine the establishment of the obesity model.After the establishment of the obesity model,the high fat diet group was divided into high fat diet group(HF group)and high fat diet group(HD group).HD group was intraperitoneally injected with orexin A,NC group and HF group were intraperitoneally injected with saline for 3 weeks:1、Body weight,Lee’s index,visceral fat mass of rats in each group were analyzed.Serum glucose and blood lipid of rats in each group were compared by automatic biochemical analyzer,to observed the effect of orexin A on above data.2、HE staining was used to observe the morphology of perirenal adipose tissue and lipid droplets in the liver of rats in each group.Immunofluorescence was used to detect the expression of occludin and ZO-1 protein in jejunum tissue of rats.Transmission electron microscopy was used to observed the distribution of tight junctions in jejunum tissue.Western blot and Real-time PCR were used to detect the expression of protein and mRNA of occludin,ZO-1 in jejunum tissue and the expression of protein and mRNA of caspase-11 P20,GSDMD-N in perirenal adipose tissue in each group,to observe the effect of orexin A on intestinal tight junctions and pyroptosis of adipose tissue.3、Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of LPS,DAO,IL-1βand IL-18 in blood,to observe the effects of orexin A in intestinal permeability and level of inflammation in rats.Section 2:Orexin A alleviates LPS-induced adipocyte pyrosis and the secretion of inflammatory cytokinesIEC-6 cells and 3T3-L1 preadipocytes cultured in vitro:1、The IEC-6 cells were treated with different concentrations of orexin A(10-9M,10-8M,10-7M,10-6M)for 48 hours,Western blot was used to detect the expression of occludin protein.Cell counting kit-8(CCK-8)assay was used to detect the cytotoxicity of orexin A to IEC-6 cells,to elect the optimal concentration of orexin A.2、The IEC-6 cells were treated with LPS of different concentrations(0.1ug/mL,1ug/mL,5ug/mL,10ug/mL,20ug/mL)for 24 hours,Western blot was used to detect the expression of occludin protein.CCK-8 assay was used to detect the cytotoxicity of LPS to IEC-6 cells,to elect the optimal concentration of LPS.3、The IEC-6 cells were divided into NC group,LPS group,orexin A group,orexin A+LPS group(orexin A was pretreated for 24 h),SB334867+orexin A+LPS group(OX1R antagonist SB334867 was pretreated for half an hour),Millicell ERS resistance tester was used to detect the permeability of IEC-6 cell layer.Western blot and Real-time PCR were used to detect the expression of protein and mRNA of occludin,ZO-1 in each group,to observe the effect of orexin A on intestinal tight junctions.4、3T3-L1 preadipocytes were induced to differentiate into adipocytes,oil red O staining was used to observe the distribution of lipid droplets in the cells to evaluate the differentiation of preadipocytes.IEC-6 cells and adipocytes were co-cultured through Transwell chamber to simulate the environment in vivo.The IEC-6 cells in apical chamber were divided into NC group,LPS group,orexin A+LPS group.Western blot and Real-time PCR were used to detect the protein and mRNA expressions of caspase-11 P20and GSDMD-N of adipocytes in basolateral chamber,and the levels of IL-1βand IL-18in culture medium of adipocytes were detected by ELISA,to observe whether orexin A can reduce the pyroptosis of adipocytes and the secretion of inflammatory factors through regulating the tight junction of intestinal epithelium.Section 3:The role of PI3K/AKT signaling pathway in orexin A regulation of tight junctions in intestinal epitheliumIEC-6 cells cultured in vitro:1、The cells were divided into NC group,LPS group,orexin A group,orexin A+LPS group,SB334867+orexin A+LPS group,Western blot and Real-time PCR were used to detect the protein and mRNA expressions of ZO-1,PI3K and AKT,to observe whether PI3K/AKT signaling pathway is involved in the regulation of tight junctions in intestinal epithelium.2、IEC-6 cells were divided into NC group,LPS group,orexin A+LPS group,LY294002+orexin A+LPS group(PI3K inhibitor LY 294002 was pretreated for half an hour).Immunofluorescence was used to detect the expression of ZO-1 protein to observe the effect of LY 294002 on intestinal tight junctions.3、The cells were divided into NC group,LPS group,orexin A group,orexin A+LPS group,LY 294002+orexin A+LPS group.Western blot and Real-time PCR were used to detect the protein and mRNA expressions of ZO-1,to observe the effect of PI3K/AKT signaling pathway in regulation of tight junctions in intestinal epithelium by orexin A.Results:Section 1:The effect of orexin A on tight junctions of intestinal epithelium,pyroptosis of adipocytes and secretion of inflammatory cytokines in obese rats1、There were significant differences in body weight,Lee’s index and visceral fat mass between rats in the normal diet group(NC group)and those in the high fat diet group(HF and HD groups).After orexin A intervention,the body weight and Lee’s index of rats in HD group were not significantly different from those in HF group,but visceral fat mass of rats in HF group was decreased.The serum glucose,TC,LDL-C and TG of rats in HF and HD group were significantly higher than those in NC group,but TG in HD group was significantly lower than that in HF group after orexin A intervention,suggesting that orexin A can reduce the proportion of adipose tissue and improve the metabolism of TG in obese rats.2、Adipocytes around the kidney of high-fat feeding rats got bigger,more lipid droplets could be seen in liver.After orexin A intervention,adipocytes in HD group were smaller than those in HF group,and lipid droplets in liver were reduced.It suggested that orexin A can alleviate the adipocyte volume and reduce the formation of lipid droplet induced by high fat diet.3、Compared with the NC group,the occludin,ZO-1 protein and mRNA expression in jejunum tissue of rats in HF group decreased,after intraperitoneal injection of orexin A3 weeks,the occludin,ZO-1 protein and mRNA expression in HD group increased compared with those in HF group,suggesting that orexin A can improve the permeability of intestinal epithelium and protect tight junctions.4、The expressions of caspase-11 P20 and GSDMD-N protein and mRNA in the perirenal adipose tissue of rats in HF group were significantly increased,and after the intervention of orexin A,the expressions of caspase-11 P20 and GSDMD-N protein and mRNA in HD group were significantly decreased compared with those in HF group.It suggested that orexin A can reduce the pyroptosis of adipocytes.5、The levels of LPS,DAO,IL-1βand IL-18 in the blood of rats in HF group were abnormally increased,on the other hand the above indexes were significantly decreased in HD group compared with HF group,suggesting that orexin A can improve intestinal permeability and reduce inflammation in obese rats.Section 2:Orexin A alleviates LPS-induced adipocyte pyrosis and the secretion of inflammatory cytokines1、Appropriate concentrations of orexin A(10-7M)and LPS(1ug/mL)were detected by Western blot and CCK-8.2、The IEC-6 cells were divided into NC group,LPS group,orexin A group,orexin A+LPS group,and SB334867+orexin A+LPS group,the expression of occludin and ZO-1 in LPS group decreased significantly compared with NC group,and the permeability of IEC-6 cell layer increased abnormally.After the intervention of orexin A,the permeability of ICE-6 cell layer and the expression of occludin and ZO-1 were significantly improved,and its effect could be partially blocked by the OX1R antagonist SB334867,suggesting the protective effect of orexin A on the tight junctions of intestinal epithelium.3、Differentiation of 3T3-L1 preadipocytes was successfully induced.In the co-culture experiment,the IEC-6 cells in apical chamber were divided into NC group,LPS group,orexin A+LPS group,the expression of caspase-11 P20 and GSDMD-N of adipocytes in basolateral chamber were abnormally increased in LPS group.The levels of IL-1βand IL-18 in the culture medium in basolateral chamber were increased in LPS group.In the orexin A+LPS group,LPS entry into basolateral chamber was reduced,and the expression of caspase-11 P20 and GSDMD-N and the secretion of inflammatory factors in adipocytes were reduced through the protective effect of tight junctions.It suggested that orexin A can reduce the pyroptosis of adipocytes and the secretion of inflammatory factors through its protective effect on intestinal barrier.Section 3:The role of PI3K/AKT signaling pathway in orexin A regulation of tight junctions in intestinal epithelium1、The IEC-6 cells were divided into NC group,LPS group,orexin A group,orexin A+LPS group,SB334867+orexin A+LPS group,the expressions of ZO-1,PI3K and AKT in LPS group decreased.After the intervention of orexin A,the expressions of ZO-1,PI3K and AKT in IEC-6 cells were increased compared with those in the LSP group,suggesting that PI3K/AKT signaling pathway is involved in the regulation on tight junctions of intestinal epithelium.2、In NC group,LPS group,orexin A+LPS group,LY 294002+orexin A+LPS group,the expression of ZO-1 in LPS group was significantly decreased compared with NC group,and the expression of ZO-1 after orexin A intervention was increased compared with LPS group.LY 294002 can partially block the protective effect of orexin A of tight junctions,suggesting that PI3K/AKT signaling pathway is involved in orexin A’s regulation of intestinal tight junction.3、The IEC-6 cells were divided into NC group,LPS group,orexin A group,orexin A+LPS group,LY 294002+orexin A+LPS group,the expression of ZO-1 protein and mRNA decreased in LPS group,and the above indexes could be up-regulated after orexin A intervention.The addition of LY 294002 partially blocks the protective effect of orexin A on tight junctions of intestinal epithelium,suggesting that orexin A protects tight junctions of intestinal epithelium through PI3K/AKT signaling pathway.Conclusion:1、orexin A can increase the expression of tight junctions of intestinal epithelium in obese rats,reduce pyroptosis of adipose tissue and secretion of inflammatory factors.2、orexin A can reduce LPS entry into the blood,reduce pyroptosis of adipose tissue and alleviate systemic inflammation through its protective effect on the tight junctions of intestinal epithelium.3、PI3K/AKT signaling pathway is involved in the regulation of orexin A on the tight junctions of intestinal epithelium. |
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