PFKFB3 Plays Critical Role In Garlic-derived Exosome-like Nanoparticles Regulating The Reprogramming Of Glucose Metabolism In Macrophages

Obesity has become a worldwide epidemic disease,which is closely related to the development of metabolic diseases such as diabetes,non-alcoholic fatty liver disease and atherosclerosis.The increasing evidence indicates that the accumulation of macrophages and its released cytokines and chemokines are important pathological features of obesity,but the mechanism and function associated with those need to be further explored.Our previous studies revealed that garlic-derived exosomes-like nanoparticles（GELNs）showed obvious anti-inflammatory activity,but the mechanisms about that are not clear.At present,our data showed that GELNs significantly attenuated glucose uptake and the production of lactate,but increased the production of ATP and the content of mitochondria in LPS-treated Raw264.7 macrophages,suggesting that GELNs might play an important role in the reprogramming of glucose metabolism.To probe the molecular mechanisms of GELNs regulating the reprogramming of glucose metabolism,we determined the effect of GELNs on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3（PFKFB3）,a key enzyme of glycolysis in LPS-treated RAW264.7macrophages,the results from q RT-PCR and western blot assays revealed that GELNs distinctively prevented the increase of PFKFB3 induced by LPS in RAW264.7macrophages.In order to clarify the relationship between GELNs regulating of PFKFB3 expression and the reprogramming of glucose metabolism in macrophages,we utilized si RNA method to interfere the expression of PFKFB3 gene in RAW264.7 macrophages,and determined the effect of GELNs on the expression of M1 cytokines and M2 cytokines.Our data demonstrated that,although GELNs significantly inhibited the expression of M1cytokines（including TNF-α、IL-1β、IL-6、i NOS、CLCX10）and partially up-regulate M2 cytokines（including Arg1,CD206,Ym1,Mgl1）in RAW264.7 macrophages induced by LPS,but once PFKFB3 gene was interrupted,these effects of GELNs on the expression of these cytokines was obviously prohibited in LPS-treated RAW264.7 macrophages,suggesting that PFKFB3 is involved in GELNs regulating the reprogramming of glucose metabolism in macrophages.To gain investigate the function of GELNs regulating macrophage reprogramming,we incubated the differentiated 3T3-L1 adipocytes with conditioned medium of LPStreated RAW264.7 macrophage in the presence or absence of GELNs.The results showed that,compared with the macrophage conditioned medium,the conditional medium from GELNs could significantly reduce the m RNA levels of proinflammatory factors including TNF-α,IL-6,IL-1β,i NOS,CXCL10 and noticeably inhibited the accumulation of lipid droplets by attenuating the e expression of FAS and ACC1,the two key genes for the biosynthesis of fat acids,in the differentiated 3T3-L1 adipocytes.Furthermore,once PFKFB3 gene in RAW264.7 cells was interfered with RNAi,these effects of GELNs were distinctively inhibited.To confirm the anti-inflammatory activity of GELNs,we evaluated the effect of GELNs on the inflammatory response in adipose tissue of high-fat diet（HFD）-fed C57BL/6 mice,our data showed that,although GELNs administration for 6 weeks had no significant effect on their body weight,food intake and water intake,but significantly improved the insulin resistance and reduced the level of TNF-α,L-10 and increased the level of IL-10 in serum.Interestingly,GELNs administration also significantly reduced the expression of PFKFB3 gene in the liver of HFD-fed obese mice.Taken together,all the findings of this study show that PFKFB3 might play a critical role in GELNs regulating the reprogramming of glucose metabolism to attenuate the inflammatory response in LPS-treated differentiated 3T3-L1 adipocytes and HFD-fed mice.All these findings do not only clarify the anti-inflammatory mechanisms of GELNs,but also provide new ideas and targets for the prevention and treatment of obesity and relative metabolic syndromes.