Effects And Mechanisma Of Isobavachromene On Improving Insulin Resistance In 3T3-L1 Adipocytes

More than 90% of the diabetic patients are type 2 diabetes mellitus(T2DM),and the main pathological features are dysfunction of islet ?-cell secretion and/or target organ resistance to insulin.Recently,the inflammation theory of T2 DM has attracted much attention,whose principle is that T2 DM is a chronic low-grade inflammatory disease.The continuous low-grade inflammatory response runs through the occurrence and development of T2 DM.Low-grade inflammation is a subclinical inflammation of the body,which is below the level of infectious inflammation and autoimmune inflammation,without clinically visible local symptoms such as redness,swelling,heat,pain,and systemic symptoms.In susceptible populations of T2 DM,with the effects of environmental factors such as aging and excess nutrition,the innate immune system is activated.Along with this,macrophages,adipocytes,and other cells secrete a variety of inflammatory factors,which in turn causes or worsens insulin resistance(IR).A large number of studies have shown that traditional Chinese medicine could increase insulin sensitivity through different pathways,and had a certain improvement effect on IR.In this thesis,compounds with ameliorated IR activity were screened from 12 compounds(psoralen,angelicin,bakuchiol,isobavachromene,bavachalcone,4,5–dehydroisopsoralidin,bavachinin,corylin,corylifolinin,psoralidin,licochalcone,neobavaisoflavone)isolated from Psoralea corylifolia Linn by our research team.Of them,isobavachromene(IB)could significantly improve glucose uptake in insulin-resistant 3T3-L1 adipocytes.Therefore,IB was used to explore the role of IB in improving IR and its mechanism.This article would explore the role and mechanism of IB in improving glucose uptake of insulin-resistant of 3T3-L1 adipocytes.(1)Effect of IB on glucose uptake of insulin-resistant 3T3-L1 adipocytes.Establishment of 3T3-L1 adipocyte insulin resistance model: culture 3T3-L1 preadipocytes,and then add an induced differentiation solution containing dexamethasone,insulin,and3-isobutyl-1-methylxanthine to differentiate them into mature 3T3-L1 adipocytes.Dexamethasone was used to establish an insulin-resistant adipocyte model.Set the blank control group,model group,positive control group,and drug treatment groups.After the cells were treated,the cell supernatant was collected,and the glucose content of the cell culture supernatant was detected by the glucose oxidase-peroxidase(GOD-POD)method.(2)The effect of IB on the expression of PI-3K/Akt insulin signalingpathway-related proteins,including IRS-1,p-IRS-1,PI-3K,Akt,p-Akt,and the cell membrane protein GLUT4 were studied by the method of Western blotting.At the same time,real-time quantitative PCR method was used to detect GLUT4 gene transcription level.(3)Wortmannin(PI-3K inhibitor)was used to further detect the effect of IB on the PI-3K/Akt signaling pathway.At the same time,we investigated the effect of IB on 3T3-L1 adipocyte inflammation and its mechanism.(1)An inflammatory model was established by RAW264.7 cells,and the effect of IB on RAW264.7 cells inflammation was studied.RAW264.7 cells were cultured,and after 1 h of IB pretreatment,an inflammation model was established by LPS with the blank control group,model group,and drug treatment groups.Supernatants were collected,and the effects of IB on the contents of NO,TNF-?,IL-6,and IL-1? in the supernatant were measured by a nitric oxide kit and ELISA method.Real-time PCR method was used to determine the transcription level of inflammatory factor m RNA.Western blotting was used to detect the effect of IB on the expression of key proteins in NF-?B and MAPK signals.Immunofluorescence experiment to observe the nuclear translocation of p65 protein after IB was administered to RAW264.7 cells.(2)The effect of IB on migration of macrophages to adipocytes was investigated.The Transwell chamber was used to examine the effect of IB on the migration ability of macrophages to adipocytes.The effects of IB on chemokines MCP-1 and MCP-1? produced by inflammatory adipocytes were detected by ELISA and real-time PCR.(3)The model of 3T3-L1 adipocytes inflammation was established and the effect of IB on adipocyte inflammation was assayed.The above method for establishing the insulin resistance model was used to differentiate preadipocytes into mature adipocytes,while TNF-? was used to establish a model of inflammatory adipocytes.The blank control group,model group,and drug treatment groups were set.After the cells were given different treatments,the supernatants were collected,and the effects of IB on inflammatory factors secreted by inflammatory adipocytes were detected by ELISA and real-time PCR.We studied the effects of IB on the major signaling pathways of inflammation JNK,NF-?B and SOCS-3 through western blotting.The results showed that IB significantly promoted glucose uptake in insulin-resistant adipocytes(P <0.001)compared with the model group.Western blotting analysis showed that IB could increase the expression of PI-3K/Akt insulin signaling pathway key proteins(PI-3K,p-IRS-1 and p-Akt)and promote GLUT4 membrane translocation,which means that IB could activate PI-3K/Akt signaling pathways to regulate glucose transport and affect glucose uptake,thereby ameliorating IR.The result of inflammationexperiments showed that IB could inhibit the infiltration of RAW264.7 cells into insulin-resistant adipocytes in vitro,the secretion of RAW264.7 celle inflammatory factors,and the phosphorylation of the key proteins in NF-?B and MAPK signaling(NF-?B/p65,ERK1/2,JNK and P38).At the same time,IB also significantly inhibited the secretion of inflammatory factors(TNF-?,IL-6 and IL-1?)and chemokines(MCP-1 and MCP-1?)in adipocytes,as well as the expression of JNK,NF-?B and SOCS-3-related proteins,which were the main signaling pathways of inflammation.In summary,IB could reduce the production of insulin resistance in adipocytes by improving glucose uptake and inflammation of adipocyte.Meanwhile,IB could improve the glucose uptake of IR adipocytes by activating PI-3K/Akt signaling pathway.And through the inhibition of JNK,NF-?B and SOCS-3 inflammation signaling pathways to improve 3T3-L1 adipocytes inflammation,and then play a role in improving 3T3-L1 adipocytes insulin resistance.