Bactrian Camel Milk-derived Exosomes Improving The Insulin Resistance Of L6 Rat Myoblasts

The therapeutic effect of camel milk on type 2 diabetes mellitus(T2DM)has been demonstrated and camel milk has been shown to improve various diseases and health outcomes,presumably in relation to exosomes(EXO).However,there are no reports or studies on the effect of Bactrian camel milk-derived exosomes on the improvement of T2 DM,so the following experiments were conducted to reveal the potential mechanisms underlying the regulation of T2 DM by Bactrian camel milk-derived exosomes.The methodological results are as follows.(1)Bactrian camel milk exosomes were isolated by ultra-high speed centrifugation,and their morphological characteristics were identified by transmission electron microscopy(TEM)and total protein concentration was determined by BCA.The Bactrian camel milk exosomes were observed to be enriched with a large number of nano-vesicles with a double-layered lipid membrane structure,averaging 70-100 nm in diameter,round or ovoid in the shape of a cupola,with a homogeneous black stained centre and a lightly stained white membrane with clear edges.The total exosome protein concentration was determined to be 18.6404±1.7297 μg/μL(2)Insulin resistance(IR)L6 rat myogenic cell model was established by using different concentrations of palmitic acid(PA),and validated by using the Glucose Oxidase(GOD)method and adding insulin to the control group to detect the remaining glucose concentration in the supernatant,and the MTT method to detect cell The survival rate of the cells was verified by using the Glucose Oxidase(GOD)method,adding insulin to the supernatant to test the remaining glucose concentration in the control group and MTT to test the survival rate.(3)The isolated exosomes were diluted in a gradient manner and incubated for 4,8,12 and 24 h.The remaining glucose concentration in the supernatant was measured by GOD,the mean fluorescence intensity of 2-NBDG was observed under fluorescence microplate reader to quantify the glucose uptake and the cell survival rate was measured by MTT.Compared with the control group,cellular glucose consumption and mean fluorescence intensity,i.e.cellular glucose uptake,were significantly higher(P<0.05)after 12 h of exosome concentration of 200 ng/m L(40 ng/well)acting on IR L6 cells,and both cell culture medium and intracellular glucose were significantly decreased,thus successfully achieving IR slow release.(4)The EXO-treated and control groups were subjected to RNA-Seq to screen for genes and metabolic pathways regulated by EXO,and differentially expressed genes(DEGs)were screened,and GO enrichment and KEGG enrichment analyses were used to annotate gene functions and gene pathways of the differentially expressed genes.The study was carried out by using GO enrichment and KEGG enrichment analysis to annotate gene functions and gene pathways.Screened out 24 genes related to glucose metabolism and IR,namely Fos,Gbe1,Rock1,Pcm1,ND2,Inhbe,ND4,ND5,Clec4 e,ND4L,Glut3,E2 B,Rxra,Senp1,Hipk2,Selenon,Vash1,Erbb2,Pim1,Ksr1,Sall2,Klf13,Foxo1,Gstm3.Combined with KEGG pathway analysis,it can be found that EXO improves IR pathways including: AGE-RAGE signaling pathway,TGF-βsignaling pathway,MAPK signaling pathway,insulin signaling pathway,etc.