| Objective Type 2 diabetes(T2DM)is an increasingly common metabolic disease.With the continuous improvement of people’s living standards,more and more patients have been diagnosed with T2 DM,which is a very important public health issue.T2 DM usually leads to an increase in blood lipid levels,especially triglyceride(TG)levels,which are further exacerbated by the secretion of triglyceride rich chylomicrons(CM)after meals.At present,the main research on dyslipidemia in T2 DM has focused on liver lipid metabolism,with little research on the metabolism of intestinal chylomicrons.Meanwhile,metformin(Met)has been found to have a positive effect on blood lipid abnormalities through numerous studies.Therefore,this topic is mainly to explore the effect of metformin on the assembly of intestinal chylomicrons in type 2 diabetes mice and the preliminary exploration of its related mechanisms.Methods(1)The mice used in this experiment were C57BL/6J and C57BLKS/J-db/db.Eight C57BL/6J mice were selected as normal control group(C57).Sixteen C57BLKS/J-db/db mice were randomly divided into two groups of eight mice each: type 2 diabetes control group(db/db)and metformin intervention group(db/db met).Referring to the literature of Memmott RM et al.,the dose of metformin per mouse is 250 mg/(kg · d),and the intervention time is 12 weeks.During the 12 weeks,the fasting blood glucose,body weight,diet and drinking water of mice were measured and recorded every week until the end of the experiment.After the intervention of metformin,the mice were fasted for 6 to8 hours,and then the mice were dissected to retain the corresponding samples.Serum samples were collected to detect relevant blood indicators,while intestinal tissues were collected to use Western blotting technology to detect the expression of relevant proteins affecting the assembly of chylomicrons.(2)Using HIEC-6 human intestinal epithelial cells,the intervention concentrations of metformin were 0.5,1,1.5,2,and 3 m M respectively,and HIEC-6 human intestinal cells were interfered for one day.At the end of 24 hours of drug intervention,samples were collected to detect the content of total cholesterol(TC),triglyceride TG,apolipoprotein B48(Apo B48)and non-esterified fatty acids(NEFA)in cells and culture medium.Western blotting technology was used to detect the expression of related proteins affecting the assembly of chylomicrons.(3)In vitro,HIEC-6 human normal intestinal epithelial cells were transfected with WDR12 si RNA.After WDR12(WDR12)knockdown,Western blotting was used to detect the effect of WDR12 knockdown on chylomicrons assembly-related proteins.(4)The WDR12 plasmid was transfected into HIEC-6 human normal intestinal epithelial cells in vitro.At the same time,the overexpression cells were treated with metformin for24 hours on this basis,and then the samples were taken.Western blotting was used to detect the expression level of the proteins related to the chylomicrons assembly under the conditions of transfection of the WDR12 overexpression plasmid and treatment with metformin after the WDR12 overexpression plasmid.Results(1)According to the animal experiment results,after 12 weeks of metformin treatment,the body weight and fasting blood glucose of type 2 diabetes mice,and the levels of TG,TC,NEFA,Apo B48 in serum and intestinal tract were significantly reduced.Western blot experiment showed that metformin could significantly reduce the protein levels of CD36(fatty acid transporter CD36,CD36),microsomal triglyceride transporter(MTTP),Niemann Pick C1 like protein 1(NPC1L1),Apo B48 in the intestinal tract of type 2diabetes mice.(2)The levels of TG,TC,NEFA and Apo B48 in the cells and culture medium were significantly decreased after the intervention of metformin in human intestinal epithelial cells at the sixth stage.The Western blot experiment showed that the intervention of metformin would reduce the protein levels of CD36,MTTP,NPC1L1 and Apo B48 in the cells.(3)At the 6th stage in vitro,human intestinal epithelial cells transfected with WDR12 si RNA were detected by Western blotting,and the protein levels of WDR12,CD36,MTTP,NPC1L1,and Apo B48 were decreased.(4)At the 6th stage in vitro,human intestinal epithelial cells were transfected and overexpressed at the 12 th week.The protein levels of WDR12,CD36,MTTP,NPC1L1,and Apo B48 were all increased by Western blotting.However,the elevated levels of these proteins would be significantly weakened after treatment with metformin on the basis of the 12 th week of overexpression.According to the results of animal experiments,compared with the mice in the normal control group,the weight,fasting blood glucose,serum TG,TC,NEFA,Apo B48 and other indicators of the type 2 diabetic mice increased.The proteins fatty acid transporter CD36,microsomal triglyceride transfer protein,Niemann pick C1 like 1,Apo B48 expression also increased.After the intervention of metformin,a series of indexes such as body weight,fasting blood glucose and blood lipids of diabetes mice have been improved,and the expression of related proteins has also been reduced.The results of cell experiments showed that metformin could also improve the corresponding biochemical indicators in cells,and the expression of proteins related to the assembly of chylomicrons also decreased.Conclusion(1)Metformin can effectively reduce the assembly of intestinal chylomicrons in type 2diabetes mice.(2)Metformin may reduce the expression of CD36,MTTP,NPC1L1,Apo B48 and other proteins by down-regulation of WDR12,and ultimately reduce the assembly of chylomicrons in type 2 diabetes mice... |
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