Effect Of Liraglutide And PFGF21 Via NH2-EDMSNs Nanoparticles On Glucose Meta Bolism In Insulin Resistance Mice

Part1 Biological efficiency of NH2-EDMSNs/pFGF21 and the safety of nanoparticles in vivoObjective: To explore the best quality of NH2-EDMSNs/pFGF21 and the best time to exert its effect in vivo.To compare the transfection efficiency of NH2-EDMSNs with hydrodynamic delivery in vivo.Validate the biological safety of nanomaterials in vivo.Methods: The injection of NH2-EDMSNs/pFGF21 was performed to 20-week-old C57/BL6 J mice through tail vein.PCR and Western Blot were used to find out the best plasmid usage and the best time.The mice were divided into the following five groups: saline group,NH2-EDMSNs group,pFGF21 group,pFGF21 with hydrodynamic delivery group,NH2-EDMSNs/pFGF21 group to explore the transfection efficiency of NH2-EDMSNs in vivo.The biosafety of the nanomaterial was evaluated by circulating AST and ALT and HE staining of liver and kidney in saline group,NH2-EDMSNs group,Lira + pFGF21 group and NH2-EDMSNs/Lira + pFGF21 group.Results: The optimal transfection efficiency was achieved when the amount of FGF21 overexpression plasmid was used at 50?g.The m RNA of FGF21 reached the highest on the 5th day,but the protein level reached the highest on the 7th day.There was no statistical comparison between the 9th day and the 7th day(P>0.05).The 7th day is chosen as the best time.The results showed that the m RNA and protein expression of FGF21 in mice transfected by NH2-EDMSNs/pFGF21 was higher than those with the hydrodynamic delivery of pFGF21.Therefore,these data indicated that the transfection efficiency of NH2-EDMSNs/pFGF-21 was significantly higher than that of the hydrodynamic delivery of plasmid in vivo.There was no statistical difference of AST and ALT values of each group and no tissue damage was found in HE staining.Conclusion: NH2-EDMSNs is a safer transfection vector in vivo.Part 2 Effects of NH2-EDMSNs/Lira/pFGF21 on glucose metabolism in insulin resistant miceObjective: To study the improvement of energy metabolism and glucose metabolism in insulin-resistant mice by the combined use of liraglutide and the over-expressing FGF21 plasmid,and to study the feasibility of using a new type of porous silica nanomaterial NH2-EDMSNs.Methods: The insulin resistance model mice were fed with high-fat diet for 12 weeks,and then divided into the following 8 groups for tail vein injection: saline group(saline),saline and NH2-EDMSNs group(NH2-EDMSNs/saline),liraglutide group(Lira),liraglutide and NH2-EDMSNs group(NH2-EDMSNs/Lira),FGF21 overexpression plasmid group(pFGF21),pFGF21 and NH2-EDMSNs group(NH2-EDMSNs/pFGF21),liraglutide and pFGF21 combined group(Lira + pFGF21),and liraglutide and pFGF21 with NH2-EDMSNs group(NH2-EDMSNs/Lira + pFGF21).Changes in body weight,food intake,and rectal temperature were monitored after injection for 9 days,and the respiratory quotient was measured at the optimal time to explore its effect on energy metabolism.Also,fasting blood glucose was measured and GTT and ITT were performed to explore its effect on glucose metabolism.Results: Under HFD feeding,compared with the saline group,the liraglutide group can significantly increase rectal temperature,reduce weight and food intake(P <0.05 or P <0.01);and the group with NH2-EDMSNs shows more significance reduction in body weight and food intake(P <0.05 or P <0.01).In the pFGF21 group,the group using only NH2-EDMSNs showed an increase in rectal temperature and a decrease in body weight,and there was no difference between the changes in food intake and the saline group.In the combination of liraglutide and pFGF21,there were significant statistical differences in rectal temperature rise,food intake and weight loss,and the group with NH2-EDMSNs shows greater significance(P <0.05 or P <0.01).In the measurement of indirect energy metabolism,except for the mice that were given the pFGF21 naked plasmid,the other groups show a significant increase in VO2 and a decrease in RER compared with the control group(P <0.05 or P <0.01).In each of the different drug interventions,the use of NH2-EDMSNs can show a more obvious trend(P <0.05 or P <0.01),especially the pFGF21 group and the combination of liraglutide and pFGF21 group.In the fasting blood glucose measurement,except for the pFGF21 naked plasmid group,the other intervention groups were significantly reduced compared with the control group(P <0.01),and liraglutide and/or pFGF21 carried by NH2-EDMSNs were more directly injected than They showed a more significant reduction trend(P <0.01).During GTT and ITT,these groups showed significantly lower blood glucose curves and areas under the curve than the control group(P <0.01).Conclusion: NH2-EDMSNs can be an effective carrier of liraglutide and pFGF21 in vivo,and the combined treatment shows better improvement of insulin resistance.Part 3 The effect of NH2-EDMSNs/Lira/pFGF21 on important factors of hepatic glucose metabolism in insulin resistant miceObjective: To study the effect of NH2-EDMSNs loaded with liraglutide and/or pFGF21 on insulin resistance mice in the liver's important glycolmetabolism pathway IRS-1/PI3K/AKT and key gluconeogenesis enzyme G-6-Pase.Understand the molecular biological effects of nanomaterial transfection.Methods: After the second part of C57/BL6 J mice completed the measurement of energy metabolism and glucose metabolism,the mice were sacrificed.Western Blot was used to observe the differences in protein expression of IRS-1,PI3 K,AKT,and G-6-Pase in the livers of mice in each group.Results: After intervention treatment,the phosphorylation level of IRS-1,PI3 K and AKT increased,the expression level of G-6-Pase decreased,and the trend change after the use of nanomaterials was more significant(P <0.05 or P <0.01).In addition,the combined use of liraglutide and pFGF21 showed the most obvious molecular biological effects(P <0.01).Conclusion: The use of NH2-EDMSNs to carry liraglutide and pFGF21 has a greater effect on insulin-related signaling pathways than unused materials.Among them,the biological effect of combined use of liraglutide and pFGF21 is stronger than single use.