| The incidence and fatality rate of Type ? diabetes mellitus(T2DM)shows a growth trend worldwide year by year.It is recognized as the world's third largest fatal disease and has been a serious threat to human health.Recently,GLP-1 has attracted much attention as a new target of antidiabetic drugs.Synthetic Exendin-4(Exenatide)was approved as the first GLP-1 analog for T2DM treatment by the Food and Drug Administration(FDA).However,as a kind of chemical synthetic drug,it is very expensive.And continuous subcutaneous injection is required,which will bring great pain to the patient,however,until now,no oral form of this product on the market.Lactic acid bacteria(LAB)are safe and natural,and have been regarded as the most desirable oral delivery vector for functional peptides or proteins.Using Lactococcus lactis as oral vaccine delivery vector to express antigenic genes of virus or bacteria has shown promising prospects in recent years,while the use of LAB as oral Exendin-4 delivery vector for the management of type ? diabetes has not yet been reported.In this study,genetic engineering technology was employed to construct engineered LAB for the recombinant Exendin-4 expression and the bioactivities of oral delivery Exendin-4 were further verified in vitro and in vivo.This study provides new evidences for using probiotics as the oral delivery system for diabetes management.(1)The feasibility of using L.lactis as the Exendin-4 delivery vector was explored.The results showed that Exendin-4 was successfully expressed and secreted by L.lactis and its bioactivities were verified in vitro.The recombinant Exendin-4 significantly enhanced the insulin secretion of INS-1 cells and also promoted proliferation and inhibited apoptosis by activating the PI3K/AKT signal pathway to upregulate the protein expression of AKT and p-AKT.(2)The potential synergistic hypoglycemic probiotic lactobacilli were screened from Lactobacillus in the laboratory.21 Lactobacillus strains were screened for potential novel new probiotics based on DPP-? and a-glucosidase inhibitory activity,and also some basic features of probiotics including GIT tolerance and intestinal epithelial cells adhesion.The results indicated that strains L.paracasei L.plantarum ZF06-1,ZF06-3,IF2-14 and L.brevis IF2-17 showed better potential probiotic properties and antidiabetic effects.(3)The strain L.paracasei L14 with potential hypoglycemic effect was used as a delivery vector of Exendin-4 and the functions of recombinant Exendin-4 were verified.The results showed that heterologous expression and secretion of functional Exendin-4 was successful in L.paracasei L14.Recombinant Exendin-4 expressed by L.paracasei L14 showed significant increase in insulin biosynthesis and secretion of INS-1 cells by activating the transcription factor PDX-1 and also enhanced proliferation and inhibited its apoptosis by activating the PI3K/AKT signal pathway.The transport and absorption ability of Lactobacillus delivered Exendin-4 through cell monolayer was also investigated.The results showed that the transport rate of Exendin-4 delivered by the live Lactobacillus delivery system was significantly increased,which was 34-fold higher than that of the standard Exendin-4 solution,indicating that recombinant Exendin-4 delivered by the live Lactobacillus delivery system can be transported and absorbed by intestinal epithelial cells.(4)The preventive and therapeutic effects of Exendin-4 expressed engineered strain(L.paracasei L14/pMG76e-Exendin-4)on a T2DM rat model were studied.The results showed that the engineered strain could significantly improve the T2DM metabolic syndrome in the prevention and treatment groups of T2DM rats,including lowering blood glucose,increasing glucose tolerance,decreasing insulin resistance and improving the function of islet ? cells.These results indicated that the recombinant Exendin-4 delivered by L.paracasei L14 was functional in vivo and it has been confirmed for the first time that the live Lactobacillus strain can be used as functional Exendin-4 delivery vector to play a key role in the prevention and regulation of T2DM rat model.In addition,L.paracasei L14 itself has good preventive effect on diabetes in prevention groups,for example,it significantly decreased blood glucose,and regulated the balance of blood lipid,and also improved the body inflammation state and antioxidant status and exerted beneficial effect for the protection of liver and pancreas against the impairment.However,the regulation effects of L.paracasei L14 itself on the treatment group rats were not significant. |
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