| Background:The number of patients with obesity and diabetes is increased year by year,so it is very important how to effectively prevent them.Traditional hypoglycemic drugs can effectively have hypoglycemic effects and increase body sensitivity to insulin,but they cannot improve islet function.The function could also decrease even with long term use.Glucagon-like peptide 1(GLP-1)and GLP-1-like peptide were the factors that could improve islet function and have better hypoglycemic effects at present.GLP-1 is a short peptide hormone secreted by intestinal epithelium L cells and is intestinal promoting pancreatic hormones.It has been proved that GLP-1 can promote the proliferation of islet ? cells and secretion of insulin,inhibit the apoptosis of islet ?cells and increase the sensitivity of muscle and liver cells to insulin.The Liraglutide and Exenatide(Extendin-4,EX4),GLP-1-like peptide,have long half-life,and their sequence were 53%homology with GLP-1 and they cannot be degraded by DPP-4.The EX4 is obtained from the poison gland of Helodema suspectum.They only have four times food one year,while their blood glucose is stable after engorgement.Some studies had revealed that EX4 had important roles in the controlling of blood glucose and appetite.Liraglutide and Exenatide have been applied in clinical and the effects are significant.While the GLP-1-like peptide are produced using chemical synthesis,and isolation and purification are needed.They can not be oral administration but only delivery by injection.Besides,their production has disadvantages as following:complex production technique,high cost and inconvenience.Bifidobacterium is one of the safest and most beneficial probiotics in the mammalian body,and it is the first batch residents in the human intestine and the main bacteria of infant intestine.Bifidobacterium can not only improve the microenvironment in intestine,inhibit the growth of pathogenic bacteria and improve the intestinal absorption,but also modify immune system,decompose toxic metabolism intermediate and prevent incidence of tumor.what GLP-1-like peptide expressed using Bifidobacterium might overcome the disadvantages of current production technique.Objective:The Bifidobacterium is as the host bacteria to secret the GLP-1-like peptide and treat diabetes by oral administration to confirm the hypoglycemic effects of transformed Bifidobacterium with GLP-1-like peptide.And we investigated the molecular mechanism of absorption and transport of GLP-1-like peptide in intestine.Methods:The expression vector was constructed on the basis of having constructed Bifidobacterium expressing system,and transformed Bifidobacterium were obtained with GLP-1 and EX-4 gene.And the expression of GLP-1 and EX4 and their biological activities were analysized in vitro.The diabetes mice model were used to analysis the hypoglycemic effects of transformed Bifidobacterium with GLP-1-like peptide in vivo;and the "U"-shape intestine model was used to investigate the molecular mechanism of absorption and transport of GLP-1-like peptide with fluorescent labeling in vivo.So it was especially important whether The GLP-1 and EX4 could be uptake by intestine.Besides,the transportation and the signal regulation were also investigated after observation and combination of GLP-1 and its receptor(GLP-1 R).The experimental methods were as following:(1)We selected shuttle plasmid pBBADs-EV that was constructed before to synthesize the GLP-1 and EX4.The pBBADs-GLP-1 and pBBADs-EX4 were constructed and identified,then plasmid power transformed into Bifidobacterium longum(NCC2705)and anaerobically cultured for 48h,then the positive B.longum was selected using MRS plates containing 60 mg/ml ampicillin.The positive colon of GLP-1 was named BL-GLP-1 and with EX4 was BL-EX4,and the empty vector was named BL-EV.The GLP-1(8G)and EX4 expression in vitro was identified by western blot and enzyme-linked immuniosorbent assay(ELISA)assay after L-arabinose induction for 12h,24h and 36h to find the best induction time.(2)The 50 T2DM model mice were selected that was induced using combination high-fat diet and STZ.They were randomly divided into five groups:T2DM group,Bifidobacterium with empty vector group(BL-EV group),the BL-GLP-1 transformed Bifidobacterium group(BL-GLP-1 group),the BL-EX4 transformed Bifidobacterium group(BL-EX4 group)and commercial EX4 group(10nmol/kg).The 0.2ml PBS were administered to NC group and T2DM group by gavage every day.The 0.2ml PBS with BL-EV,BL-GLP-1 and BL-EX4 induced by 0.2%L-Arb for 24 were administered respectively to BL-EV group,BL-GLP-1 group and BL-EX-4 group every day.The all groups were treated for 1 month.And 5 mice of each group were randomly selected to analysis the fasting glucose and OGTT.After 1 month,all mice were sacrificed after taking blood sample by extirpating eyealls after 12-hour fasting.The part normal colon,liver,kidney and islet were obtained in each group.(3)The NCM460 cells were used to detect the observation function of the mouse cells to GLP-1,in which the commercial GLP-1 and EX4 were used as positive control andCLP-1R was as inhibitor.The laser confocal was used to detect the absorption effects and ability of intestine to GLP-1.(4)The NCM460 cells were treated with GLP-1,EX4 and GLP-1 R for some time,and the changes of downstream signal factors,such as AKT-2 and cAMP,in CLP-1R signaling pathway were detected.Besides,the changes of PTP-1B were also detected.(5)The rat intestinal mucosa was made into U-type pond to stimulate the observation test of intestine to drugs.The GLP-1,EX4,GLP-1 R and GFP were added to detect whether they could be uptake by rat intestinal mucosa.(6)The GLP-1,EX4 and GFP were used to treat mice,then they were detected in other organs(intestine,liver,kidney,islet and hypothalamus)to show whether they could be transferred and all over the body.Results:(1)The transgenic NCC2705 with CLP-1 and EX4 was successfully constructed,and the selected and identified BL-GLP-1 and BL-EX4 had no significant changes in biology and morphology.The protein secreted by transgenic Bifidobacterium with induction of L-arabinose,and the best induction time was 24h.(2)The detection of mice fasting blood:the fasting blood glucose were detected by blood glucose machine after 1 week,2 week,3 week and 4 week.At 14 day,the fasting blood glucose was lower in the BL-GLP-1 group and T2DM group,comparing with BL-EV group.At 21 day,the fasting blood glucose of BL-GLP-1 group and T2DM group were lower than BL-EV group,and the BL-EX-4 group,EX-4 group were also lower than T2DM group and BL-EV group.At 28 day,comparing with T2DM group and BL-EV group,the fasting blood glucose was lower in the BL-GLP-1 group,BL-EX-4 group and EX group.The results showed that the fasting blood glucose was decreased after treating with BL-GLP-1,BL-EX-4 and EX-4.(3)The OGTT test:the weights of mice were weighted after detecting fasting blood glucose test.The OGTT was detected after agastric administration with 2g/kg glucose for 30min,60min,90min and 120min.The blood glucose values of each group were detected at Omin,30min,60min and 120min.At 14d,the results of OGTT suggested that the blood glucose values were lower in the BL-GLP-1 group and EX-4 group than BL-EV group and T2DM group at 1.5h,and BL-GLP-1 group and EX4 group are lower than BL-EV group and T2DM group at 2h.The BL-EX-4 group was lower than BL-EV and T2DM group.Its area under AUC curve suggested that the sensitivity to blood glucose of BL-GLP-1 group,EX-4 group and BL-EX-4group was improved;after 28d,the results of OGTT test showed that the blood glucose of in BL-GLP-1 group was decreased than BL-EV group and T2DM group;and the BL-EX-4 group and EX-4 group were lower than BL-EV group and T2DM group at 1h.Comparing with BL-EV group and T2DM group,the blood glucose value of BL-GLP-1 group and EX-4 group and BL-GLP-1 group and BL-EX-4 group were also decreased at 1.5h.Comparing with BL-EV group and T2DM group,the blood glucose values of BL-GLP-1 group and EX4 group were decreased as well as BL-EX-4 group at 2h.Its area under AUC curve suggested that the sensitivity to blood glucose have improved in the BL-GLP-1 group,BL-EX-4 group and EX-4 group.(4)Absorption results of intestine cells in vitro:the GLP-1 and EX4 labeled with rhodamine could uptake into NCM460 while GFP could not,which confirmed that the GLP-1 and EX4 were uptake by combining with the GLP-1R.(5)The western blot results of GLP-1 signal patway moleculars:the P-AKT-2(s473)was increased,while PTP1B was decreased.Besides,the cAMP was increased detected by ELISA.These results showed that GLP-1 and EX4 repaired islet,promoted the proliferation of islet ? cells and inhibited the apoptosis of islet ?cells.Besides,they inhibited the expression of the PTP-1B.So we guessed that CLP-1 and EX4 might promote the secretion of the insulin by inhibiting the activity of the PTP-1B.(6)The results of U-type pond absorption and transport:the GLP-1 and EX4 could be detected at two side of the U-type pond.And the fluorescent of rhodamine was detected under laser scanning confocal microscope.These results revealed that GLP-1 and EX4 transferred by combining with the GLP-1 R.(7)The small intestine,liver,kidney,pancreas and hypothalamus of mice were obtained to detect the fluorescent of rhodamine.We found that the red fluorescent of rhodamine was detected in these organs.These results suggested that the GLP-1 and EX4 could be transported to every organ of body to play their roles.Conclusion:In this study,we succeeded constructing and identifying the Bifidobacterium with GLP-1 and EX4.And the target peptides could express after induction in vitro.The expression products could promote the proliferation of the INS-1 cells and stimulate the secretion of insulin.Besides,they could decrease blood glucose level and insulin sensitivity of diabetes mice model in vivo.The GLP-1 and EX-4 might be uptake depending on medication of GLP-1R through intestine mucosa.The production technique was simple that was by Bifidobacterium to express GLP-1-like peptide,and the bacteria could be taken by oral.The GLP-1-like peptide could not only effect on the intestine mucosa,but also uptake depending on medication of GLP-1R through intestine mucosa to play its roles in body.Therefore,it might be a new way to treat diabetes. |
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