The Mechanism Research Of Glucagon-like Peptide-1Receptor Agonist Exendin-4on Decreasing Osteoporosis In Old Rats

Objectives:Osteoporosis (OP) is a common disease occurs in the older people.1in3women and1in5men are at risk of osteoporotic fracture around the world in people above50yearsold. Various types of fractures are the most common and serious complications ofosteoporosis. It can not be ignored that the number of osteoporotic fractures are more thanthe sum of myocardial infarction and stroke in United States, in addition the mortality rateof hip fractures was up to24%. With the increasingly serious situation of the populationages, osteoporosis has become the global public health problem. It is the pathological mechanism of osteoporosis that the broken balance between bone formation and boneabsorption, which promotes the ability of bone absorption leading to bone loss andfracture. At present, most drugs in osteoporotic treatment just inhibit the bone absorptionbut can’t restore the lost bone mass and bone structure. Therefore, it is essential to explorea new drug to prevent the osteoporosis.Glucagon-like peptide-1(GLP-1) is an essential hormone. Our previous study showsthat GLP-1receptor agonists Exendin-4(Ex-4) can significantly increase bone mineraldensity in ovary (OVX) rat osteoporotic model. But GLP-1receptors can’t be found inosteoblast and osteoclast suggesting that GLP-1may promotes bone formation by indirectmechanism. Considering that the bone marrow mesenchymal stem cells (BMSCs) candifferentiate into osteoblasts, BMSCs have GLP-1receptors, thus we hypothesize thatGLP-1can promote the osteoblast differentiation of BMSCs, and promotes boneformation improving the osteoporosis by GLP-1receptors on the surface of theBMSCs.In order to verify the hypothesis, the molecular mechanism of GLP-1receptor onosteoblast differentiation of BMSCs was tested, and the effect on inhibiting osteoporosis.Methods:1. The determination of rat bone biomechanics and morphology: Male SD rats (4monthsand12months,20months) given Ex-4for12weeks were analyzed by Micro-CT toobserve the changes of bone mineral density (BMD) and bone structure. Three pointbending test is adopted to test the bone biomechanics; calcein and tetracyclinedouble-labeled fluorescence were used to observe the mineral deposit rate (MAR) andbone formation rate (BFR); HE staining and toluidine blue staining were used todeterminate the number of osteoblasts and adipocytes.2. The detection of biochemical markers on rat bone metabolism: Rats were put inmetabolic cages before death, fasting, but water is available, urine was collected for24h, centrifuged to collect the supernatant; blood was collected for5ml throughabdominal aorta after death, centrifuged to collect serum. Automatic biochemicalanalyzer to determine the levels of serum Ca, P, urine Ca, P; Serum levels of bonealkaline phosphatase (BALP) and osteocalcin (OCN) were measured by ELISA kits. 3. BMSCs isolation and cell culture: The femurs and tibias were separated from the3weeks old SD rat, and bone marrow mesenchymal stem cells (BMSCs) were isolatedfrom the femurs and tibias. The cells were collected in α-MEM culture mediumsupplemented with10%fetal bovine serum (FBS, Gibco),100U/ml penicillin,100mg/ml streptomycin in a humidified atmosphere of95%air and5%CO2at37°C. TheBMSCs were a relatively pure population of stromal cells that were negative for CD45and positive for CD29, and the osteogenic and adipogenic induction was to observe thedifferentiation ability of BMSCs.4. The analysis of GLP-1receptors: GLP-1receptors antibodies and Hoechst were usedto analyze the BMSCs by double-labeled fluorescence, the laser confocal microscopewas used to detect the GLP-1receptors; RT-PCR, Western blot were used to detect thegene and protein expression of GLP-1receptors.5. The effect of Ex-4on the proliferation and differentiation of BMSCs: Differentconcentrations (10-9,10-8,10-7M) of GLP-1agonists were given to BMSCs forproliferation analysis, which was determined by MTT; BMSCs cultured in theosteogenic culture medium (containing10%FBS,10mm beta glycerin, sodiumphosphate,10nm dexamethasone50μg/L of vitamin C alpha MEM) for21d to detectthe calcified nodules by alizarin red staining, the expression of Runx-2and Oc weredetected by RT-PCR to observe the differentiation of BMSCs; BMSCs cultured in theadipogenic induction medium (containing10%FBS,5ug/ml insulin,1mMdexamethasone, IBMX0.5mM,100microns of indomethacin alpha MEM) for17d todetermine the lipid droplets by oil red O staining, the expression of Pparγ and C/ebpαwere detected by RT-PCR to observe the inhibition effect of Ex-4on adipogenicdifferentiation.6. The detection of signaling molecules on BMSCs differentiation: The BMSCs weretreated with10-8M Ex-4for0h,1h,2h,4h,8h,24h, and the expression ofbeta-catenin in cytoplasm and nuclei were detected by Western Blot; GLP-1receptor(GLP-1R) was knocked down by infected with lentiviruses encoding small hairpinRNA (shRNA).The mRNA and protein levels of GLP-1R and beta-catenin were detected by RT-PCR and Western Blot; adenylate cyclase activator Forskolin, PKAinhibitors H-89, GLP-1r antagonists Ex(9-39) were given with Exendin-4to test theexpression of β-catenin and PKA.Results:1. Exendin-4inhibits the osteoporosis in old rats: Micro-CT analysis results showedthat the bone mineral density decreased significantly, the bone trabecularmicrostructure was serious damaged in the older group of rats (20months) comparedwith young rats (4months),but Ex-4can significantly increased bone mineral density,improved the destruction of the bone trabecular microstructure; Exendin-4cansignificantly improve the biomechanical characteristics of the older group of ratsfemur, enhance the mechanical strength of femur determined by three point bending;biochemical analysis results showed that Ex-4can significantly reduce urine calciumphosphorus loss, elevate serum BAP and OCN levels, inhibit osteoclast activity,promote the osteoblast activity in the older group of rats; the bone tissue morphologyshowed that Ex-4can improve the BFR and MAR, increased the number of osteoblastsin bone marrow, reduced the number of adipocytes in the bone marrow, promoted boneformation.2. Exendin-4promote the proliferation and the osteogenic differentiation of BMSCs,inhibit the adipogenisis of BMSCs: RT-PCR, Western Blot and immunofluorescencemethod were used to confirmed the expression of GLP-1receptor on BMSCs, theresults showed that GLP-1R expresses on BMSCs. Compared with the control group(OIM), the treated group showed that GLP-1and Ex-4can obviously promote theproliferation of BMSCs in dose dependent manner by MTT. Adipogenisis relatedgenes Pparγ, C/ebpα were inhibited by Exendin-4according to the results of RT-PCR,osteogenesis related gene Runx2, Oc were increased, suggested that it promoted theosteogenic differentiation of BMSCs, inhibited the adipogenic differentiation.3. The regulation of GLP-1receptor signaling pathways in differentiation andmolecular mechanism: With the extension of time, the expression of beta-catenin incytoplasm gradually increased, the expression of beta-catenin in nucleus increased gradually, shows that Exendin-4can promote beta-catenin transposition into thenucleus. Adenylate cyclase activator Forskolin promote the expression of PKA incytoplama and the expression of beta-catenin in nucleus; PKA inhibitors H-89orGLP-1R antagonist Ex(9-39) decreased the expression of PKA in cells andbeta-catenin in nucleus. The expression of PKA in cytoplama and beta-catenin innucleus was decreased in GLP-1R knockdown cells. The results showed that Ex-4canactivate Wnt signaling pathways by activated PKA, promote beta-catenin to transportinto nuclear, activate the downstream osteogenetic differentiation related genes,accelerate the osteogenic differentiation of BMSCs.Conclusions:1. Ex-4can significantly inhibit the osteoporosis of rats caused by age, increase bonemineral density, bone biomechanical properties, serum ALP and OCN, osteoblastnumber in bone marrow, indicating that Exendin-4plays a role in anti-osteoporosisand promotes bone formation.2. GLP-1receptors express on BMSCs. Activated GLP-1receptors can accelerate theosteogenic differentiation of BMSCs, inhibit adipogenic differentiation.GLP-1receptors regulate osteogenic differentiation may be through the Wnt/beta-cateninsignaling pathway.