Effects And Mechanism Of RhGLP-1 On Osteogenic Differentiation In HPDLSCs Induced By AGEs

Objective:The purpose of the study was to examine the reversal effect of rhGLP-1 on the decreased osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)induced by advanced glycation end products(AGEs)and the regulation of PKC and PKA signaling pathwaysMaterials and Methods:(1)The activity of the hPDLSCs was identified using osteoplastic differentiation.The sternness of hPDLSCs was characterized by scanning cell surface markers(CD34,CD73,CD90,CD105,Vimentin,and CK19)through flow cytometric analysis.(2)CCK-8 was used to detect the proliferation effect of AGEs on hPDLSCs and to screen the appropriate AGE concentration.Different concentrations of AGEs induced osteogenic differentiation of hPDLSCs according to ELISA detection of Col-I expression and real-time PCR,and Western blot detection of AGEs induced 7 days of BSP,runx2 and OPN expression;ALP quantitative analysis was used to analyze the ALP expression level.(3)CCK-8 was used to detect the proliferation effect of rhGLP-1 on hPDLSCs and to screen the appropriate rhGLP-1 concentration.ELISA was used to detect the expression of Col-I on 7 days of rhGLP-1 intervention.Real-time PCR and Western blot were used to detect the expression of BSP,Runx2 and OPN on 7 days of rhGLP-1 intervention.ALP quantitative analysis,alkaline phosphatase staining and alizarin red staining were used to analyze the mineralization.The expression of key proteins including RAGE,pPKA,PKA,pPKCβ1/2,and PKCβ1/2 in the PKC signaling pathway and the PKA signaling pathway was simultaneously detected.(4)PKC inhibitors and agonists were used to perform ALP quantitative analysis,ELISA was used to detect the expression of Col-I after 7 days,and real-time PCR,Western blot and immunofluorescence detection were used to determine the interference with the 7-day expression of BSP,Runx2 and OPN.Real-time PCR detected PKC,the PKA signaling pathway-related gene expression,while Western blot and immunofluorescence were used to detect the expression of key proteins such as RAGE,pPKA,PKA,pPKCβ1/2,and PKCβ1/2.Results:(1)hPDLSCs were cultured,and osteogenesis was successfully induced.The mesenchymal stem cell surface markers were positive,CD90 was positive(98.80%%±1.16%),Vimentin was positive(98.96%±0.89%),CD73 was positive(95.10%±2.81%),CD105 was positive(91.70%±3.50%),hematopoietic stem cell surface markers were negative,CK19 was negative(3.51%±1.92%),and CD34 was negative(6.83%±1.57%).(2)AGEs could significantly inhibit the proliferation of hPDLSCs in a concentration-time-dependent manner.AGEs could significantly inhibit the osteogenic differentiation ability of hPDLSCs.The expression levels of ALP,Col-I,BSP,Runx2 and OPN were significantly lower than those in the simple induction group after 7 days of osteogenic induction(p<0.05).The higher the concentration of AGEs was,the lower the osteogenic-related gene and protein expression levels were(3)After adding 10-8 M rhGLP-1,we found that rhGLP-1 could partially reverse the decline in the osteogenic differentiation ability of PDLSCs caused by AGEs,increase the expression of ALP,Col-I,BSP,Runx2 and OPN osteogenic-related genes and proteins,and increase mineralization deposits(p<0.05).RhGLP-1 has a certain protective effect on the osteogenic differentiation ability of hPDLSCs.(4)With the effect of AGEs,the expression of RAGE in hPDLSCs increases,the phosphorylation of PKC-β2 is enhanced,and the phosphorylation of PKA is blocked(p<0.05).In contrast,the addition of rhGLP-1 can significantly increase the phosphorylation of PKA in hPDLSCs,reduce the expression of RAGE,and inhibit PKC-β2 phosphorylation.(5)Administration of PKC inhibitors can mimic the effect of rhGLP-1,while PKC agonists can block the effect of rhGLP-1,suggesting that rhGLP-1 may improve the osteogenic differentiation of hPDLSCs induced by AGEs through the PKC and PKA pathways.Conclusions:(1)AGEs can inhibit the proliferation and osteogenic differentiation of hPDLSCs and reduce the expression of the osteogenic factors ALP,Col-I,BSP,Runx2 and OPN in hPDLSCs.(2)AGEs may inhibit the osteogenic differentiation of hPDLSCs by enhancing PKC-β2 phosphorylation.(3)rhGLP-1 can promote the osteogenic differentiation of hPDLSCs and increase the expression of osteogenic factors ALP,Col-1,BSP,Runx2 and OPN in hPDLSCs.(4)rhGLP-1 can partially reverse the osteogenic differentiation of hPDLSCs caused by AGEs.The main mechanism is that rhGLP-1 simultaneously inhibits the PKC signaling pathway and activates the PKA signaling pathway.