| Backgroud and ObjectivesPeriodontal disease is a chronic inflammatory disease with high incidence,bacterial infection often causes the destruction of periodontal soft and hard tissues,which leads to the loosening and falling out of teeth,which seriously affects the chewing function,quality of life,and physical and mental health of patients.The existing traditional periodontal treatment methods,such as cleaning,scraping,root surface leveling and other basic treatments,can only eliminate the etiology,but fail to make the lost periodontal tissue get ideal regeneration.Periodontal tissue regeneration is an effective measure for the treatment of periodontal disease and is the common goal of multidisciplinary field research in stomatology.In recent years,although the clinical application of guided tissue regeneration and periodontal bone grafting has improved the regeneration ability of periodontal tissue to a certain extent,the effectiveness of these treatment methods depends on the number and function of remaining mesenchymal stem cells.for this reason,they have a certain degree of their limitations.With the rapid development of stem cells and tissue engineering,periodontal tissue engineering technology dominated by dental stem cells has provided a more scientific and effective method for the treatment of periodontal disease.Human periodontal ligament stem cells(hPDLSCs)were successfully isolated and cultured since 2004 by Seo and other scholars using the technology of single cell cloning technology.hPDLSCs are a group of mesenchymal stem cells derived from the periodontal ligament tissue.Because of their biological advantages of easy drawn and expansion,as well as their physiological self-replication ability,self-renewal and multi-directional differentiation potential,they have been defined as promising alternative cell sources for repairing missing periodontal tissues.The ability of hPDLSCs on improving the biological regeneration of periodontal tissues has provided important clinical application value for various oral clinical specialties,such as periodontology,prosthetics,orthodontics and maxillofacial surgery.hPDLSCs have also received extensive attention from researchers in various fields of stomatology.As is known to all,periodontal disease and diabetes mellitus are two diseases which are harmful to health with a high incidence.Meanwhile,they are related to each other,and the relationship between them is a hot topic for researchers in Stomatology and other related fields.Diabetes mellitus,which was in high incidence of periodontal disease and severes damage to periodontal tissues in diabetics,has been proved to be a major risk factor for periodontal disease.Perfect periodontal treatment can improve or delay the occurrence and development of diabetes mellitus to some extent.Advanced glycation end products(AGEs)is one of the irreversible non-enzymatic proteins formed by persistent hyperglycemic stimulation in diabetic patients.It has been reported that AGEs plays an important role in many inflammatory responses and can significantly increase accumulation levels in diabetic patients’plasma.Given that the biological properties of hPDLSCs are affected by different environmental factors,it is suggested that AGEs may aggravate the damage and delay repairment of periodontal tissues in diabetic patients by affecting the osteogenic differentiation potential of hPDLSCs.When the osteogenic differentiation potential of hPDLSCs is affected,it is the focus of this study to promote the osteogenic differentiation ability of cells and thus promote the repair and regeneration of damaged periodontal tissues.At present,berberine hydrochloride(BBR)has attracted more and more attentions in the field of medicine.BBR,with its 2,000 years of medicinal history,is the novel drug used in this study.It can exert the biological activities of antibacterial,anti-inflammation,lipid-lowering and anti-diabetic.In recent years,scholars at home and abroad have conducted a large number of studies on BBR’s ability to promote and improve osteogenic differentiation of bone marrow mesenchymal stem cells and other cells,but there have no reports on BBR’s function and molecular regulatory mechanism of osteogenic differentiation of hPDLSCs under the disturbance of AGEs.The multi-directional differentiation potential of cells can be affected by the influence of different signaling pathways.Since the osteogenic related genes such as Runx2 are downstream target genes of the canonical Wnt/β-catenin pathway,it can be proved that the canonical Wnt/β-catenin pathway plays a crucial role in bone formation process and regulation.Therefore,we speculated that in the diabetic microenvironment,the canonical Wnt/β-catenin pathway may be involved in BBR’s promotion for osteogenic differentiation potential of hPDLSCs,which finally leads to the restoration of damaged periodontal tissues.In this study,hPDLSCs were cultured in vitro as the basis,the hPDLSCs were firstly subjected to osteogenic induction and osteogenic differentiation ability identification,which proved that the cells had the potential to differentiate into osteoblasts.Then,alkaline phosphatase(ALP)staining,ALP activity assay,alizarin red staining,enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot analysis were conducted to investigate the related indexes of osteogenic differentiation of hPDLSCs,in order to further explore the effect of AGEs on the osteogenic differentiation potential of hPDLSCs and the effect of BBR’s application on the osteogenic differentiation potential of hPDLSCs.Finally,in order to further explore its related molecular regulation mechanism,qRT-PCR,Western blot analysis and immunofluorescence technique were applied to detect the expression of the canonical Wnt/β-catenin pathway-related indicators in each group,as well as the expression of osteogenic differentiation-related indicators in each group after the application of small molecule inhibitor XAV-939 and small molecule activator CHIR-99021.Through this study,it is expected to clarify the regulatory function and relevant molecular mechanisms of BBR on the osteogenic differentiation potential of hPDLSCs under AGEs disturbance.Meanwhile,this study provided important theoretical basis and experimental guidance for the further understanding of the osteogenic differentiation of hPDLSCs and the method of promoting periodontal tissue regeneration,which would promote the development of new ideas and treatment methods for the regeneration of damaged periodontal tissues in diabetic patients by using BBR.Materials and Methods1.The cultivation and osteogenic identification of hPDLSCs.The hPDLSCs used in this study were provided by ORAL STEM CELL BANK which was running by Beijing Tason Biotech Co.,Ltd.In order to ensure the purity and stability of the biological properties of hPDLSCs,this part of the experiment conducted subculture and preliminary identification of hPDLSCs,including observing the growth status of the cells and drawing the growth curves.The expression of stem cell surface markers CD73,CD90,CD 105,CD34,CK-19 and Vimentin were observed by flow cytometry,and the character of stem cells of the obtained cells were identified.Finally,the potential of osteogenic differentiation was detected by conventional,mature osteogenic induction methods,ALP staining and alizarin red staining.2.The effect of AGEs on osteogenic differentiation of hPDLSCs was observed.In this part,the 3rd generation of hPDLSCs with good growth condition were selected firstly,and cell proliferation changes of hPDLSCs stimulated by AGEs at different concentrations(100μg/mL,200μg/mL and 400μg/mL)for different lengths of time(0 day,1 day,3 days,5 days and 7 days)were detected by CCK-8 cell proliferation assay kit.The optimal concentration of AGEs was selected(200μg/mL)In order to explore the effect of AGEs on the osteogenic differentiation potential of hPDLSCs,the cells were divided into two groups:OSTEO and OSTEO+AGEs According to the requirements of different detection techniques,osteogenic induction was performed for each group at different times.ALP staining and ALP activity assays were used to evaluate the changes of ALP in early osteogenic differentiation in each group(osteogenic induction 14 days).Alizarin red staining was used to evaluate the mineralization degree of calcium matrix in each group(osteogenic induction 21 days).ELISA was used to detect the expression level of Collagen I in the osteogenic differentiation of cells in each group(osteogenic induction 14 days)qRT-PCR was used to investigate the mRNA expression levels of osteogenic related genes,such as,Runx2,Osterix,ALP,OPN,Collagen I and OCN in each group(osteogenic induction 7 days).Western blot analysis was used to detect the expression levels of osteogenic related proteins,such as,Runx2,OPN,BSP and OCN in each group(osteogenic induction 7 days).The effect of AGEs on osteogenic differentiation of hPDLSCs was further analyzed.3.The effect of BBR on osteogenic differentiation of hPDLSCs under AGEs disturbance was observed.In this part,the 3rd generation of hPDLSCs with good growth condition were selected firstly,and cell proliferation changes of hPDLSCs stimulated by BBR at different concentrations(0.1μmol/L,1μmol/L,3μmol/L and 10μmol/L)under AGEs disturbance for different lengths of time(0 day,1 day,3 days,5 days and 7 days)were detected by CCK-8 cell proliferation assay kit.The optimal concentration of BBR was selected(1μmol/L).In order to explore the effect of BBR on the osteogenic differentiation potential of hPDLSCs under AGEs disturbance,the cells were divided into three groups:OSTEO,OSTEO+AGEs and OSTEO+AGEs+BBR.According to the requirements of different detection techniques,osteogenic induction was performed for each group at different times.The same techniques as the second part were used to detect the expression levels of osteogenic related indicators in each group.ALP staining and ALP activity assays were used to evaluate the changes of ALP in early osteogenic differentiation in each group(osteogenic induction 14 days).Alizarin red staining was used to evaluate the mineralization degree of calcium matrix in each group(osteogenic induction 21 days).ELISA was used to detect the expression level of Collagen I in the osteogenic differentiation of cells in each group(osteogenic induction 14 days).qRT-PCR was used to explore the mRNA expression levels of osteogenic related genes,such as,Runx2,Osterix,ALP,OPN,Collagen Ⅰ and OCN in each group(osteogenic induction 7 days).Western blot analysis was used to investigate the expression levels of osteogenic related proteins,such as,Runx2,OPN,BSP and OCN in each group(osteogenic induction 7 days).The effect of BBR on osteogenic differentiation of hPDLSCs under AGEs disturbance was further analyzed.4.The molecular mechanism of the canonical Wnt/β-catenin pathway regulating BBR’s promoting the osteogenic differentiation of hPDLSCs under AGEs disturbance was explored.In this part,the 3rd generation of hPDLSCs with good growth condition were selected firstly.In order to explore the expression levels of the canonical Wnt/β-catenin pathway related indicators under the effect of AGEs and BBR,the cells were divided into three groups:OSTEO,OSTEO+AGEs and OSTEO+AGEs+BBR.qRT-PCR was applied to detect the mRNA expression levels of the canonical Wnt/β-catenin pathway related genes,such as,βcatenin、LRP5、GSK-3β and DKK-1 in each group(osteogenic induction 7 days).Western blot analysis was applied to explore the expression levels of the canonical Wnt/β-catenin pathway related proteins,such as,β-catenin,wnt3a and GSK-3β in each group(osteogenic induction 7 days).Immunofluorescence technique was applied to detect the distribution of β-catenin,the core component of the pathway,in each group(osteogenic induction 7 days).Thus,the molecular regulation mechanism of the canonical Wnt/β-catenin pathway in BBR’s promoting of osteogenic differentiation of hPDLSCs under AGEs disturbance was further exploredIn order to further validate the canonical Wnt/β-catenin pathway involved in the inhibition of AGEs on osteogenic differentiation of hPDLSCs and the promotion of BBR on osteogenic differentiation of hPDLSCs under AGEs disturbance,small molecule inhibitor XAV-939(1μmol/L)and small molecule activator CHIR-99021(1μmol/L)were added in cell culture.The optimal concentrations of XAV-939 and CHIR-99021 were selected by CCK-8 cell proliferation assay kit.In the inhibitor and activator group,the cells were pretreated with inhibitor or activator for 30 minutes.After that,the cells were cultured in corresponding osteogenic induction medium for 7,14 or 21 days.And the same detection techniques as those used in the second and third parts were used to complete the detection of various osteogenic related indicators of cells in each group.The regulation mechanism of the canonical Wnt/β-catenin pathway in osteogenic differentiation of hPDLSCs promoted by BBR under AGEs disturbance was further clarifiedResults1.After cultivated by the conventional method in vitro,the cells showed a long spindle or spindle shaped fibroblasts with full cell bodies under optics and electronic microscope,which grew in monolayer,and the proliferation and growth of the cells flourished in the first 6～8 generations.The cells could cover 90%of the bottom of the culture bottle in about 3～5 days,and the entered the next passage cycle.The growth curve of the 3rd-generation of hPDLSCs presented an "S" shape,showing four obvious periods of incubation,exponential growth,plateform and decay.hPDLSCs were detected positively expressed mesenchymal stem cell surface markers CD73(99.21%),CD90(99.96%),CD105(95.6%)and Vimentin(99.12%)by flow cytometry;while cells were negatively expressed endothelial markers CD34(6.33%)and cytokeratin CK-19(2.82%).After osteogenic induction and cultivation,the cells showed obvious morphological changes similar to osteoblasts.The cells were obviously thickened and agglomerated into pieces locally.The cells were diverse and irregular in shape,with short spindle shape,polygonal shape or irregular triangle shape.Moreover,the granular substance of the cells in the cytoplasm increased,so did the density.ALP staining showed that blue granular or patchy precipitates appeared in the cytoplasm of the osteogenic induced group and negative staining in the control group.Alizarin red staining showed orange mineralized nodules of different sizes appeared in the osteogenic induced group,while it was negative in the control group.The above results indicated that hPDLSCs used in this study had the ability to differentiate into osteoblasts after osteogenic induction cultivation.2.In the experiment to explore the effect of AGEs on the osteogenic differentiation potential of hPDLSCs,we found that the ALP staining and ALP activity of cells in the OSTEO+AGEs group were significantly weaker than those in the control group after 14 days of osteogenic induction under AGEs disturbance(P<0.01).After 21 days of osteogenic induction,the mineralized nodules formed by cells in the OSTEO+AGEs group were smaller,lighter in color,less in number and scattered in distribution compared with the control group.After 14 days of osteogenic induction,the expression level of Collagen I decreased significantly in the OSTEO+AGEs group compared with the control group(P<0.01).After 7 days of osteogenic induction,the mRNA expression levels of osteogenic related genes,Runx2,Osterix,ALP,OPN,Collagen I and OCN in the OSTEO+AGEs group were significantly lower than those in the control group(P<0.01),After 7 days of osteogenic induction,the expression levels of osteogenic related proteins,Runx2,OPN,BSP and OCN in the OSTEO+AGEs group were lower than those in the control groupFrom the above results,we concluded that AGEs could weaken the ALP activity of hPDLSCs,inhibit mineralized nodule formation of hPDLSCs,inhibit the synthesis of Collagen I,inhibit the expression of osteogenic related genes and proteins.Therefore,this study showed that AGEs could partially inhibit the osteogenic differentiation potential of hPDLSCs3.In the experiment to explore the application of BBR on the osteogenic differentiation potential of hPDLSCs under AGEs disturbance,we discovered that the expression levels of osteogenic related indicators of hPDLSCs were improved after the application of BBR,details were as follows.ALP staining and ALP activity of cells in the OSTEO+AGEs+BBR group were improved than those in the OSTEO+AGEs group after 14 days of osteogenic induction after the application of BBR(P<0.01),but still lower than those in the OSTEO group.After 21 days of osteogenic induction,the size,number and color of mineralized nodules formed in the OSTEO+AGEs+BBR group were improved compared with the OSTEO+AGEs group,but were still not as good as those in the OSTEO group.After 14 days of osteogenic induction,the expression level of Collagen I had a significant increase in the OSTEO+AGEs+BBR group compared with the OSTEO+AGEs group(P<0.01),but still lower than this in the OSTEO group.After 7 days of osteogenic induction,the mRNA expression levels of osteogenic related genes,Runx2,Osterix,ALP,OPN,Collagen I and OCN in the OSTEO+AGEs+BBR group were all higher than those in the OSTEO+AGEs group(P<0.05,P<0.01),but still lower than those in the OSTEO group.After 7 days of osteogenic induction,the expression levels of osteogenic related proteins,Runx2,OPN,BSP and OCN in the OSTEO+AGEs+BBR group were increased compared with the OSTEO+AGEs group,but the levels were still lower than those in the OSTEO group.All of the above experimental results reflected that BBR can improve the ALP activity of hPDLSCs,improve the formation of mineralized nodules,improve the expression of Collagen Ⅰ,promote the expression levels of osteogenic related genes and proteins to some extent,showing that the application of BBR can be partially promote the osteogenic differentiation potential of hPDLSCs under AGEs disturbance.4.In the study of the effects of AGEs and BBR on the expression of the canonical Wnt/β-catenin pathway in hPDLSCs,we found that the mRNA expression levels ofβ-catenin and LRP5 in the OSTEO+AGEs group were significantly increased after 7 days of osteogenic induction compared with the OSTEO group(P<0.01),and the mRNA expression levels of GSK-3β and DKK-1 were significantly decreased compared with the OSTEO group(P<0.05,P<0.01),after the application of BBR,the mRNA expression levels of β-catenin and LRP5 in the OSTEO+AGEs+BBR group significantly lower than those in the OSTEO+AGEs group(P<0.01),but still higher than those in the OSTEO group,the mRNA expression levels of GSK-3β was significantly higher than that in the OSTEO+AGEs group(P<0.05),but still lower than that in the OSTEO group.After 7 days of osteogenic induction,the expression levels of β-catenin and wnt3a in the OSTEO+AGEs group were higher than those in the OSTEO group,while the expression of GSK-3β was lower than that in the OSTEO group.After the application of BBR,the expression levels of β-catenin and wnt3a in the group of OSTEO+AGEs+BBR were down-regulated compared with the group of OSTEO+AGEs,while the expression of GSK-3β were up-regulated compared with the OSTEO+AGEs group.After 7 days of osteogenic induction,β-catenin in the OSTEO group was mainly located in the cytoplasm of hPDLSCs,and the accumulation of β-catenin was in the cytoplasm and the nucleus of hPDLSCs in the OSTEO+AGEs group,especially in the nucleus of cells,β-catenin was found in both cytoplasm and nucleus of hPDLSCs in the OSTEO+AGEs+BBR group,but the difference in position distribution of β-catenin was not obvious compared with the OSTEO+AGEs group.Combined with the above results,it was suggested that AGEs could promote the transfer and distribution of β-catenin in hPDLSCs to the nucleus,thus activating the canonical Wnt/β-catenin pathway;BBR can inhibit the transfer and distribution of β-catenin to the nucleus of hPDLSCs under AGEs disturbance,thus inhibit the canonical Wnt/β-catenin pathway.On the other hand,in order to further clarify that the inhibition of hPDLSCs’osteogenic differentiation ability caused by AGEs is related to the activation of the canonical Wnt/β-catenin pathway,we added XAV-939,a small molecule inhibitor of the canonical Wnt/β-catenin pathway in cells’cultivation,and then we completed the detection of various osteogenic related indicators after osteogenic inductive cultivation for 7 days,14 days and 21 days.The results showed that compared with the group of OSTEO+AGEs,after 14 days of osteogenic induction,ALP in the group of OSTEO+AGEs+XAV-939 was stained deeper than that in the group of OSTEO+AGEs,but still lighter than that in the OSTEO group.After 14 days of osteogenic induction,the ALP activity of the cells in the OSTEO+AGEs+XAV-939 group was enhanced,but was still lower than that in the group of OSTEO(P<0.01).After 21 days of osteogenic induction,the mineralized nodules formed by cells in the OSTEO+AGEs+XAV-939 group were larger,darker in color and more in number,but they were still less than those in the OSTEO group.After 14 days of osteogenic induction,the expression level of Collagen I in the OSTEO+AGEs+XAV-939 group was obviously increased(P<0.01),but still lower than that in the OSTEO group.After 7 days of osteogenic induction,the mRNA expression levels of most osteogenic related genes(Runx2,Osterix,ALP,Collagen I and OCN)were obviously increased in the OSTEO+AGEs+XAV-939 group compared with those in the OSTEO+AGEs group(P<0.05,P<0.01),but still lower than those in the OSTEO group,it is worth noting that the application of XAV-939 had no significant effect on the expression of OPN in cells mediated by AGEs.After 7 days of osteogenic induction,the expression levels of osteogenic related proteins Runx2,OPN,BSP and OCN of hPDLSCs were increased in the group of OSTEO+AGEs+XAV-939 compared with the group of OSTEO+AGEs.At the same time,in order to further clarify the improvement of hPDLSCs’osteogenic differentiation ability caused by BBR under AGEs disturbance is related to the inhibition of the canonical Wnt/β-catenin pathway,we added CHIR-99021,a small molecule activator of the canonical Wnt/β-catenin pathway in cells’ osteogenic inductive cultivation.And then we completed the detection of various osteogenic related indicators after osteogenic inductive cultivation for 7 days,14 days and 21 days.The results showed that after 14 days of osteogenic induction,the ALP staining in the group of OSTEO+AGEs+BBR+CHIR-99021 were lower than that in the group of OSTEO+AGEs+BBR,but still higher than that in the group of OSTEO+AGE.After 14 days of osteogenic induction,the ALP activity of cells in the group of OSTEO+AGEs+BBR+CHIR-99021 was significantly lower than that in the group of OSTEO+AGEs+BBR(P<0.01),but was still higher than that in the group of OSTEO+AGEs(P<0.01).After 21 days of osteogenic induction,mineralized nodules formed by cells in the OSTEO+AGEs+BBR+CHIR-99021 group were smaller than those in the OSTEO+AGEs+BBR group,but the number and size of mineralized nodules were still higher than those in the OSTEO+AGEs group.After 14 days of osteogenic induction,the expression level of Collagen I by cells in the OSTEO+AGEs+BBR+CHIR-99021 group decreased obviously than that in the OSTEO+AGEs+BBR group(P<0.01),but still higher than that in the OSTEO+AGEs group(P<0.01).After 7 days of osteogenic induction,the mRNA expression levels of osteogenic related genes(Runx2,Osterix,ALP,OPN,Collagen I and OCN)decreased significantly in the OSTEO+AGEs+BBR+CHIR-99021 group compared with the OSTEO+AGEs+BBR group(P<0.05,P<0.01),but the expression levels of part genes were still higher than those in the OSTEO+AGEs group(P<0.01).After 7 days of osteogenic induction,the expression levels of osteogenic related proteins Runx2,OPN,BSP and OCN in the OSTEO+AGEs+BBR+CHIR-99021 group were decreased compared with the group of OSTEO+AGEs+BBR,but were still higher than those in the group of OSTEO+AGEs.The results above showed that the inhibition effect of AGEs on the osteogenic differentiation potential of hPDLSCs was partially regulated by the activation of the canonical Wnt/β-catenin pathway,and the positive improvement effect of BBR on the inhibited osteogenic differentiation potential of hPDLSCs mediated by AGEs was partially regulated by the inhibition of the canonical Wnt/β-catenin pathway.Conclusions1.In diabetic patients,the presence of AGEs can inhibit the osteogenic differentiation potential of hPDLSCs.2.The application of BBR can partially reverse the inhibition effect on the osteogenic differentiation potential of hPDLSCs cansed by AGEs and promote the osteogenic differentiation of hPDLSCs.3.AGEs could inhibit the expression of DKK-1 by promoting the expression of Wnt ligand wnt3a in the canonical Wnt/β-catenin signaling pathway of hPDLSCs,thereby inhibiting the specific binding of DKK-1 and Wnt co-receptor LRP5,and promoting the binding of LRP5 and Wnt ligand-Frizzled receptor complex.At the same time,it could promote the mRNA expression of LRP5 and the transduction of the Wnt/β-catenin signaling pathway.Then,the activity of GSK-3β and GSK-3β/Axin/APC degradation complex were inhibited,which promoted the accumulation of β-catenin.Next,β-catenin was transferred into the nucleus and combined with TCF/LEF to regulate the transcription of downstream target genes,thus the classical Wnt/β-catenin signaling pathway was activated which inhibited the osteogenic differentiation potential of hPDLSCs.After inhibiting the canonical Wnt/β-catenin pathway by XAV-939,hPDLSCs could partially restore their osteogenic differentiation potential.BBR could inhibit the mRNA expression of LRP5 by inhibiting the expression of Wnt ligand wnt3a of hPDLSCs mediated by AGEs,thereby inhibiting the transduction of Wnt signal.Then,the activity of GSK-3β and GSK-3β/Axin/APC degradation complex were improved,which promoted the degradation of β-catenin.Meanwhile,the transfer of β-catenin into the nucleus was inhibited,which inhibited the transcription of downstream target genes,thus the classical Wnt/β-catenin signaling pathway was inhibited which promoted the osteogenic differentiation potential of hPDLSCs.After activating the canonical Wnt/β-catenin pathway by CHIR-99021,the improvement effect of BBR on osteogenic differentiation potential of hPDLSCs under AGEs disturbance was partially inhibited. |
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