| Background and purposePeriodontitis is an inflammatory disease characterized by destruction of periodontal tissues including attachment loss of connective tissue and resorption of alveolar bone,resulting in tooth loss eventually.Bacterial infections have been shown to contribute to the development of periodontitis.Lipopolysaccharide(LPS)is the main component of Gramnegative bacteria and participates in the pathogenesis of periodontitis.Nuclear factor-kappa B(NF-κB)is a transcriptional molecule that regulates the expression of genes associated with congenital and acquired immune responses.Under the stimulation of LPS,the classical NFκB pathway is activated to produce inflammatory factors and cytokines.Inhibition of NF-κB pathway to change the inflammatory microenvironment which helps osteogenic differentiation is the key to prevent bone loss in inflammatory diseases.1.25-diydroxyviatamin D3(VD3)is the functional active form of vitamin D and plays an important role in bone metabolism.Meanwhile,VD3 initiates a variety of biological reponses through vitamin D receptor(VDR),including inhibiting inflammation and protecting autoimmune diseases.However,the therapeutic effect of VD3 on periodontitis remains to be studied.Human periodontal ligament stem cells(hPDLSCs)are a kind of undifferentiated mesenchymal stem cells,which can differentiate into multifunctional cell lines and have great potential for periodontal regeneration,so hPDLSCs are considered to be the key cells in tissue engineering for the treatment of periodontal disease.Periodontal regeneration surgery is a treatment to achieve periodontal regeneration.However,due to its uncertain curative effect,trauma and other reasons are clinical constraints,regenerating the loss of periodontal structure and functions is still challenging.Based on the above research background,hPDLSCs were used as the experimental model in vitro,and LPS stimulation was used to mimic the inflammatory microenvironment of patients with periodontitis to study the effect of VD3 on osteogenic differentiation of hPDLSCs stimulated by LPS and its possible mechanism.Experimental methodPart 1 The effect of VD3 on the proliferation and osteogenic differentiation of hPDLSCs stimulated by LPS.The primary hPDLSCs were obtained from the premolars extracted by 14-26-year-old healthy patients due to orthodontic needs cultured with periodontal ligament tissue mass method and cloned with limited dilution.hPDLSCs were identified by alizarin red staining and oil red O staining after osteogenic and adipogenic induction.The surface molecules CD34,CD44,CD45 and CD90 of the cells were identified by flow cytometry,and the clone formation test was used to identify them by crystal violet staining.hPDLSCs were cultured in LPS(0,1,10,50 μg/mL),VD3(0,1,10,100 nmol/L)and LPS+VD3(10 μg/mL LPS+0,1,10,100 nmol/L VD3)for 24,48 and 72 hours,respectively.The effects of LPS and VD3 on the proliferation of hPDLSCs were detected by CCK-8 technique.The 3rd-5th passage of hPDLSCs were cultured in LPS medium(0,1,10,50 μg/mL)to induce osteogenic differentiation.The activity of ALP was detected on the 3rd day.The mRNA and protein expression of ALP,Runx2 and OPN were detected on the 7th and 14th day.On the 21st day,the mineralized nodules were detected by alizarin red staining.The lowest concentration(10 μg/mL)of inhibiting osteogenic differentiation of hPDLSCs were selected as the working concentration of LPS.hPDLSCs were divided into five groups:control group(osteogenic induction solution),LPS group(osteogenic induction solution+10 μg/mL LPS)and LPS+VD3 group(osteogenic induction solution+10 μg/mL LPS+1、10、100 nmol/L VD3).The results of osteogenic differentiation were detected by the same method mentioned above.The lowest concentration(10 nmol/L)to promote the osteogenic differentiation of hPDLSCs was selected as the working concentration of VD3.Part 2 The mechanism of the effect of VD3 on osteogenic differentiation of hPDLSCs stimulated by LPS.hPDLSCs were divided into control group(osteogenic induction solution),NF-κB pathway inhibition group(osteogenic induction solution+10 μg/mL LPS+40 ng/mL PDTC)and LPS group(osteogenic induction solution+10 μg/mL LPS).After 7 days of culture,total mRNA and total protein were extracted to detect the levels of ALP,Runx2 and OPN.hPDLSCs were divided into three groups:control group(osteogenic induction solution),LPS group(osteogenic induction solution+10 μg/mL LPS)and LPS+VD3 group(osteogenic induction solution+10 μg/mL LPS+10 nmol/L VD3).After 15 minutes of stimulation,total protein and nuclear protein were extracted.Western blot was used to detect the expression of IkB-α,p-IkB-α,p65 and p-p65 in total protein and p65 protein in nuclear protein.The distribution of p65 in cells was detected by immunofluorescence.Use the same way to divide groups.After 3,7 and 14 days of culture,total mRNA was extracted to detect expression of VDR.VDR gene in hPDLSCs was knocked down by small interference RNA transfection.hPDLSCs were divided into LPS group,LPS+VD3 group,si-VDR+LPS group and si-VDR+LPS+VD3 group.After 3 days of culture,the activity of ALP was detected.After 7 days of culture,the gene and protein expression levels of ALP,Runx2 and OPN were detected to determine the effect of si-VDR on osteogenic differentiation.After culturing for 15 min,total protein was extracted to detect the protein expression level of IκB-α、p-IκB-α、p65、p-p65,and nuclear protein was extracted to detect the expression level of p65 protein.Experimental resultsPart 1 The effect of VD3 on the proliferation and osteogenic differentiation of hPDLSCs stimulated by LPS.The hPDLSCs were consistent in shape and showed long fusiform shape in the third passage.Alizarin red staining showed mineralized nodule formation after osteogenic induction culture for 21 days,and oil red O staining showed lipid droplet formation after adipogenic induction culture for 21 days,indicating that the cultured cells had multifunctional differentiation potential.Flow cytometry showed that the positive rates of CD45,CD34,CD44 and CD90 were 0.6%,2.5%,98.1%and 98.6%,respectively,according with the phenotypic characteristics of hPDLSCs.Multiple clones were observed by crystal violet staining after low density inoculation for 10 days.The results of CCK-8 detection showed that compared with the control group,the OD values of 10 and 50 μg/mL LPS decreased significantly after 48 and 72 hours(P<0.05),indicating that the cells proliferative ability decreased,and the OD values of 10 and 100 nmol/L VD3 increased significantly after 48 and 72 hours(P<0.01),indicating that the cells proliferative ability increased.In LPS+VD3 culture medium,10 and 100 nmol/L VD3 could significantly increase the OD value(P<0.01),suggesting that 10-100 nmol/L VD3 could reverse the effect of LPS-induced decrease in proliferation of hPDLSCs.When hPDLSCs were cultured with 1,10,50 μg/mL LPS in osteogenic induction medium,the ALP activity of 1,10,50μg/ml LPS significantly decreased on day 3(P<0.01),the mRNA and protein expression of ALP,Runx2 and OPN significantly decreased at 10 and 50 μg/mL LPS on day 7 and 14(P<0.05).After 21 days,the alizarin red staining in the control group was the darkest and thickest.Semi-quantitative analysis showed that the OD values of 1,10 and 50 μg/mL LPS compared were lower than those in the control group(P<0.05).In summary,10μg/mL LPS was selected as the working concentration.Compared with LPS group,10 and 100 nmol/L VD3 significantly increased ALP activity(P<0.05)on day 3.10 and 100 nmol/L VD3 significantly increased the expression of ALP,Runx2 and OPN mRNA on day 7 and 14 compared to LPS group.After 21 days,alizarin red semi-quantitative analysis showed that the OD values of 1,10 and 100 nmol/L VD3 in LPS+VD3 group were higher than those in LPS group(P<0.05),and the levels in LPS group and LPS+VD3 group decreased compared to control group(P<0.05).10 nmol/L VD3 was selected as the working concentration.Part 2 The mechanism of the effect of VD3 on osteogenic differentiation of hPDLSCs stimulated by LPS.After the cells were treated with 40 ng/mL PDTC,the mRNA and protein levels of ALP,Runx2 and OPN were increased in NF-κB pathway inhibition group compared with LPS group(P<0.05)on day 7.hPDLSCs were divided into control group,LPS group and LPS+VD3 group.The cells were cultured for 15 minutes.The protein results showed that the phosphorylation degree of IkB-α and p65 in LPS+VD3 group was significantly lower than that in LPS group,and the nuclear translocation of p65 in LPS+VD3 group was significantly lower than that in LPS group(P<0.05).After 3,7 and 14 days of culture,the VDR level in LPS+VD3 group increased compared to LPS group(P<0.05).After si-VDR transfection into hPDLSCs,the activity of ALP in transfection group decreased significantly on day 3(P<0.05),but there was no significant difference between the two transfection groups.On the 7th day,the mRNA and protein expression levels of ALP,Runx2 and OPN in the transfection group were significantly lower than those in the nontransfection group(P<0.05).The results of western blot showed that the phosphorylation degree of IκB-α and p65 was significantly increased in the transfection group,and the nuclear translocation of p65 was also significantly increased(P<0.05).Conclusion(1)VD3 significantly increases the expression of osteogenic differentiation of hPDLSCs inhibited by LPS.(2)Inhibition of NF-κB pathway significantly increases the expression of osteogenic differentiation of hPDLSCs inhibited by LPS.(3)VD3 significantly inhibites the activation of NF-κB pathway in hPDLSCs stimulated by LPS.(4)VD3 significantly increases the expression of VDR inhibited by LPS.After the silencing of VDR expression,the regulatory effect of VD3 on the expression of osteogenic differentiation is significantly decreased which suggests that the mechanism may be achieved through the regulation of its receptor VDR. |
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