| Objective: In recent years,it is considered that oxidative stress is one of the most important causes of diabetes with periodontitis.It plays a crucial role in the pathogenesis of diabetes-induce or aggravate periodontitis.Advanced glycation end products,as longlasting and chronic hyperglycemic products in patients with type 2 diabetes,are localized to the paradentium and accumulate and activate the host's innate immune system by activating oxidative stress and affecting cell signaling,triggering or amplifying the local inflammatory tissue immune response,which induce or aggravate periodontitis.However,whether this effect can lead to the damage of periodontal ligament stem cells(PDLSCs),making it unable to exert self-renewal and multiple differentiation potential in time is still unknown.We isolated the PDLSCs from healthy people and simulated the microenvironment of periodontitis,diabetes and periodontitis in vitro,investigated whether oxidative stress can affect the performance of PDLSCs.if so,what way is it caused by? What will be the result?Methods: In this study,normal periodontal ligament cells were cultured in vitro by enzymatic digestion method.PDLSCs was cloned,purified and subcultured through limited dilution method.The phenotype of stem cells was identified by flow cytometry.By loading the corresponding stimulus(10ng/m L TNF-? and /or 100?g/m L AGEs),the four kinds of microenvironment conditions(Stimulation groups)of healthy(Control group),simple periodontitis(TNF-?-PDLSCs group),type 2 diabetes mellitus(AGEs-PDLSCs group)and type 2 diabetic with periodontitis(AGEs+TNF-?-PDLSCs group)were simulated in vitro.Pretreatment interventions of the pathway inhibitor SP600125(blocker)can be divided into three inhibitor groups: TNF-?+blocker-PDLSCs group,AGEs+blocker-PDLSCs group and AGEs+TNF-?+blocker-PDLSCs group.The four stimulation groups were used as experimental subjects:(1)(1)Detection of cell proliferation by CCK-8;(2)The osteogenic,adipogenic and chondrogenic induction mediums were grouped according to the experiment,PDLSCs of each group were induced for 21 days after adding TNF-? and AGEs respectively.The formation of bone nodules was observed with Alizarin red stainin in each group.Osteogenic induction was observed by alkaline phosphatase staining in each group.The formation of cartilage induction was tested by Toluidine blue staining in each group.Oil red O staining observed the formation of lipid droplets in each group.(3)Osteoblasts,adipocytes and cartilage were induced by experimental groups for 21 days.The expression of genes related to osteogenesis,lipid and chondrogenic differentiation was detected by RT-PCR.(4)Detection of ROS level in each group by flow cytometry.(5)Detection of the formation of malondialdehyde(MDA)in each group.(6)Detection of the production of total superoxide dismutase(T-SOD)in each group.(7)Measurement of the expression of calcium ion in each group by laser confocal microscopy.(8)Changes of mitochondrial membrane potential(JC-1)detected by flow cytometry.(9)Detection of structure changes of PDLSCs mitochondria by transmission electron microscopy.(2)Four stimulation groups and three inhibitor groups were used as experimental subjects:(1)Flow cytometry was used to detect cell apoptosis in each group.(2)The gene expression of JNK,Cyt-c,Caspase-3,Bax and BCL-2 in each group was detected by RT-PCR.(3)Western Blot was used to detect the protein expression of P-JNK,Cyt-c,Caspase-3,Bax and BCL-2 in each group.Results: Isolation,Purification and Identification of Periodontal Ligament Stem Cells: The PDLSCs extracted and purified from the periodontal ligament showed long fusiform,bipolar and adherent growth,the morphology approached fibroblasts,the cytoplasm was abundant,and the nucleus was located in the center.Flow cytometry showed that the surface antigens of PDLSCs on the 3rd generation were identified as STRO-1(6.97 ± 0.012)%,CD146(39.77 ± 0.028)%,CD90(93.10 ± 0.009)%,CD44(83.27 ± 0.050)%.This confirms the stem cell source and stem cell characteristics extracted and cultured in this experiment.(1)(1)CCK-8 results showed that although TNF-? can promote the proliferation of PDLSCs to a certain extent,compared with control group,TNF-? can inhibit the proliferation of PDLSCs in general.AGEs and the combination of AGEs with TNF-? significantly inhibited the proliferation of PDLSCs,and the inhibitory effect was the strongest at the third day.(2)Alizarin red staining shows: The mineralized nodules were induced in all groups,but the size of the mineralized nodules in the Control group was the largest and the degree of density was high.The volume of mineralized nodules in the TNF-?-PDLSCs group decreased and the density was weakened.The mineralized nodules in AGEs-PDLSCs group and AGEs+TNF-?-PDLSCs group showed the smallest and more dispersed nodules with lower density.Alkaline phosphatase staining showed: Control group had the strongest ability to form bone,while the capacity of bone formation by TNF-?-PDLSCs was weakened.The ability of bone formation by AGEs-PDLSCs group and AGEs+TNF-?-PDLSCs group was significantly weakened.Toluidine blue staining showed: In the control group,a large amount of glycosaminoglycan was secreted in the cell matrix,the secretion of TNF-?-PDLSCs was decreased compared with the control group,the secretion of AGEs-PDLSCs was greatly reduced,and the secretion of AGEs+TNF-?-PDLSCs was the least.Oil red O staining shows: There were a large number of lipid droplets in the control group and a few lipid droplets in the TNF-?-PDLSCs group.No lipid-dropping substance was found in the AGEs-PDLSCs and AGEs+TNF-?-PDLSCs groups.(3)After 21 days of osteogenic,chondrogenic and adipogenic induction,the expression of related genes in each group was the highest in control group,the expression of TNF-?-PDLSCs group,AGEs-PDLSCs group and AGEs+TNF-?-PDLSCs group were decreased in turn.(2)The level of ROS in mitochondria of control group was the lowest,and the levels in the other three stimulation groups were significantly increased,especially in the combination of AGEs with TNF-?.(3)Changes of mitochondrial structure of PDLSCs in each group: Compared with the Control group,the mitochondrial internal and external membrane of the TNF-?-PDLSCs group disappeared,the mitochondrial crista was altered and arranged in disorder,and the substructure of the cell was basically normal.A large number of cells in AGEs-PDLSCs group showed apoptotic state,cytoplasm concentration,sub-structure damage,endoplasmic reticulum swelling and apoptotic body formation,part of the mitochondrial inner and outer membrane survived and part destroyed and disappeared,mitochondrial cristae structure completely destroyed.A large number of cells in AGEs+TNF-?-PDLSCs group also showed apoptotic state,decreased organelles,mitochondrial membrane gradually dissolved and dissolved,most mitochondrial cristae disappeared.The levels of cytoplasmic calcium ion in PDLSCs of control group,TNF-?-PDLSCs group,AGEsPDLSCs group and AGEs+TNF-?-PDLSCs group increased in turn,but there was no statistical significance between control group and TNF-?-PDLSCs group(P > 0.05).Cytoplasmic calcium overload can indirectly cause calcium overload in the mitochondria,resulting in mitochondrial membrane potential depolarization,the membrane potential of the control group was much higher than the three stimulation groups,but there was no statistical significance between TNF-?-PDLSCs group(P > 0.05).The membrane potentials of AGEs-PDLSCs group and AGEs+TNF-?-PDLSCs group decreased significantly and the depolarization were severe,which indicated the apoptosis of the two groups of cells.AV/PI double staining also confirmed that the apoptosis rate of AGEsPDLSCs group and AGEs+TNF-?-PDLSCs group was significantly higher than that of control group and TNF-?-PDLSCs group,and the expression of related genes except antiapoptotic protein BCL-2 decreased in turn,the other genes in turn increased the expression.WB confirms this result.After pretreatment of the cells with the inhibitor SP600125,the apoptosis rates of the three stimulation groups decreased,but there was no statistical significance between TNF-?-PDLSCs group and TNF-?+blocker-PDLSCs group(P >0.05),and AGEs+TNF-?+blocker-PDLSCs group was the most significant reduction.In the three inhibitor groups,the expression of BCL-2 increased in turn and the expression of other genes decreased in turn,compared with the corresponding stimulation group.WB results corroborate with it.Conclusion: 1.Both AGEs and TNF-? could inhibit the proliferation and differentiation of PDLSCs.2.The damage of AGEs on PDLSCs is more serious than that of TNF-? on PDLSCs.3.TNF-? and AGEs can induce oxidative stress in PDLSCs to generate endogenous ROS,which leads to oxidative damage of PDLSCs.4.Endogenous ROS generated by PDLSCs induced by TNF-? and AGEs can activate JNK signaling pathway.5.Phosphorylation of JNK induces apoptosis of PDLSCs by triggering the mitochondriamediated endogenous apoptotic pathway and regulating the expression of Bax and Bcl-2. |
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