The Role Of Endoplasmic Reticulum Stress On Low-intensity Pulsed Ultrasound Inhibiting Inflammation And Promoting Osteogenic Differentiation In Periodontal Ligament Stem Cells

Objective: Periodontitis can affect bone tissue regeneration and repair ability.The reason may be that the inflammation affects its biological characteristics by changing the microenvironment of periodontal ligament stem cells(PDLSCs),which leads to inhibition of its osteogenic differentiation ability.Endoplasmic reticulum stress(ERS)is a new-found a kind of widespread protective response in eukaryotic cells.It can induce unfolded protein response(UPR)to maintain homeostasis of cells by enhancing protein folding,adjusting protein translation,reducing protein synthesis and promoting endoplasmic reticulum associated degradation.Numerous studies have shown that ERS is closely related to the occurrence and development of inflammation.In periodontal tissue engineering,periodontal ligament stem cells(Periodontal PDLSCs,P-PDLSCs)derived from patients with periodontitis are one of the most promising and easily obtained stem cells for repairing damaged alveolar bone and other connective tissues around the teeth.However,the inflammatory microenvironment of chronic periodontitis affects the osteogenic differentiation capacity of P-PDLSCs.Low-intensity pulsed ultrasound(LIPUS)can enhance the osteogenic differentiation of healthy periodontal ligament stem cells(Healthy PDLSCs,H-PDLSCs)cultured in vitro and reduce the expression of lipopolysaccharide(LPS)-induced inflammatory factors.However,the functional mechanism of LIPUS is unclear.Therefore,in this experiment,P-PDLSCs and H-PDLSCs were cultured in vitro to study the generation of ERS and the activation of UPR in both cells,and to explore the effects of LIPUS on the osteogenic differentiation ability of inflammatory-derived P-PDLSCs in vitro and the repair of alveolar bone in periodontitis in vivo.Expect to provide a more solid basis for the application of LIPUS in the periodontal tissue regeneration project,and promote the widespread application of LIPUS in the field of stem cell therapy.Methods: 1.Isolation,culture and identification of P-PDLSCs and H-PDLSCs in vitro: periodontal ligament derived from periodontitis and healthy periodontal ligament were scraped,and P-PDLSCs and H-PDLSCs were isolated and cultured by tissue block combined with enzyme digestion.Then the expression of cell surface markers and osteogenic differentiation ability of the two PDLSCs were detected.2.Observe the changes of ERS and osteogenic differentiation ability of P-PDLSCs: Compare the endoplasmic reticulum morphology in two PDLSCs by transmission electron microscopy.CCK-8 detected cell proliferation,then qRT-PCR and Western Blot were used to detect the expression of genes(GRP78,CHOP,IL-6,RUNX2)and proteins(IRE1?,RUNX2,ALP)related to ERS,inflammation and osteogenic differentiation,respectively.3.Observe the role of ERS in the inhibition of P-PDLSCs inflammation and promotion of osteogenic differentiation by LIPUS: In order to optimize the intensity of LIPUS and the dose of 4-PBA,LIPUS at different times(30 min,60 min,90 min,120 min)and different concentrations of 4-PBA(from 1 mM to 10 mM)treatment,the proliferation of PDLSCs was detected by CCK-8.P-PDLSCs are divided into four groups:(1)P-PDLSCs group;(2)P-PDLSCs + LIPUS group;(3)P-PDLSCs + 4-PBA group;(4)P-PDLSCs + LIPUS + 4-PBA group,the endoplasmic reticulum morphology of each group was observed by TEM,and the expression of inflammatory factors(IL-6,IL-8)in each group was detected by ELISA.Then qRT-PCR and Western Blot were used to detect the expression of UPR,inflammation and osteogenesis-related genes(GRP78,IRE1?,ATF4,IL-6,IL-8,RUNX2)and proteins(PERK,GRP78,IRE1?/P-IRE1?,PDI,RUNX2,ALP).The osteogenic differentiation ability of each group was compared by ALP staining and alizarin red staining at 7 and 21 days after osteogenesis induction.4.The repairing effect of LIPUS and ERS on experimental periodontitis: Constructed a periodontitis model by ligating wire ligation and local injection of LPS in the rat mouth.Intervened with LIPUS or 4-PBA,and observed the absorption of alveolar bone in each group using Micro-CT and H&E staining.Results: 1.The primary P-PDLSCs and H-PDLSCs showed fibroblastic appearance and adhered to the dish.Both PDLSCs positively expressed mesenchymal stem cell surface markers,including CD73 and CD146,and negatively expressed hematopoietic cell surface marker CD34.While P-PDLSCs were found a reduced osteogenesis by Alizarin red staining.2.Transmission electron microscopy showed changes in endoplasmic reticulum morphology of P-PDLSCs;CCK-8 test showed that the proliferation rate of P-PDLSCs was significantly higher than that of H-PDLSCs after three days,qRT-PCR and Western Blot showed that the expression of genes and proteins associated with UPR(GRP78,CHOP,IRE1?)and inflammation(IL-6)in P-PDLSCs increased.The decrease of osteogenesis-related genes and proteins(RUNX2,ALP)indicates that increased inflammatory factors and decreased osteogenesis in P-PDLSCs may be related to UPR activation caused by chronic inflammation.3.CCK-8 results showed that LIPUS treatment for 30 minutes could significantly promote cell proliferation,while 4-PBA(5mM)pretreatment for 8 hours had little effect on cell proliferation,and the level of toxicity was low.ELISA test found that both LIPUS and 4-PBA reduced the expression of IL-6 and IL-8 in P-PDLSCs;qRT-PCR and Western Blot showed that LIPUS and 4-PBA reduced the UPR(GRP78,IRE1? / P-IRE1?,ATF4,PERK,PDI)and inflammation(IL-6,IL-8)related genes and proteins expression,while enhanced osteogenesis related genes and proteins expression(RUNX2,ALP);ALP staining and alizarin red staining also proved that LIPUS and 4-PBA promote the osteogenic differentiation of P-PDLSCs.4.Micro-CT results in vivo showed that alveolar bone was significantly absorbed in experimental periodontitis,while alveolar bone loss rate(ABLs / RBLs)after LIPUS and 4-PBA treatment was lower than that in experimental periodontitis group.H&E staining showed After treatment with LIPUS and 4-PBA,the alveolar bone height can be partially restored,and periodontal tissue regeneration could be promoted.Conclusions: 1.P-PDLSCs derived from periodontitis were in the endoplasmic reticulum stress state,resulting in increased inflammation and decreased osteogenic ability compared to H-PDLCs.2.Both LIPUS and endoplasmic reticulum stress inhibitor 4-PBA can significantly reduce the expression of UPR signaling molecules,significantly enhance the osteogenic differentiation ability of P-PDLSCs and reduce the expression of inflammatory factors,suggesting that LIPUS can decrease inflammation and increase osteogenesis of P-PDLSCs by reducing endoplasmic reticulum stress.3.Both LIPUS and 4-PBA can partially restore the alveolar bone height of damaged periodontal tissues in vivo,further promoting periodontal tissue regeneration.