LIPUS Regulates Proliferation, Apoptosis And Autophagy Of Periodontal Ligament Cells By YAP In Inflammatory State

Objective:The present study aims to explore if low intensity pulsed ultrasound(LIPUS)inhibits human periodontal ligament cells(hPDLCs)apoptosis and autophagy induced by lipopolysaccharide(LPS),and whether the molecular mechanism is related to Yes-associated protein(YAP).Method:1.The hPDLCs were isolated and cultured by tissue block enzyme digestion.Periodontal ligament tissue was isolated and cultured.When the cells crawled out 3-5 days after culture and the fusion degree of the cells reached 80%,the cells began to be subcultured to provide a cellular resource for subsequent experiments.2.The effects of LPS on hPDLCs activity were detected and LPS concentrations were screened.The experiments were divided into three groups:the control group,the low concentration LPS(1μg/mL)and the high concentration LPS(10μg/mL).When the fusion degree of the cells reached80%,CCK-8 kit was used to detect the change in cell proliferation,and flow cytometry was used to detects changes in cell cycle and apoptosis.An electron microscopy was used to observe the formation of autophagosomes.3.The effects of LIPUS on the proliferation or apoptosis of hPDLCs were to be detected,and the effects of LIPUS on the changes of hPDLCs proliferation or apoptosis were also observed in an inflammatory state.The hPDLCs were divided into two groups according to different cell states,ie.good state or poor state.When the cell fusion reached 70%,ultrasound(90mW/cm~2)was applied every day.When the cell fusion degree reached80%,hPDLCs proliferation and apoptosis levels were detected by flow cytometry.Cells under normal condition were further divided into four groups:the control group,the LIPUS irradiation group,the LPS treatment group,the LPS+LIPUS treatment group.When the fusion degree of hPDLCs reached 70%,LIPUS was used to irradiate the cells for 30 min every day after LPS at high concentration of 10μg/mL treated hPDLCs for 12hours.Afterwards,the cell fusion degree reached 90%,apoptosis was detected by flow cytometry,TUNEL staining,and western-blot(WB).4.The effects of LIPUS on the expressions of YAP,autophagy related proteins and apoptosis related proteins were detected in an inflammatory state.When the cell fusion degree of hPDLCs reached 40%,LPS treatment was performed,and after 12 hours,LIPUS treatment was performed.Next,when the cell fusion degree reached 70%,the YAP protein localization change was detected by immunofluorescence.When the cell fusion degree of hPDLCs reached 70%,LPS treatment was performed,and after 12 hours,LIPUS treatment was performed.Subsequently,when the cell fusion degree reached 90%,protein samples were collected and western-blot was used to detect the effects of LPS and LIPUS on the expressions of YAP,apoptosis-related proteins and autophagy-related proteins.The effects of LIPUS and LPS on autophagosomes in hPDLCs were identified with an electron microscope.Results1.Tissue block enzyme digestion method was successfully used to obtain periodontal ligament cells.2.The growth of hPDLCs were tested by CCK-8 method.It was found that LPS derived-E.coli at different concentrations(1μg/mL,10μg/mL)to stimulate hPDLCs did not significantly affect cell proliferation.Flow cytometry showed that stimulation of hPDLCs with E.coli-derived LPS at different concentrations(1μg/mL,10μg/mL)had no significant effects on the cell cycle of hPDLCs.Flow cytometry also showed that when hPDLCs were stimulated with E.coli-derived LPS at different concentrations(1μg/mL,10μg/mL),LPS at high concentration promoted the early apoptosis of hPDLCs.An electronic microscopy observation showed that LPS could promote hPDLCs to form autophagosomes.3.LIPUS has a significant effect on the promotion of the proliferation of hPDLCs under poor condition,but has no obvious positive effect on hPDLCs in good condition.After LIPUS irradiation,flow cytometry showed that LIPUS significantly reduced the early apoptosis of hPDLCs.Flow cytometry,TUNEL cell staining,and caspase-3 protein detection revealed that LIPUS can reduce the number of apoptotic hPDLCs in the inflammatory state to a certain extent,which suggested the inhibition of cell apoptosis to a certain extent.4.Through immunofluorescence and western-blot detection,the LIPUS treatment group significantly increased the expression of YAP,and YAP was found to be transported into the nucleus;LPS had an inhibitory effect,but could promote the phosphorylated YAP expression.It was observed under an electron microscope that LPS promoted the formation of autophagosomes.Western-blot detection showed that LPS promoted the expression of LC3-Ⅱprotein,and LIPUS could attenuate this effect on autophagy.After the YAP gene was knocked down,the inhibitory effect of LIPUS on hPDLCs apoptosis was significantly weakened,and the role of LPS in promoting autophagy was restored.ConclusionsLPS at high concentration promotes apoptosis of hPDLCs,while LIPUS inhibits apoptosis.LPS can inhibit YAP activity and promote YAP phosphorylation,while LIPUS activates YAP.It was concluded that LIPUS might inhibit the apoptosis and the autophagy of hPDLCs under the inflammatory environment through the promotion of YAP expression.