ERK5 Signaling Pathway Mediates Periodic Stretching Stress Promotes Osteoblasts Proliferation Under Pg-LPS Stimulation

Objective: Periodontal disease is one of the most common oral diseases in adults,and its incidence rate is relatively high.The incidence rate of periodontal disease in adult patients with malocclusion should be more serious,besides,most orthodontic patients suffer from periodontal tissue inflammation of different degrees due to the difficulty in cleaning appliances.At present,the research on osteogenic pathway has been in-depth,but there is little research on the mechanism of changes after applying force to osteoblasts under inflammatory condition.This experiment was to study the effect of periodic stretch stress on osteoblast proliferation under the stimulation of Pg-LPS,and to further explore the molecular biological mechanism of this effect.Methods: After 1 μg/mL Pg-LPS was added to osteoblasts for 24 hours,then 8% periodic stretching stress was applied for 8 hours,and 1)The proliferation activity of osteoblasts was detected by CCK-8 techniques;2)Detection of osteoblast differentiation ability by ALP activity;3)Osteoblasts MC3T3-E1 were cultured with 1 μg/mL Pg-LPS for 24 h,then 8% periodic stretch stress was applied for 8h,and the skeleton changes of the osteoblasts were observed under confocal microscope after phalloidin staining;4)Detect the expression changes of Runx-2,Cyclin D1,p-ERK5 and ERK5 protein in cells with Western blot technology,and qRT-PCR was used to detect the expression changes of Runx-2,Cyclin D1,and ERK5 mRNA;5)Osteoblasts were cultured with 1 μg/mL Pg-LPS for 24 h,treated with 5 μmol/L XMD8-92 for 1h and then subjected to periodic stretch stress with 8% elongation at 0.5 Hz for 8h.The expression changes of Runx-2,Cyclin D1,p-ERK5,ERK5 protein was detected by Western blot while the expression of Runx-2,Cyclin D1,ERK5 mRNA was detected by qRT-PCR;Results: 1)After applying tension,the osteoblast proliferation activity was enhanced;after 24 h of osteoblast stimulation with 1 μg/mL Pg-LPS,the cell proliferation activity decreased;while under the stimulation of Pg-LPS,the cell proliferation activity increased after the application of stretching stress;2)The ALP activity of osteoblasts composed of stretched cells was significantly higher than that of the normal group,the ALP activity of the LPS group was lower than that of the normal group,and the ALP activity of the “LPS + force” group was higher than that of the LPS group;3)Results of phalloidin-TRITC staining showed that the osteoblasts in the normal group were polygonal,and the F-actin microfilaments were interwoven with each other,with different directions and scattered and irregular arrangement.After 8 hours of treatment with 8% 0.5Hz stretch stress,the skelemins of osteoblasts were obviously remodeled,the cell morphology was changed,and the F-actin microfilaments were depolymerized and rearranged,showing obvious directivity,bundle-like arrangement,palisade-like,and nearly parallel to the long axis of cells.Under the stimulation of Pg-LPS,skelemins were remodeled and their morphology was also changed.It could be seen that after applied with stretch stress and stimulated by Pg-LPS,F-actin microfilaments in the cells were arranged in bundles,and the arrangement direction was similar to the long axis of the cells.4)Compared to the control group,the expression of Runx-2 and Cyclin D1 both increased and the ratio of p-ERK5 to ERK5 increased(P <0.05)in osteoblasts after periodic stretch stress was applied.After adding XMD8-92,a specific inhibitor of ERK5,the expression of all proteins decreased significantly,and the ratio of p-ERK5 to ERK5 also decreased(P <0.05).Under the stimulation of Pg-LPS,the protein expressions of Runx-2 and Cyclin D1 in osteoblasts decreased,and the ratio of p-ERK5 to ERK5 decreased;Compared with the LPS group,p-ERK5/ERK5 increased after Pg-LPS stimulation and subjected to periodic stretch stress,and the expression of each protein was increased(P <0.05);Compared with the“ LPS + force” group,the“ LPS + force + XMD8-92” group had lower p-ERK5/ERK5,and the protein expressions of Runx-2 and Cyclin D1 were down-regulated(P <0.05).4)The expression of Runx-2,Cyclin D1 and ERK5 mRNA in osteoblasts were all up-regulated after 8% periodic stretch stress was applied for 8h,compared to the normal group(P <0.05).The expression of each mRNA decreased significantly after the inhibitor XMD8-92 was added(P <0.05).Under Pg-LPS stimulation,the expression of Runx-2,Cyclin D1,and ERK5 mRNA in osteoblasts was down-regulated.Compared with the LPS group,the expressions of Runx-2,Cyclin D1 and ERK5 mRNA were all up-regulated after tension was applied(P <0.05).Compared with the “LPS + force” group,in the “LPS + force + XMD8-92” group,the expression of each mRNA increased(P <0.05).Conclusion: 1)After 1 μg/mL Pg-LPS was applied to osteoblast MC3T3-E1 for 24 hours,osteoblast proliferation and differentiation activity were reduced;0.5 Hz 8% periodic stretch stress was applied to osteoblast MC3T3-E1 for 8 h,proliferation and differentiation activity of cells increased;2)Propriate periodic tensile stress has protective effect on the decrease of osteoblast proliferation and differentiation activity induced by Pg-LPS;3)Periodic stretch stress can promote ERK5 Phosphorylation and activate ERK5 signaling pathway;4)Periodic stretch stress can mediate ERK5 signaling pathway to promote Runx-2 and Cyclin D1 protein and mRNA expression,thereby promoting osteoblast proliferation;5)Periodic stretch stress can reverse the down-regulation of Runx-2 and Cyclin D1 mRNA induced by Pg-LPS through the ERK5 signaling pathway,thereby protecting the reduced proliferation activity of osteoblasts induced by Pg-LPS.