Effect Of Porphyromonas Gingivalis Lipopolysaccharide On The Biological Behaviors Ofbone Marrow-derived Macrophages From Osteoporotic Rats

Periodontitis is a chronic infectious disease characterized by periodontal support tissue destruction.Menopausal women are prone to postmenopausal osteoporosis(PMO)with decreasing estrogen levels.Periodontitis and PMO are mutually dangerous factors.Porphyromonas gingivalis lipopolysaccharide are the main virulence factor causing periodontitis,which induces immune responses in macrophages,leading to connective tissue destruction and alveolar bone resorption.In addition,P.gingivalis LPS can enter the blood circulation and affect various body organs.Bone marrow-derived macrophages(BMMs),as precursor cells of osteoclasts(OCs)in bone tissue,play an important role in the bone resorption activities motivated OCs.Objective To isolate and culture BMMs from osteoporotic rats in vitro and explore the effect of P.gingivalis LPS on the biological behaviors of BMMs from osteoporotic rats.Methods 1.The PMO model of SD rats was established by bilateral ovariectomy.BMMs from osteoporotic rats were isolated and cultured in vitro.2.Inverted microscope,Wright Giemsa staining and cytoskeletal staining were employed for morphological observation of BMMs.Cell surface markers were identified by flow cytometry on BMMs.3.The proliferation of BMMs from osteoporotic rats were detected by CCK-8 and Ed U flow cytometry.4.The polarization and inflammation-related factors of BMMs from osteoporotic rats were evaluated by immunofluorescence staining,Real-time PCR,Western Blot and ELISA.5.The osteoclastogenesis ability of BMMs from osteoporotic rats were evaluated by cytoskeletal staining and Real-time PCR.6.The effects of P.gingivalis LPS and E.coli LPS on the proliferation ability of BMMs from osteoporotic rats were detected by CCK-8 assay.7.Real-time PCR and Western Blot were used to detect the effects of P.gingivalis LPS and E.coli LPS on the polarization of BMMs from PMO rats.8.Real-time PCR and ELISA were used to detect the effects of P.gingivalis LPS and E.coli LPS on the expression of inflammation-related factors in BMMs from PMO rats.Results 1.PMO model was established successfully.The weight of rats increased significantly and bone resorption appeared in femur.2.Flow cytometry showed that BMMs expressed macrophage surface marker CD11 b.BMMs were mainly polygonal and omelette-like in osteoporotic rats.Compared with Sham group,the proliferation ability of osteoporotic rats BMMs was significantly elevated.3.Immunofluorescence staining,Real-time PCR and Western Blot showed that BMMs from osteoporotic rats had higher expression of M1 macrophage marker CCR7 and lower expression of M2 macrophage marker MRC1.4.Real-time PCR and ELISA demonstrated that the expression of pro-inflammatory factors of TNF-?,IL-6 and IL-1? increased in osteoporotic rats BMMs,while the expression of anti-inflammatory factors of IL-10 and TGF-? decreased.5.Cytoskeletal staining and Real-time PCR results showed that BMMs from PMO rats had stronger ability to differentiate into OCs under the action of M-CSF and RANKL.6.CCK8 assay results showed that P.gingivalis LPS and E.coli LPS promoted the proliferation ability of BMMs from PMO rats in the early stage,while inhibited the proliferation of BMMs in the late stage.7.Real-time PCR and Western Blot results showed that BMMs from PMO rats had higher expression of CCR7 and lower expression of MRC1 after stimulation with P.gingivalis LPS and E.coli LPS.8.Real-time PCR and ELISA demonstrated that the expression of pro-inflammatory factors of TNF-?,IL-6 and IL-1? increased in osteoporotic rats BMMs,while the expression of anti-inflammatory factors of TGF-? decreased after stimulation with P.gingivalis LPS and E.coli LPS.Conclusions 1.BMMs from osteoporotic rats tended to polarize toward M1 type and had stronger ability to differentiate into OCs.2.P.gingivalis LPS can affect the proliferative of BMMs from PMO rats;stimulate the polarization of BMMs to M1 type;upregulate the pro-inflammatory factors of TNF-?,IL-6 and IL-1? and reduce the expression of anti-inflammatory factor of TGF-?.