Studies On The Effect Of LPS From Escherichia Coli On Osteoblasts And Bone Marrow-derived Monocytes In Periodontal Tissue, And Its Mechanism

Bone tissue is composed of two distinct coll populations, namely the bone-forming ostcoblasts (OBs) derived from the mesenchymal stromal lineage, and the bone-resorbing osteoclasts (OCs), which arc of haematopoietic stem cell origin. Previous studies indicated that alveotar bone destruction results from the increase of bone resorption by OCs, or the reduction of bone formation by OBs, and or an imbalance between the two aspects. Then, the gingival giant cells such as osteoblasts and bone marrow monocytes (BMMs) play important roles in periodontitis. In addition, lipopolysaccharide (LPS) from gram-negative bacteria such as Porphyromonas gingival (Pg), Actinobacillus actinomycctemcomitan (Aa) has been considered a key factor in the development of chronic inflammation leading to the destruction of periodontal tissue. However, the effect of LPS from Escherichia coli on the above cells remains unknown. This study was perfomed to observe the role of osteoblasts and BMMs in gingival resorption, and periodontal disease, and to understand its possible mechanism. And anti-resorptive drug was also investigated in this study.In the present study, confocal laser scanning microscopy, flow cytometry, and immunofluroscence and western Blots were used to observe the effect of E.coli-LPS on ostcoblasts differentiation and ostcoclasts resorption in vitro, and further to understand the role of phosphatidylinositol 3-kinasc/Akt (PI3K/Akt) pathway in the process. In addition, we examined the effects of puerarin on bone formation and LPS-induced osteolysis and studied their possible molecullar mechanisms.Calvaria ostcoblasts isolated from newborn SD rats were used to observe the effect of LPS on osteoblastic growth and differentiation. Data showed that LPS significantly inhibited osteoblastic bone formation, and resulted in osteoblastic apoptosis and F-actin rearrangment. This dysfuction was accompanied by loss of mitochondrial membrane potential (ΔΨm) and dephosphorylation of Akt. Inhibition of PI3K with LY294002 enhanced LPS-induced inhibition of ostcoblasts growth and dephosphorylation of Akt.Furthmore, the PI3K stimulator IGF-Ⅰwas found to significantly weaken LY294002-triggercd dephosphorylation of Akt and growth inhibition. But LPS effect on Akt was not affected by IGF-Ⅰ. This meant that LPS inhibited osteoblasts differentiation and induced cell apoptosis might be through inactivation of Akt, but independently of PI3K.To study whether LPS induced bone resorption, osteoclasts were obtained from bone marrow monocytes (BMMs) of 6-9 weeks SD rats, and found evidence of PI3K/Akt pathway in the process. Results indicated that LPS significantly stimulated osteoclasts formation, bone resorption and activation of Akt at Ser473 and Thr308, which was obviously blocked by another PI3K specific inhibitor Wortmanin. Furthermore, Wortmanin triggered rapid apoptosis of mature osteoclasts and Akt inavtivation. These findings suggested that LPS stimulated osteoclasts differentiation, resorption and survival, which might be mediated by activation of the PI3K/Akt signaling pathway.In addition, this study examined the effects of puerarin from Chinese Pueraria lobata (Wild.) Ohwi on bone formation and on LPS-induced osteolysis. Data suggested that puerarin caused a significant increase in cell viability, alkaline phosphatase (ALP) activity and mineral nodules formation in osteoblasts, suggesting that puerarin had a stimulatory effect on osteoblastic bone formation. The functional improvement by puerarin was accompanied by activation and translocation of Akt. Furthermore, the activation of Akt and its redistribution were blocked by LY294002. These results indicated that puerarin stimulated activation of Akt in a PI3K-dependent manner. Then, puerarin can promote bone formation in cultured rat osteoblasts, which might be mediated by activation of the PI3K/Akt pathway. On the other hand, puerarin significantly inhibited LPS-induced osteolysis, and triggered rapid apoptosis of mature osteoclasts and their precursor cells. And puerarin blocked LPS-stimulated Akt activation, and caused an obvious decrease in Akt phosphorylation at Ser473 and Thr308, implying an important role for Akt pathway in the anti-osteolysis action. These resuts indicated puerarin could protect against LPS-stimulated alveolar bone loss by triggering cell apoptosis and inactivation of Akt. Taken together, puerarin from Chinese Pueraria lobata (Wild.) Ohwi might stimulate bone formation and inhibit bone resorption by activation or inactivation of PI3K/Akt pathwy. Based on the above results, some innovative points have been drawn as below:1. LPS from Escherichia coli inhibited osteoblastic growth and differentiation, suggesting that E. coli-LPS may prevent new bone formation in gingival bone tissue.2. E. coli-LPS induced osteoclasts formation and bone resorption, indicating that E.coli-LPS can regulate periodontal BMMs, and result in the reduction of alveolar bone resorption.3. We studied the roe of PI3K/Akt pathway in the E.coli LPS-triggered osteoblasts apoptosis and osteolysis, and offerd a new signaling pathway for understanding periodontal bone resorption, and some academic data for developing anti-resorption drugs.4. Puerarin from Pueraria lobata (Wild.) Ohwi might stimulate bone formation and inhibit LPS-inducd bone resorption, which have therapeutic value in effective in treating alveolar bone destruction and periodontitis, and offer some data for its application in these diseases.