Preliminary Study On The Changes Of Skeleton Of Mice With Endotoxemia And Its Mechanism

Current studies on critical illness are mainly focused on several major vital organs, such as heart, lung, renal, blood and immune system, while there are few studies on the roles of skeletal system in critical illness such as inflammation, shock, sepsis. Recent studies demonstrated that bone cells have secretory functions similar to those of endocrine cell, bone cells can secrete osteocalcin to regulate blood sugar level. Furthermore, bone marrow is the source of a variety of stem cells and immune cells, and there is complicated interactions amomg bone cells and immune cells or stem cells. For example, bone cells can regulate the differentiation and mobilization of bone marrow stem cells, which reveals the critical role of bone in homeostasis. Actually, recent studies showed that systemic changes especial neuroendocrine system could affect the functions of osteoclasts and osteoblasts in patients with stress. Endotoxemia is the important factor which is responsible for sepsis even MODS pathogenesis, research on pathogenesis of endotoxemia is very important. Recently, more and more studies have been focusing on the functional injuries to organs caused by endotoxemia. Because of the interplay between stress and sepsis, we presumed that bone would have changes in endotoxemia, and these changes might affect the process and prognosis in critical illness.ObjectTo investigate the mechanisms for the endotoxin-caused injuries,we first made the endotoximia mouse model by injecting with lipopolysaccharide(LPS), then the gene expression and histilogical changes of bone(bone cells) were detected to explore the underlying mechanisms of LPS related damages.Materials and Methods1.To make and evaluate the endotoxemia mouse model ,twenty-four mice were separated into 8 groups: normal control, LPS4h, LPS6h, LPS8h, LPS12h, LPS24h, LPS48h and LPS72h, 3 mice per group,serum were used to detect liver and renal function respectively by automatic biochemistry analyzer, and the organs were taken out to evaluate inflammation by means of histological methods. Blood of three mice were draw in mouse tail at 0h , 4h, 6h, 8h, 12h, 24h, 48h and 72h after injection of LPS to count the number of the leucocytes .Eighteen mice were used to measure respiratory rate and mortality in a week follwoed.2. To measure the alterations of bone in endotoxemia mice models, the mice which were separated into 8 groups: normal control, LPS4h, LPS6h, LPS8h, LPS12h, LPS24h, LPS48h and LPS72h, 3 mice per group, among which were taken out for observation of bone pathological change by means of HE, the activity of osteoclasts were measured with TRAP staining. Tibiae were taken out to extract the whole RNA, and then the mRNA Levels of TLR4, HIF-1α, caspase 3, osteocalcin and cathepsin K were detected with realtime RT-PCR. The mice were divided into 4 groups: normal control group, 6h, 24h, 5d, 3 mice per group. The changes of bone cells were observed by means of scanning electron microscope and transmission electron microscope.Results1. General evaluation of endotoxemia model of mice: the 7 days mortality was 22.2%(4/18).Compared with the normal control , the respiratory rate was elevated at 30 min after injection of LPS (P <0.05), after 1 h,the respiratory rate was increased significantly, and recuperated to normal level at 72h. The number of leucocytes in LPS4h group was decreased compared with normal control group (P <0.01), then the leucocyte number was increased gradually, and reached to a high level at 72h (P <0.05).Compared with normal control, ALT level in LPS groups increased significantly from 4 to 8 h (P <0.05), and then decreased gradually after 8h (P <0.05). The increase of BUN start at 6h (P <0.05), keeping a higher level at 8h(P <0.05), then decreased gradually, and reached to a normal level at 12h(P >0.05).2. Changes of bone cells in endotoxemia mice :Compared with normal control group, osteoclast was activated begin at 4h after injection of LPS(P <0.05), increased significantly at 6h(P <0.01)and increased gradually. Through the SEM results on osteocyte, we found that there were slight collapse and reduced cytodendrite of cells at 6h after LPS administration. The obvious collapse and decrease of cytodendrite was found at 24h and both severe lesions were found at 5d after LPS administration. Normal cells appear to be full and cylinder shaped. But when LPS was administrated for 6h and 24h, cylinder shape became thiner and flat at 5d; at 6h, the osteoblast enlarged and cytodendrite increased, expanded and invested on the surface of bone. After 5d, there remains trails and pitting which were formed due to the absorption of bone matrix by osteoclast.Under the TEM, normal osteocytes exhibit clear cellular organelle. After 6h administration of LPS, the chromatin margination and slight collapse of cells were found; after 24h, collapse of osteocyte, widen low mineralization band and reduction of organelle were also found. 5d after injection of LPS, osteocytes present a morphology of apoptosis, collapse of osteocyte, widen low mineralization band and chromatin pyknosis were found. In normal cells, clear organelles, big nuclei and obvious nucleolus, and abundant RER were found. In LPS treated group, cell organelles were reduced, RER swelled. There were less nucleolus found in 5d treatment group. Compared with control, osteoclast spreading, cellular organelle increase and mitochondria swelling were observed in osteoclast after injection of LPS.3 .Gene expression change of bone tissuse in endotoxemia mice: the mRNA levels of TLR4 which is the receptor of LPS in LPS groups were higher than normal control group at 6h after injection of LPS(P <0.05), reaching the summit at 24h (P < 0.01), and a high level could still be found at 72h(P <0.05). The levels of caspase 3 which is related to apoptosis increased at 4h after injection of LPS(P <0.05), recuperations were observed at 24h. There was no significant difference compared with normal control group, the same changes were found in expressions of HIF-1αwhich is a hypoxia respond gene. The statistics results indicate there is a direct correlation between the expression of HIF-1αand caspase 3,the peason's coefficient of correlation r=0.71(P <0.05). Compared with normal control group, the mRNA expression levels of osteocalcin which is a bone matrix protein decreased obviously at 4h after injection of LPS, and there was nearly no expression at 12h, a extreme low level was still kept at 72h(P <0.01), cathepsin K mRNA increased in 4h, retrieving gradually at 6h after injection of LPS.Conclusions1. The endotoxemia model of mice performed successfully in this study, which provides a reliable basement for the subsequent experiments.2. Endotoxin can exert influences on bone cells by resulting in apoptosis of osteocytes, furthermore, it can cause not only the increase in numbers and functions of osteoclast but also the collapse and decrase of function of osteoblast.3. Bone can be impaired by endotoxin. The expressions of TLR4, HIF-1αand caspase 3 can increase in sepsis, moreover, there is a close relationship between HIF-1αand caspase 3, which demonstrate that there should be an intimate relationship between the increase of HIF-1αand bone cells apoptosis in sepsis.