Abstract: BACKGROUND: Sepsis-induced systemic inflammation leads to rapid bone mass loss; however, there is a lack of effective treatments. Ulinastatin is an anti-inflammatory drug, but its protective effect and mechanism on bone under sepsis-induced systemic inflammation are still unclear. 
OBJECTIVE: To explore whether ulinastatin can relieve acute bone loss caused by lipopolysaccharide. 
METHODS: (1) Animal experiment. Thirty male C57BL/6 mice were randomly divided into three groups (n=10 per group): control group, model group and experimental group. The control group was injected intraperitoneally with normal saline, the model group was injected intraperitoneally with lipopolysaccharide, and the experimental group was injected intraperitoneally with lipopolysaccharide and ulinastatin. In the experimental group, ulinastatin was injected continuously for 3 days. After intraperitoneal injection of ulinastatin for 14 days, femoral tissues were taken for CT scanning and pathological observation. (2) Cell experiment. C57BL/6 mouse primary osteoblasts were isolated and divided into three groups: the control group was routinely cultured, lipopolysaccharide was added to the model group, and lipopolysaccharide with ulinastatin was added to the experimental group. Cell proliferation and osteogenic differentiation were detected. C57BL/6 mouse bone marrow mononuclear cells were isolated and divided into three groups: the control group was routinely cultured, lipopolysaccharide was added to the model group, and lipopolysaccharide and ulinastatin were added to the experimental group. Osteoclast differentiation was detected.
RESULTS AND CONCLUSION: (1) Animal experiment. CT scanning and hematoxylin-eosin staining showed that bone mass in lipopolysaccharide-treated mice was reduced but increased after treatment with ulinastatin.  Tartrate resistant acid phosphatase staining showed that the number of osteoclasts in bone tissue increased in the model group, but significantly decreased in the experimental group compared with the model group. (2) Cell experiment. Cell counting kit-8 assay showed that lipopolysaccharide treatment inhibited the proliferation of osteoblasts, and ulinastatin elevated the proliferation of osteoblasts after lipopolysaccharide treatment. Alkaline phosphatase staining, alizarin red staining and osteogenesis-related gene (alkaline phosphatase, Runx2, osteocalcin, osteoblastin, nuclear factor κB receptor-activating factor ligand, osteoprotegerin) detection showed that lipopolysaccharide treatment inhibited osteogenic differentiation of osteoblasts and elevated the nuclear factor κB receptor-activating factor ligand/osteoprotegerin ratio; ulinastatin did not have any significant effect on the reduction of osteoblast function induced by lipopolysaccharide but decreased the nuclear factor κB receptor-activating factor ligand/osteoprotegerin ratio. Tartrate resistant acid phosphatase staining and osteoclast-related gene (tartrate resistant acid phosphatase and matrix metalloproteinase 9) detection showed that lipopolysaccharide treatment could promote osteoclast differentiation of bone marrow monocytes, while ulinastatin could inhibit lipopolysaccharide-induced osteoclast differentiation of bone marrow monocytes. (3) Overall, ulinastatin can significantly inhibit lipopolysaccharide-induced bone loss, mainly through promoting osteoblast proliferation and directly or indirectly inhibiting osteoclast differentiation to alleviate bone loss and achieve osteoprotective effects.

Key words: lipopolysaccharide, ulinastatin, sepsis, osteoblast, osteoclast, bone loss