乌司他丁对脓毒症急性骨丢失的保护作用

Protective effect of ulinastatin on acute bone loss in sepsis

背景:在脓毒症引发的全身炎症情况下机体会出现骨量快速丢失的现象,目前尚缺乏有效的治疗办法。乌司他丁作为一种抑炎类药物在全身炎症情况下对骨骼的具体保护作用和机制尚不明确。目的:探讨乌司他丁对脂多糖导致的急性骨丢失是否有缓解作用。方法:(1)动物实验:将30只雄性C57BL/6小鼠随机分为3组,对照组腹腔注射生理盐水,模型组腹腔注射脂多糖,实验组腹腔注射脂多糖与乌司他丁,其中乌司他丁连续注射3 d,每组10只。乌司他丁注射14 d后,进行股骨CT扫描与病理切片观察。(2)细胞实验:分离培养C57BL/6小鼠颅骨原代成骨细胞,分3组处理,对照组常规培养,模型组加入脂多糖,实验组加入脂多糖与乌司他丁,检测细胞增殖与成骨分化情况。分离培养C57BL/6小鼠骨髓单核细胞,分3组处理,对照组常规培养,模型组加入脂多糖,实验组加入脂多糖与乌司他丁,检测破骨分化情况。结果与结论:(1)动物实验:CT扫描与苏木精-伊红染色显示,脂多糖处理后小鼠骨量减少,乌司他丁可提升脂多糖处理后小鼠的骨量。抗酒石酸酸性磷酸染色显示,模型组骨组织破骨细胞数量增加,实验组骨组织破骨细胞数量较模型组显著减少。(2)细胞实验:CCK-8检测显示脂多糖处理抑制了成骨细胞的增殖,乌司他丁可提升脂多糖处理后成骨细胞的增殖。碱性磷酸酶染色、茜素红染色及成骨相关基因(碱性磷酸酶、Runx2、骨钙素、骨桥素、RANKL、OPG)检测显示,脂多糖处理可抑制成骨细胞的成骨分化,提升RANKL/OPG比值;乌司他丁对脂多糖诱导的成骨细胞功能降低无明显作用,但可降低RANKL/OPG比值。抗酒石酸酸性磷酸酶与破骨相关基因(抗酒石酸酸性磷酸酶、基质金属蛋白酶9)检测显示,脂多糖处理可促进骨髓单核细胞的破骨分化,而乌司他丁可抑制脂多糖诱导的骨髓单核细胞破骨分化。(3)乌司他丁可明显抑制脂多糖引起的骨丢失,主要通过促进成骨细胞增殖、直接或间接抑制破骨细胞分化来缓解骨丢失,达到骨保护的作用。

BACKGROUND:Sepsis-induced systemic inflammation leads to rapid bone mass loss;however,there is a lack of effective treatments.Ulinastatin is an antiinflammatory 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 factorκB receptor-activating factor ligand,osteoprotegerin)detection showed that lipopolysaccharide treatment inhibited osteogenic differentiation of osteoblasts and elevated the nuclear factorκB 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 factorκB 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.