IL-37通过调控M-CSF和抑制IL-6-JAK2/STAT3信号通路抑制骨质疏松的机制研究

IL-37 inhibits osteoporosis via regulating M-CSF and inhibiting IL-6-JAK2/STAT3 signaling pathway

目的:探讨IL-37在抑制骨质疏松过程中的作用机制。方法:选取本院2013年1月至2015年12月收治的97例骨质疏松患者及在本院行骨折手术的81例无骨质疏松患者(对照组)为研究对象,检测两组血清中IL-37及IL-6的水平。构建IL-37转基因小鼠,将C57BL/6J小鼠、IL-37转基因小鼠分别设置假手术(Sham)组,手术组(卵巢切除术,OVX组)。8周后,取小鼠血清,检测血清中雌激素水平、碱性磷酸酶水平(ALP)、血钙和血磷水平;同时取小鼠的双侧股骨、脊柱,病理切片分析股骨组织形态结构,骨密度仪检测脊柱骨密度变化。分离培养各组小鼠骨髓基质细胞(Bone marrow stromal cells,BMSCs),检测BMSCs的体外增殖能力,M-CSF及IL-6的表达及STAT3的激活。IL-37转染小鼠成骨细胞MC3T3-E1,转染后72 h,ELISA检测上清中M-CSF及IL-6,流式细胞术检测MC3T3-E1细胞的凋亡,Western blot检测STAT3的激活。结果:骨质疏松患者血清中IL-37水平显著低于对照组(P<0.05),而IL-6则显著高于对照组;C57BL/6J小鼠、IL-37转基因小鼠OVX组血清中雌激素、血钙和血磷显著低于假手术组,而ALP水平显著高于假手术组(P<0.05),但IL-37转基因小鼠OVX组血钙和血磷则显著高于C57BL/6J小鼠OVX组(P<0.05)。股骨病理切片及脊柱骨密度结果显示,C57BL/6J小鼠、IL-37转基因小鼠OVX组均出现组织形态结构的破坏和骨密度下降,但IL-37转基因小鼠明显优于C57BL/6J小鼠(P<0.05)。IL-37转基因小鼠OVX组BMSCs增殖能力显著高于C57BL/6J小鼠OVX组,而STAT3的激活和M-CSF的表达则显著低于C57BL/6J小鼠OVX组(P<0.05)。MC3T3-E1细胞转染IL-37后能明显抑制M-CSF及IL-6的表达,而STAT3的激活也明显被抑制,流式细胞检测显示转染IL-37后能显著抑制MC3T3-E1细胞的凋亡。结论:骨质疏松患者血清IL-37水平显著降低,IL-37可能是通过调控M-CSF及IL-6-JAK2/STAT3信号通路促进BMSCs增殖和抑制成骨细胞的凋亡从而抑制骨质疏松的进展。

Objective： To investigate the mechanism of IL-37 in inhibiting osteoporosis. Methods： Ninety-seven patients with osteoporosis and eighty-one fracture surgery patients without osteoporosis in our hospital from Jan 2013 to Dec 2015 were selected as study subjects. The serum level of IL-37 and IL-6 were detected. Construction of IL-37 transgenic mice,the C57BL/6J mice,IL-37 transgenic mice were set in sham operation group（ Sham）,operation group（ ovariectomy,OVX） respectively. The serum level of estrogen,alkaline phosphatase（ ALP）,calcium and phosphorus were detected after 8 weeks. The bilateral femur and spine of mice were collect after sacrifice,the morphology and structure of the femur were analyzed,and the bone density was measured by bone density meter. The bone marrow stromal cells（ BMSCs） were isolated and cultured in vitro. The proliferation ability of BMSCs,expression of M-CSF and IL-6,and the activation of STAT3 were detected. IL-37 was transfected into mouse osteoblast MC3T3-E1 cell,M-CSF and IL-6 in cultured supernatant were measured by ELISA. Apoptosis of MC3T3-E1 cells were detected by flow cytometry. The activation of STAT3 was detected by Western blot. Results： The serum level of IL-37 in patients with osteoporosis were significantly lower than control group（ P0. 05）,while IL-6 was significantly higher than control group（ P0. 05）. The serum level of estrogen,calcium and phosphorus in OVX group of C57BL/6J mice and IL-37 transgenic mice were significantly lower than the sham operation group（ P0. 05）,while the level of ALP was significantly higher than sham operation group（ P〈0. 05）,but the serum level of calcium and phosphorus in OVX group of IL-37 transgenic mice were significantly higher than C57BL/6J mice（ P0. 05）. The pathological section of femur and spine BMD showed that the bone tissue in C57BL/6J mice and IL-37 transgenic mice in OVX group were damaged and the bone density decreased significantly,but IL-37 transgenic mice was significantly better than C57BL/6J mice（ P〈0. 05）. The proliferation ability BMSCs in OVX group of IL-37 transgenic mice was significantly higher than C57BL/6J,while the activation of STAT3 and expression of M-CSF were significantly lower than C57BL/6J（ P0. 05）. The expression of M-CSF and IL-6 were inhibited after MC3T3-E1 cell was transfected with IL-37,and the activation of STAT3 were also inhibited after IL-37 transfection. The results of flow cytometry showed that IL-37 could significantly inhibit the apoptosis of MC3T3-E1 cells. Conclusion： The serum level of IL-37 in patients with osteoporosis decreased significantly. IL-37 may inhibit the proliferation of BMSCs and inhibit the apoptosis of osteoblasts by regulating the expression of M-CSF and the activation of IL-6-JAK2/STAT3 signaling pathway.