地黄梓醇对新生大鼠炎性成骨细胞的保护作用

Protection of inflammatory osteoblasts in neonatal rats using catalpol from the root of Rehmannia glutinosa

背景:有研究表明成骨细胞代谢异常也可能是膝骨关节炎中软骨下骨矿化异常的原因。目的:验证地黄梓醇对成骨细胞增殖及药物毒性研究,以及对脂多糖诱导的炎性成骨细胞的抗炎保护作用。方法:①新生SD大鼠成骨细胞进行原代提取、培养、传代,通过观察成骨细胞的形态、碱性磷酸酶染色以及矿化结节染色法(茜红素S法)染色,确定成骨细胞活性及增殖情况;②CCK-8法观察不同质量浓度地黄梓醇(0,1,10,100 mg/L)对成骨细胞活性的影响;③采用不同质量浓度脂多糖(0,10,20,40,80,160 mg/L)诱导胎鼠炎性成骨细胞模型,CCK-8法验证筛选最佳浓度;④实验分为正常组、模型组、低浓度地黄梓醇组、中浓度地黄梓醇组、高浓度地黄梓醇组,各组成骨细胞在80 mg/L脂多糖诱导为炎性成骨细胞后,分别给药8 h,CCK-8法观察不同质量浓度地黄梓醇对炎性成骨细胞的影响。实验方案经南京市六合区人民医院医学伦理委员会批准。结果与结论:①将成骨细胞培养至第4代,经碱性磷酸酶染色后可见阳性细胞胞浆内有灰黑色颗粒,细胞体形态呈不规则形;②地黄梓醇质量浓度低于1 mg/L时,对成骨细胞无明显影响;高于10 mg/L时,地黄梓醇对成骨细胞具有促进其增殖作用且无药物毒性作用(P<0.05);③不同质量浓度脂多糖对成骨细胞均有炎性损害,当其质量浓度高于80 mg/L时,对成骨细胞的炎性损伤呈显著趋势(P<0.01),因此实验选择80 mg/L为最佳损伤浓度;④地黄梓醇质量浓度低于1 mg/L时,对成骨细胞炎性反应无明显保护作用;高于10 mg/L时,对成骨细胞炎性反应能起到明显保护作用(P<0.05);⑤结果说明,地黄梓醇达到一定浓度时对成骨细胞无药毒作用且能促进增殖,对脂多糖诱导的炎性成骨细胞有抗炎保护作用。

BACKGROUND:Studies have shown that abnormal osteoblast metabolism may cause abnormal subchondral bone mineralization in knee osteoarthritis.OBJECTIVE:To verify the effects of catalpol from the root of Rehmannia glutinosa on osteoblast proliferation and drug toxicity,and the anti-inflammatory protective effect on inflammatory osteoblasts induced by lipopolysaccharide.METHODS:(1)Osteoblasts from newborn Sprague-Dawley rats were primarily extracted,cultured,and passaged.The activity and proliferation of osteoblasts were determined by observing the morphology of osteoblasts alkaline phosphatase staining and staining of mineralized nodules.(2)Cell counting kit-8(CCK-8)method was used to observe the effect of different concentrations of catalpol from the root of Rehmannia glutinosa on osteoblast activity.(3)Different concentrations of lipopolysaccharide(0,10,20,40,80,160 mg/L)were used to induce inflammatory osteoblasts in fetal rats.CCK-8 method was used to verify the best concentration.(4)The experiment was divided into three groups:blank group,model group,low-,medium-and high-concentration catalpol groups.Inflammatory osteoblasts were induced by lipopolysaccharide at 80 mg/L,and then treated for 8 hours.CCK-8 method was used to observe the effect of different concentrations of catalpol on inflammatory osteoblasts.The study protocol was approved by the Medical Ethics Committee of Liuhe District People’s Hospital in Nanjing.RESULTS AND CONCLUSION:When osteoblasts were cultured to the fourth generation,followed by alkaline phosphatase staining,black grey granules were found in the cytoplasm of the positive cells,and the morphology of the cells was irregular.When the concentration of catalpol was lower than 1 mg/L,it had no significant effect on osteoblasts;when it was higher than 10 mg/L,it had a significant effect on the proliferation of osteoblasts but no drug toxicity(P<0.05).When the concentration of lipopolysaccharide was higher than 80 mg/L,there was a significant trend of inflammatory damage to osteoblasts(P<0.01).Therefore,80 mg/L was selected as the best injury concentration in this experiment.When the concentration of catalpol from the root of Rehmannia glutinosa was lower than 1 mg/L,it had no significant protective effect on the inflammatory response of osteoblasts(P<0.05);when it was higher than 10 mg/L,it had significant protective effect on the inflammatory response of osteoblasts(P<0.05).Therefore,when the concentration of catalpol from the root of Rehmannia glutinosa reaches a certain level,it has no toxic effect on osteoblasts,promotes proliferation,and has anti-inflammatory protective effect on inflammatory osteoblasts induced by lipopolysaccharide.