肾阳虚大鼠血清对OB-OC共育体系中β-catenin/OPG/RANKL表达的影响及淫羊藿苷的调控作用

Effect of Serum from Kidney Yang Deficiency Rats on Expression of β-catenin/OPG/RANKL in OB-OC Co-culture System and Regulatory of Icariin

目的:观察肾阳虚大鼠血清对共育体系中骨保护素(OPG),胞质蛋白(β-catenin)及核转录因子-κB受体活化因子配体(RANKL)蛋白表达的影响及淫羊藿苷对其的调控,进一步探究其诱导骨质疏松发生的可能机制。方法:取16只雄性SD大鼠,随机分为空白组和模型组,每组8只,模型组以10 mL·kg^-1腺嘌呤灌胃建立肾阳虚模型,造模成功后收集血清。体外分离培养成骨细胞(OB)和破骨细胞(OC),采用茜素红、碱性磷酸酶(ALP)及姬姆萨染色观察并鉴定OB,OC用抗酒石酸酸性磷酸酶(TRAP)染色鉴定,transwell小室建立OB-OC共育体系,设置淫羊藿苷组(100μmol·L^-1),空白组,淫羊藿苷(100μmol·L^-1)+血清组(10%肾阳虚血清),血清组(10%肾阳虚血清)及Dickkopf1(DKK-1)药物组(100μg·L^-1),干预共育体系2 d后,对OC进行计数,微板酶标法检测培养液中TRAP及ALP的含量,蛋白免疫印迹法(Western blot)检测各组OPG,β-catenin及RANKL蛋白的表达。结果:与空白组比较,血清组ALP活性,β-catenin及OPG蛋白表达均明显降低(P<0.05),而OC数量,TRAP活性及RANKL蛋白的表达明显升高(P<0.05);与血清组比较,淫羊藿苷组ALP活性显著降低(P<0.01),与淫羊藿苷组比较,淫羊藿苷+血清组ALP活性及OPG蛋白表达明显降低(P<0.05),TRAP活性及RANKL表达明显升高(P<0.05)。结论:肾阳虚大鼠血清能诱导骨质疏松的发病,其作用机制可能是通过降低ALP活性,下调OPG及β-catenin蛋白的表达,同时提高TRAP活性,上调RANKL蛋白的表达来实现的。

Objective:To observe the effect of serum of kidney Yang deficiency rats on the expression of β-catenin,osteoprotegerin(OPG)and nuclear transcription factor-κB receptor activator ligand(RANKL)in the co-culture system and regulatory of icariin on it,and to explore the possible mechanism of inducing osteoporosis. Method:The 16 male SD rats were randomly divided into blank group and model group,8 rats in each group. 10 mL·kg^-1 adenine was administrated to stomach to establish kidney yang deficiency model. Serum was separated and extracted after the model was established successfully. Isolation and culture of osteoblast(OB)and osteoclast(OC)in vitro,OB was observed and identified by alkaline phosphatase(ALP),alizarin red and Giemsa staining,OC was identified by tartrate resistant acid phosphatase(TRAP) staining,OB-OC coculture system was established in transwell cell,icariin group(100 μmol·L^-1),blank group,icariin(100 μmol·L^-1)+ serum group,serum group and Dickkopf1(DKK-1)drug(100 μg·L^-1)were set up in group,2 days after intervention of co-culture system,OC was counted,ALP and TRAP in supernatant were detected by microplate enzyme labeling,and the expression of OPG,β-catenin and RANKL in each group was detected by Western blot. Result: Compared with blank group,the ALP activity,β-catenin and OPG protein expression in serum group were significant reduction(P<0. 05), while the OC quantity,TRAP activity and RANKL protein expression were marked increase(P<0. 05). Compared with serum group,ALP activity of icariin group decreased significantly(P<0. 01),Compared with icariin group,ALP activity and OPG protein expression decreased(P<0. 05),trap activity and RANKL expression increased(P <0. 05) in icariin + serum group.Conclusion: The serum of kidney Yang deficiency rats can induce the occurrence of osteoporosis,and the mechanism of action may be through inhibition of ALP activity, down regulating the expression of β-catenin and OPG protein,increasing the activity of TRAP and the expression of RANKL protein.