淫羊藿苷通过RANKL-p38/ERK-NFAT通路抑制硫代乙酰胺诱导的破骨分化  

Icariin inhibits thioacetamide-induced osteoclast differentiation through RANKL-p38/ERK-NFAT pathway

作　　者：程琳燕 金晓丽 陈煊威 陈瑾[1] 任军[1] 黄慧[1] 许健[1] CHENG Lin-yan;JIN Xiao-li;CHEN Xuan-wei;CHEN Jin;REN Jun;HUANG Hui;XU Jian(Schoo of Medical Technology and Information Engineering,Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学医学技术与信息工程学院,浙江杭州310053

出　　处：《中国中药杂志》2022年第21期5882-5889,共8页China Journal of Chinese Materia Medica

基　　金：浙江省自然科学基金项目(LY19H060001);浙江省中医药科技计划项目(2022ZB093)。

摘　　要：探究淫羊藿苷(icariin, ICA)对硫代乙酰胺(thioacetamide, TAA)诱导的大鼠股骨骨溶解的治疗作用和机制。用TAA和ICA干预RAW264.7细胞,CCK-8法检测TAA和ICA对细胞增殖活力的影响,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色检测破骨细胞形成情况,Western blot和免疫荧光法检测细胞中破骨相关蛋白TRAP、cathepsin K、c-Fos和NFATc1的表达水平。SD大鼠32只,随机分为对照组、TAA组(TAA腹腔注射300 mg·kg-1)、ICA组(ICA灌胃600 mg·kg-1),TAA+ICA组(TAA腹腔注射300 mg·kg-1同时ICA灌胃600 mg·kg-1),隔天给药1次,持续6周。处死时记录体质量和股骨长度,苏木精-伊红(hematoxylin-eosin, HE)和TRAP染色观察股骨病理损伤和破骨细胞分化情况,检测血清中骨代谢相关指标碱性磷酸酶(alkaline phosphatase, ALP)、钙(Ca)、磷(P)、镁(Mg)和Ⅰ型胶原N末端肽(typeⅠcollagen crosslinked N-telopeptides, NTX-I)的变化,三点弯曲实验和micro-CT评估股骨质量,Western blot法检测破骨相关蛋白TRAP、cathepsin K、RANK、RANKL、p38、p-p38、ERK、p-ERK、JNK、p-JNK、c-Fos和NFATc1的表达水平。结果发现,ICA可以抑制TAA诱导的TRAP阳性细胞的产生,抑制破骨相关蛋白的表达并抑制NFATc1的核易位。ICA可以缓解TAA诱导的SD大鼠体质量和股骨长度减少及生长受抑,改善TAA造成的股骨弹性模量的下降,使骨小梁骨密度(bone mineral density, BMD)、骨小梁形态因子(trabecular pattern factor, Tb.Pf)、骨小梁数量(trabecular number, Tb.N)、骨小梁厚度(trabecular thickness, Tb.Th)和结构模型指数(structure model index, SMI)等参数显著恢复,从而改善骨结构。Western blot结果显示,ICA通过RANKL-p38/ERK-NFATc1信号通路抑制由TAA导致的大鼠股骨破骨分化。ICA通过下调RANKL-p38/ERK-NFAT信号通路抑制破骨细胞分化,防止TAA诱导的骨溶解。This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg), ICA group(gavage of ICA at 600 mg·kg) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kgand gavage of ICA at 600 mg·kg). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type Ⅰ collagen(NTX-Ⅰ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoc

关 键 词：硫代乙酰胺 淫羊藿苷 破骨分化 骨溶解 核因子ΚB受体活化因子配体 

分 类 号：R285.5[医药卫生—中药学]