骨碎补抑制骨髓腔内脂肪异常积累引起的脂毒性介导的成骨细胞焦亡及其机制  

The mechanism of inhibition of lipotoxicity-induced pyroptosis in osteoblasts caused by abnormal intramedullary fat accumulation with rhizoma drynariae

作　　者：高宽惠 李志超[1] 谭国庆[2] 薛海鹏[2] 汪陈莫及 张加豪 徐展望[1,2] GAO Kuanhui;LI Zhichao;TAN Guoqing;XUE Haipeng;WANG Chenmoji;ZHANG Jiahao;XU Zhanwang(Shandong University of Traditional Chinese Medicine,Ji’nan 250014,China;The Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Ji’nan 250014,China)

机构地区：[1]山东中医药大学,山东济南250014 [2]山东中医药大学附属医院,山东济南250014

出　　处：《中国骨质疏松杂志》2024年第8期1133-1139,共7页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金面上项目(82174410);山东省自然科学基金重点项目(ZR2020KH011);山东省自然科学基金面上项目(ZR2020MH362);全国名老中医药专家传承工作室建设项目(国中医药人教函[2022]75号)。

摘　　要：目的探讨骨碎补(rhizoma drynariae,Rhd)对骨髓腔内脂肪异常积累引起的脂毒性介导成骨细胞焦亡的影响及其机制。方法体内构建卵巢摘除(ovariectomy,OVX)骨质疏松小鼠模型,用Rhd进行灌胃,给药8周后称重,取股骨组织和血清。HE染色观察髓腔内脂肪堆积状态。体外构建成脂-成骨细胞共培养系统,茜素红S(alizarin red S,ARS)染色观察钙化结节的数量,碱性磷酸酶(alkaline phosphatease,ALP)染色检测碱性磷酸酶活性水平,乳酸脱氢酶(lactate dehydrogenase,LDH)检测和Hoechst33342/PI染色检测共培养下成骨细胞(osteoblasts,OBs)焦亡水平,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞培养上清液中白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-18(interleukin-18,IL-18)的释放量,Western blot检测成骨能力相关蛋白Runt相关转录因子2(runt-related transcription factor 2,RUNX2)、骨形态发生蛋白2(bone morphogenetic protein,BMP2)和1型胶原蛋白(collagen-1,COL-1)。选择NLRP3特异性抑制剂MCC950作为阳性对照检测焦亡相关蛋白NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、半胱天冬酶-1(cysteinyl aspartate specific proteinase-1,CASP-1)、效应蛋白消皮素D(gasdermin D,GSDMD)、IL-1β和IL-18的表达。结果OVX小鼠髓腔内发生骨质流失伴大量脂肪堆积,Rhd灌胃可以有效缓解这一趋势。在成脂-成骨共培养系统中,共培养环境抑制了OBs活性和OBs中ALP活性及矿化结节,抑制了RUNX2、BMP2、COL-1蛋白表达,并且诱导焦亡发生,提高了LDH释放量和PI染色细胞阳性率,以及上调NLRP3、CASP-1、GSDMD、IL-1β和IL-18蛋白表达水平。而这一趋势在Rhd干预后得到逆转。结论大量脂肪堆积引起的脂毒性可以抑制OBs活性并通过激活NLRP3炎症小体诱导OBs焦亡,而Rhd可能通过抑制NLRP3炎症小体的激活以抑制OBs焦亡和炎性反应,从而起到抗骨质疏松的�Objective To explore the effect and mechanism of rhizoma drynariae(Rhd)on lipotoxicity-mediated osteoblast pyroptosis caused by abnormal fat accumulation in the bone marrow cavity.Methods An ovariectomy(OVX)osteoporosis mouse model was constructed in vivo.Rhd was administered with gavage.After 8 weeks of administration,mice were weighed and the femoral tissue and serum were collected.HE staining was used to observe the fat accumulation status in the medullary cavity.An adipogenic-osteoblast co-culture system was constructed in vitro.Alizarin red S(ARS)staining was used to observe the number of calcified nodules.Alkaline phosphatase(ALP)staining was used to detect the alkaline phosphatase activity level.Lactate dehydrogenase(LDH)detection and Hoechst33342/PI staining were used to detect the pyroptosis level of osteoblasts(OBs)in co-culture.Enzyme-linked immunosorbent assay(ELISA)was used to detect the release amounts of interleukin-1β(IL-1β)and interleukin-18(IL-18)in cell culture supernatant.Western blotting analysis was employed to quantify the expression levels of osteogenic capacity-related proteins,including runt-related transcription factor 2(RUNX2),bone morphogenetic protein 2(BMP2),and collagen-1(COL-1).The NLRP3 specific inhibitor MCC950 was selected as a positive control to detect the pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3(NLRP3)and cysteinyl aspartate specific proteinase-1(CASP-1),Gasdermin D(GSDMD),IL-1β,and IL-18.Results Bone loss and massive fat accumulation occurred in the medullary cavity of OVX mice.Intragastric administration of Rhd could effectively alleviate this trend.In the adipogenic-osteogenic co-culture system,the co-culture environment inhibited the activity of OBs,ALP activity,and mineralized nodules in OBs,inhibited the expressions of RUNX2,BMP2,and COL-1 proteins,induced pyroptosis,and increased LDH.Release amount of LDH and PI positive cell staining rate increased.NLRP3,CASP-1,GSDMD,IL-1βand IL-18 protein expression levels were up

关 键 词：骨质疏松症 骨碎补 细胞焦亡 脂毒性 成骨细胞 

分 类 号：R681[医药卫生—骨科学] R285.5[医药卫生—外科学]