骨疏康颗粒影响破骨细胞外泌体治疗绝经后骨质疏松的机制研究

基金项目:国家自然科学基金青年项目（No. 82204829）;国家自然科学基金面上项目（No. 82274272，No. 82074183）;浙江省自然科学基金探索项目（No. LBY22H270005）;浙江省中医药科技计划重点项目（No. 2022ZZ020）;北京长江药学发展基金会沃华科研基金青年激励项目（No. BYPDF2231202）

作者	单位
唐彬彬;袁一峰;刘康;黄海;周航;史晓林	1.浙江中医药大学附属第二医院骨伤二科（杭州 310005）,2.浙江中医药大学第二临床医学院（杭州 310053）

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中文摘要:

目的探究骨疏康颗粒（GSK）通过影响破骨细胞（OC）源性外泌体治疗绝经后骨质疏松（PMOP）的潜在机制。方法细胞实验建立OC和成骨细胞（OB）模型，制备GSK含药血清和对照血清，设空白组、对照组、对照外泌体组（对照血清）、药物外泌体组（GSK含药血清）。Western Blot检测两种血清干预OC后分泌的外泌体蛋白，电镜检测外泌体形态，荧光标记检测外泌体被OB摄取情况。随后开展OC来源外泌体干预OB模型研究。采用碱性磷酸酶（ALP）试剂盒检测ALP表达，Real-time PCR技术检测骨钙素（OCN）、骨桥蛋白（OPN）、Runt相关转录因子2（RUNX2）基因表达情况。动物实验建立PMOP模型，分为假手术组，模型组和药物实验组，每组6只大鼠。模型组进行生理盐灌胃，药物实验组进行GSK灌胃。干预8周后处死，取各组大鼠股骨组织进行HE和TRAP染色，观察骨细胞分化情况。采用Western Blot检测股骨组织OCN、OPN、RUNX2、核因子κB受体活化因子配体（RANKL）、骨保护素（OPG）蛋白表达情况。结果实验成功构建OC、OB、PMOP大鼠模型。对照血清和GSK含药血清干预OC模型均表达出CD9、CD63、CD81蛋白，且两种血清处理后OC外泌体均能被OB摄取。对照外泌体组及药物外泌体组均抑制成骨分化因子ALP、OCN、OPN、RUNX2 mRNA的表达，但后者的抑制作用减弱（P＜0.01）。体内实验结果显示，与模型组比较，大鼠股骨组织染色提示药物实验组骨小梁中破骨陷窝减少，骨密质部位破损现象减轻，骨吸收减少；药物实验组大鼠股骨组织中OB分化的标记物OCN、OPN、RUNX2蛋白表达均显著增加，同时RANKL、OPG蛋白表达均降低（P＜0.01）。结论 GSK含药血清可以抑制OC分化，有效改善OC外泌体对OB分化的抑制作用。另外，GSK能有效促进PMOP模鼠OB分化，同时减少OC分化。

英文摘要:

Objective To explore the underlying mechanism of Gushukang Granule（GSK）in treating postmenopausal osteoporosis（PMOP） via tuning osteoclast（OC）derived exosomes. Methods OC and osteoblast （OB） models were established. Additionally， control serum and GSK-containing serum were prepared for experimentation. Blank group，control group，control exosome （control serum）group and drug exosome （GSK-containing serum）group were set up. Western Blot analysis was used to detect OC-derived exosome levels，while the morphology of the exosomes was examined using transmission electron microscopy. Additionally，fluorescence labeling was used to investigate the uptake of exosomes by OBs. Intervention experiments were then conducted on the OB model. Alkaline phosphatase（ALP） expression was detected by ALP kit，and the gene expression of osteocalcin（OCN），osteopontin（OPN） and RUNt-associated transcription factor 2 （RUNX2） were detected by Real-time PCR. PMOP rat model was established in animal experiments， and the rats were divided into sham operation group，model group，and drug experimental group，6 in each group. The model group was given physiological salt gavage，and the drug experimental group was given GSK gavage. After 8 weeks，all animals were euthanized，and their femoral tissues were collected. Hematoxylin and eosin（HE） and TRAP staining were used to investigate osteocyte differentiation in these tissues. Additionally，OCN，OPN，RUNX2，receptor activator of nuclear factor-κ B ligand（RANKL），and osteoprotegerin（OPG）protein expressions in the collected tissues were detected by Western Blot analysis. Results The OC，OB，and PMOP models were successfully established. Subsequently，interventions were performed on the OC models using the control serum and the GSK-containing serum. The CD9， CD63，and CD81 protein expressions were specifically examined，and it was found that OBs effectively absorbed OC-induced exosomes. Exosomes extracted from the control exosome group and dug exosome group downregulated mRNA expressions of OB differentiation factors such as ALP，OCN，OPN，and RUNX2，the inhibitory effect on OB differentiation was markedly reduced in the drug exosome group（P<0.01）. In vivo results showed that compared with the model group， the staining results revealed that the drug experimental group had fewer broken bone lacunae in the trabecular bone，less damage to the compact bone，and less bone resorption. Furthermore，compared with the model group，the drug experimental group exhibited significantly higher expression levels of OB markers such as OCN，OPN，and RUNX2 and significantly lower RANKL and OPG expression levels in the femoral tissue（P<0.01）. Conclusions The GSK-containing serum inhibited OC differentiation and effectively improved the inhibitory effect of OC-induced exosomes on OB differentiation. Furthermore，GSK was found to significantly accelerate OB differentiation while simultaneously decelerating OC differentiation in PMOP rats.

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