基于RANKL/OPG探讨桂枝芍药知母汤对骨质疏松症模型大鼠的治疗作用

Therapeutic Effect of Guizhi Shaoyao Zhimutang on Osteoporosis in Rats Based on RANKL/OPG

目的基于大鼠卵巢切除术(OVX)联合糖皮质激素注射致骨质疏松模型探讨桂枝芍药知母汤(GSZT)对骨质疏松症(OP)模型SD雌鼠的治疗作用。方法OVX手术1周后,除假手术组外,大鼠分为OP模型组、骨化醇组(0.1 mg·kg^(-1)),GSZT低、中、高剂量组(0.8,1.6,3.2 g·kg^(-1))。除假手术组,大鼠肌肉注射地塞米松(1 mg·kg^(-1),2次/周),连续6周;给药8周后,处死动物收集股骨样本,计算机微断层扫描(Micro CT)扫描股骨远端1/3,检测骨矿物质密度(BMD),骨体积(BV),组织体积(TV),结构模式指数(SMI),骨小梁连接密度(Conn.D),骨小梁数目(Tb.N),骨小梁厚度(Tb.Th),和骨小梁分离度(Tb.Sp)。采用苏木素-伊红(HE)染色和马松(Masson)染色检查股骨组织病理学改变。采用抗酒石酸酸性磷酸酶(TRAP)染色分析骨组织中破骨细胞。采用酶联免疫吸附测定法(ELISA)检测骨组织基质金属蛋白酶-9(MMP-9),组织蛋白酶K(CTSK),抗酒石酸酸性磷酸酶5b(TRAP5b)的含量;蛋白免疫印迹法(Western blot)检测骨组织中核转录因子-κB受体活化因子配体(RANKL),骨保护素(OPG),碱性磷酸酶(ALP),骨碱性磷酸酶(BALP)蛋白表达。结果与正常组比较,OP模型大鼠骨松质部位骨小梁数量明显减少,间距增宽,厚度变薄,连续性差;与模型组比较,GZST中、高剂量组对股骨骨松质的骨小梁改变均有改善作用。Masson染色表明与正常组比较,OP模型组股骨远端骨骺生长板厚度变薄,新生骨面积变小,骨化醇及GZST高剂量组可抑制新生骨的减少。TRAP染色结果表明,与正常组比较,OP组破骨细胞数目明显增加,与模型组比较,GZST中、高剂量组可减少OP大鼠破骨细胞数量。Micro CT结果表明,与正常组比较,OP大鼠BMD,BV,Tb.N,Tb.Th和Conn.D减小,SMI和Tb.Sp显著升高(P<0.01);与模型组比较,GZST中、高剂量可改善OP大鼠骨质疏松程度,BV,Tb.N显著升高,SMI,Tb.Sp显著降低(P<0.01)。ELISA结果表明,与正常组比较,OP模型组ALP含量显著减少(P<0.01),MMP-9,TRAP5b和CTSK含量显著增多(P<0.01);与模型组比较,GSZD高剂量ALP含量显著升高(P<0.01),MMP-9,TRAP5b和CTSK显著减少(P<0.01)。Western blot结果表明,与模型组比较,骨化醇组及GSZD高剂量组均抑制RANKL蛋白的表达(P<0.05),提高OPG表达(P<0.05)。结论本研究表明GZST对OVX联合激素引起的大鼠骨质疏松具有治疗作用,其机制与调节RANKL,OPG相关,为其进一步的临床应用和现代制剂开发奠定了一定基础。

Objective To investigate the therapeutic effect of Guizhi Shaoyao Zhimutang(GSZT)on the osteoporosis(OP)in SD female rats induced by ovariectomy(OVX)combined with glucocorticoid injection and its related mechanisms.Method The rats were divided into a sham operation group and an experimental group for OVX.One week later,the experimental rats were divided into an OP model group,a calciferol(positive control,0.1 mg·kg^(-1))group,and low(0.8 g·kg^(-1)),medium(1.6 g·kg^(-1)),and high-dose(3.2 g·kg^(-1))GSZT groups.Except for those in the sham operation group,the rats received an intramuscular injection of dexamethasone(1 mg·kg^(-1))twice per week for six weeks.After the rats were treated correspondingly for eight weeks,the rats were sacrificed for thighbone sample collection.The computer microtomography(Micro CT)was used to analyze the parameters of bone mineral density(BMD),bone volume(BV),tissue volume(TV),structure model index(SMI),connectivity density(Conn.D),trabecular number(Tb.N),trabecular thickness(Tb.Th),trabecular pattern factor(Tb.Pf),and trabecular separation(Tb.Sp).In addition,hematoxylin-eosin(HE)and Masson staining was used for the histopathological examination of the thighbone.Tartrate-resistant acid phosphatase(TRAP)staining was used to analyze osteoclasts in bone tissues.The enzyme-linked immunosorbent assay(ELISA)was used to determine matrix metalloproteinase-9(MMP-9),cathepsin K(CTSK),and TRAP5b in bone tissues,and Western blot was carried out to determine receptor activator of NF-kappaB ligand(RANKL),osteoprotegerin(OPG),alkaline phosphatase(ALP)in bone tissues.Result Compared with normal group,the number of bone trabeculae in cancellous part of OP model rats decreased significantly,the spacing widened,the thickness became thinner,and the continuity was poor.Compared with model group,GZST medium and high-dose groups could improve the changes of femoral bone trabecular cancellous bone.Masson staining showed that compared with the normal group,the growth plate thickness of the distal femur epiphysis was thinner and the area of new bone was smaller in the OP model group,while the reduction of new bone was inhibited in the high dose group of calcitol and GZST.TRAP staining results showed that the number of osteoclasts in OP group was significantly increased compared with the normal group,and the number of osteoclasts in medium-high dose GZST group was reduced.Micro CT results showed that compared with normal group,BMD,BV,Tb.N,Tb.Th and Conn.D of OP rats were decreased,SMI and Tb.Sp were significantly increased(P<0.01).Compared with model group,medium and high dose of GZST could improve the degree of osteoporosis in OP rats,BV,Tb.N were significantly increased,SMI,Tb.Sp were significantly decreased(P<0.01).ELISA results revealed that compared with the sham operation group,the OP model group showed decreased ALP content(P<0.01)and increased MMP-9,TRAP5b,and CTSK(P<0.01).Compared with the OP model group,the high-dose GSZT group showed increased ALP content(P<0.01)and decreased MMP-9,TRAP5b,and CTSK(P<0.01).Western blot results demonstrated that compared with the OP model group,the calciferol group and the high-dose GSZT group showed inhibited expression of the RANKL protein(P<0.05),and increased OPG expression(P<0.05).Conclusion The findings suggest that GZST has a potential therapeutic effect on OP caused by OVX combined with glucocorticoids in rats,and the underlying mechanism is associated with the regulation of RANKL/OPG in bone tissues.This study is expected to lay a preliminary foundation for its clinical application and modern preparation development.