桑白皮总黄酮联合知母总皂苷改善高脂血症伴骨质疏松症大鼠的作用机制

Effect Mechanism of Total Flavonoid of Mori Cortex Combined with Total Saponins of Anemarrhenar Rhizoma on Hyperlipidemia Rats with Osteoporosis

目的:探讨桑白皮总黄酮联合知母总皂苷对高脂血伴骨质疏松症大鼠的改善作用及其可能的机制。方法:40只SPF级雄性SD大鼠适应性喂养7 d后,随机分为正常组、模型组、骨化三醇组(45 ng·kg^(-1)),桑白皮总黄酮-知母总皂苷1∶2组(0.6 g·kg^(-1)+0.4 g·kg^(-1))和桑白皮总黄酮-知母总皂苷2∶1组(1.2 g·kg^(-1)+0.2 g·kg^(-1))。除正常组外,其余各组大鼠高脂饲喂造模9周;正常组与模型组灌胃给予生理盐水,其他组灌胃给予相应药物;给药12周后,除正常组外,其余各组给药的同时肌肉注射糖皮质激素,给药22周后测定各组大鼠的体质量,生化法检测血清中总胆固醇(TC),甘油三酯(TG),低密度脂蛋白胆固醇(LDL-C),骨钙素(BGP)及骨碱性磷酸酶(BALP)的含量;苏木素-伊红(HE)染色观察大鼠胫骨病理形态变化;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠骨组织中过氧化物酶体增殖物激活受体γ(PPARγ),Runt相关转录因子2(Runx2)mRNA的表达水平;免疫荧光法检测大鼠PPARγ,Runx2的表达。结果:与正常组比较,模型组大鼠体质量显著升高(P<0.01),血清中TC,TG,LDL-C含量显著增加(P<0.01);与模型组比较,桑白皮总黄酮-知母总皂苷1∶2组和桑白皮总黄酮-知母总皂苷2∶1组大鼠体质量显著降低(P<0.01),血清中TC,TG,LDL-C含量显著降低(P<0.01),BGP,BALP含量显著升高(P<0.01)。HE染色结果表明,与正常组比较,模型组大鼠胫骨脂肪空泡增多,成骨细胞数量减少;与模型组比较,桑白皮总黄酮-知母总皂苷1∶2组和桑白皮总黄酮-知母总皂苷2∶1组大鼠胫骨脂肪空泡减小,成骨细胞数量增加。免疫荧光与Real-time PCR结果表明,与正常组比较,模型组大鼠Runx2表达减少,PPARγ表达增加(P<0.01),与模型组比较,桑白皮总黄酮-知母总皂苷1∶2组和桑白皮总黄酮-知母总皂苷2∶1组上调Runx2的表达,下调PPARγ的表达(P<0.05,P<0.01)。结论:桑白皮总黄酮联合知母总皂苷可上调Runx2,下调PPARγmRNA和蛋白的表达,从而影响了血液中TG和TC代谢,达到对骨质疏松的治疗作用,为临床防治高脂血症伴骨质疏松症的治疗提供了实验依据。

Objective:To explore the improvement effect of total flavonoids of Mori Cortex combined with total saponins of Anemarrhena Asphodeloide on hyperlipidemia rats with osteoporosis and its possible mechanism. Method:The 40 SPF male SD rats were adaptively fed for 7 days,and then randomly divided into normal group,model group,calcitriol group(45 ng·kg^(-1)),total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloide 1∶2 group(0.6 g·kg^(-1)+0.4 g·kg^(-1))and 2∶1 group(1.2 g·kg^(-1)+0.2 g·kg^(-1)). Except for the normal group,rats in the other groups were fed with high fat for 9 weeks,the normal group and the model grouotal flavonoids of total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloip were given normal saline by gavage,and the other groups were given corresponding drugs by gavage,after 12 weeks of administration, except for the normal group, the other groups were given intramuscular injection of glucocorticoids at the same time. After 22 weeks of administration,the weight of rats with total flavonoids from Mori Cortex combined with total saponins of Anemarrhena Asphodeloide was measured. Serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),osteocalcin(BGP)and bone alkaline phosphatase(BALP)were determined by biochemical assay. Hematoxylin-eosin(HE)staining to observe the pathological changes of rat tibia. Real-time fluorescent quantitative polymerase chain reaction(Realtime PCR)was used to detect the expression levels of peroxisomal proliferators activate the receptor gamma(PPARγ)and Runt-related transcription factor 2(Runx2)mRNA in rat bone tissue,immunofluorescence was used to detect the expression of PPARγ and Runx2 in rats. Result:Compared with normal group,the body mass of rats in model group was significantly increased(P<0.01),and the contents of TC,TG,and LDL-C in the serum were significantly increased(P<0.01). Compared with model group,the body weight of rats in thet total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloide 1∶2 group and 2∶1 group were significantly reduced(P<0.01),and the contents of TC,TG,and LDL-C in the serum were significantly reduced(P<0.01), the content of BGP and BALP increased(P<0.01). HE staining results showed that compared with the normal group,the tibia fat vacuoles of the model group increased,and the number of osteoblasts decreased, compared with the model group, the total flavonoids of the Mori Cortex and the flavonoids-total saponins of Anemarrhena Asphodeloide 1∶2 group and 2∶1 group decreased in tibia fat vacuoles and increased the number of osteoblasts,the results of immunofluorescence and Real-time PCR showed that,compared with normal group,the expression of Runx2 in the model group decreased and the expression of PPARγ increased(P<0.01). Compared with model group,the total flavonoids of Mori Cortex-total saponins 1∶2 group and the total flavonoids of Mori Cortex-total saponins 2∶1 Group up-regulated the expression of Runx2 and down-regulated the expression of PPARγ(P<0.05,P<0.01). Conclusion: The total flavonoids of Mori Cortex combined with the total saponins of Anemarrhena Asphodeloide up-regulated Runx2 and down-regulated the expression of PPARγ mRNA and protein,thereby affecting the metabolism of TG and TC in the blood,achieving a therapeutic effect on osteoporosis,provides experimental basis for the clinical prevention and treatment of hyperlipidemia with osteoporosis.