补肾化痰方对OVX诱导的骨质疏松大鼠血清LPS及TLR4/MyD88/NF-κB信号通路的影响  

Effect of Bushen Huatan Prescription on Serum LPS and TLR4/MyD88/NF-κB Signaling Pathway in Rats with Ovariectomy-induced Osteoporosis

作　　者：黄诗怡 周广文[1] 朱伟[2] 谭张奎 张妍 熊梦欣 薛瑶珺 张麟 向楠[1] HUANG Shi-yi;ZHOU Guang-wen;ZHU Wei;TAN Zhang-kui;ZHANG Yan;XIONG Meng-xin;XUE Yao-jun;ZHANG Lin;XIANG Nan(Hubei University of Traditional Chinese Medicine,Wuhan 430065,China;Wuhan University,Wuhan 430072,China;Wuhan Hospital of Tranditional Chinese and Western Medicine,Wuhan 430022,China)

机构地区：[1]湖北中医药大学,武汉430065 [2]武汉大学,武汉430072 [3]武汉市中西医结合医院,武汉430022

出　　处：《中国实验方剂学杂志》2021年第9期70-76,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82074416,81904267);湖北省自然科学基金青年项目(2019CFB232);湖北省卫生健康委员会中医药科研青年人才项目(ZY2019Q006);湖北省卫生和计划生育委员会湖北中医名师向楠工作室项目(鄂卫生计生办通[2018]32号);武汉市卫生健康委员会武汉市医学科研项目(WZ19Q03,WZ20C28)。

摘　　要：目的:研究补肾化痰方对双侧卵巢切除术(OVX)诱导的骨质疏松大鼠脂多糖(LPS)及Toll样受体4(TLR4)/髓样细胞分化蛋白88(MyD88)/核转录因子-κB(NF-κB)信号通路的影响。方法:将60只SPF级6月龄的雌性大鼠随机分为假手术组,模型组,戊酸雌二醇组,补肾化痰方低、中、高剂量组,使用双侧OVX造模,造模后1周,每组中挑选8只,戊酸雌二醇组按0.184 mg·kg^(-1),补肾化痰方低、中、高剂量组分别按4.7,9.4,18.8 g·kg^(-1)灌胃,假手术与模型组给予0.9%生理盐水4 mL灌胃,干预12周后处死取材。运用酶联免疫吸附测定(ELISA)法检测血清LPS含量,蛋白免疫印迹法(Western blot)检测骨组织中TLR4,MyD88,磷酸化(p)-NF-κB p65的蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测骨组织中TLR4,MyD88,NF-κB p65 mRNA水平及通路相关炎症因子白细胞介素(IL)-1β,IL-6 mRNA的表达。结果:与假手术组比较,模型组的血清LPS水平,骨组织中TLR4,MyD88,p-NF-κB p65蛋白表达水平,TLR4,MyD88,NF-κB p65,IL-1β,IL-6 mRNA水平均明显升高(P<0.05);与模型组比较,戊酸雌二醇组和补肾化痰方高剂量组血清LPS水平,骨组织中TLR4,MyD88,p-NF-κB p65的蛋白表达,TLR4,MyD88,NF-κB p65 mRNA水平,以及下游炎症因子IL-1β,IL-6 mRNA水平有不同程度降低(P<0.05)。结论:补肾化痰方可以降低血清LPS含量,调控TLR4/MyD88/NF-κB通路中TLR4,MyD88,NF-κB p65,p-NF-κB p65的mRNA水平及蛋白表达,降低骨组织中IL-1β,IL-6 mRNA水平,改善骨微结构,抑制绝经后骨质疏松症的病情发展。Objective: To explore the effect of Bushen Huatan prescription on serum lipopolysaccharide(LPS)and Toll-like receptor 4(TLR4)/myeloid cell differentiation protein 88(MyD88)/nuclear transcription factor-κB(NF-κB)signaling pathway in rats with ovariectomy-induced osteoporosis.Method: Sixty SPF 6-month-old female rats were randomly divided into sham operation group,model group,estradiol valerate group and Bushen Huatan prescription low,medium and high dose groups. One week after modeling by bilateral ovariectomy,8 rats in each group were selected to receive intragastric administration. The estradiol valerate group was given 0.184 mg·kg^(-1) by gavage,and Bushen Huatan prescription low,middle and high dose groups were given 4.7,9.4 and 18.8 g·kg^(-1) by gavage,sham operation group and model group were given 0.9% saline 4 mL by gavage respectively. After 12 weeks of intervention,the rats were sacrificed for detection.SerumLPS was detected by enzyme linked immunosorbent assay(ELISA),while protein expressions of TLR4,MyD88 and phosphorylated(p)-NF-κB p65 in bone tissue were detected by Western blot,and the mRNA expressions of TLR4,MyD88,NF-κB p65,IL-1β,and IL-6 in bone tissue were detected by quantitative real-time polymerase chain reaction(PCR).Result: Compared with sham operation group,the serumLPS level as well as protein expression of TLR4,MyD88,p-NF-κB p65 and mRNA expression of TLR4,MyD88,NF-κB p65,IL-1β,and IL-6 significantly increased in model group(P<0.05).Compared with the model group, serumLPS level,protein expression of TLR4,MyD88,and p-NF-κB p65,mRNA levels of TLR4,MyD88,and NF-κB p65 in bone tissues as well as downstream inflammatory factors IL-1β,IL-6 mRNA expression decreased to different degrees in estradiol valerate group and Bushen Huatan prescription high dose group(P<0.05).Conclusion: Bushen Huatan prescription can reduce serumLPS content, regulate mRNA and protein expression of TLR4, MyD88, NF-κB p65 and p-NF-κB p65 in TLR4/MyD88/NF-κB pathway, and down-regulate mRNA levels of IL-1�

关 键 词：补肾化痰方 绝经后骨质疏松症(PMOP) 脂多糖 Toll样受体4(TLR4)/髓样细胞分化蛋白88(MyD88)/核转录因子-κB(NF-κB)信号通路 骨微环境 炎性细胞因子 骨免疫学 

分 类 号：R2-0[医药卫生—中医学] R22