左归丸含药血清对成骨细胞中Ihh、Ptch、Smo、GLi1蛋白表达及破骨细胞数量的影响  

Effect of Serum Containing Zuogui Pill on the Expression of Ihh,Ptch,Smo,GLi1 Proteins in Osteoblasts and the Number of Osteoclasts

作　　者：史婧儒 廖泽绮 王雨荷 赵宏艳[1] 张元月 刘梅洁[1] SHI Jingru;LIAO Zeqi;WANG Yuhe;ZHAO Hongyan;ZHANG Yuanyue;LIU Meijie(Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院医学实验中心,北京100700

出　　处：《中国中医基础医学杂志》2024年第6期998-1004,共7页JOURNAL OF BASIC CHINESE MEDICINE

基　　金：国家自然科学基金面上项目(82074290);全国中医药创新骨干人才培训项目(ZX2020002)。

摘　　要：目的 探讨左归丸含药血清对成骨细胞中Hedgehog-GLi通路关键蛋白表达及破骨细胞数量的影响。方法 选用50只雌性SD大鼠,随机分为空白对照组、假手术组、造模组。造模组大鼠切除双侧卵巢,12周后随机分为卵巢切除组(ovariectomy,OVX)、阳性对照组和左归丸组。阳性对照组大鼠灌服戊酸雌二醇混悬液,左归丸组大鼠灌服左归丸水煎液,连续干预7 d。末次给药1.5 h后,腹主动脉取血制备各组相应含药血清。建立成骨细胞、破骨细胞、骨磨片共培养体系,分为空白对照血清组、假手术血清组、OVX血清组、阳性对照血清组、左归丸含药血清组、激动剂组和激动剂+左归丸含药血清组,分别添加各组对应血清。免疫荧光法检测成骨细胞印度刺猬蛋白(Indian hedgehog,Ihh)、碎片蛋白(patched,Ptch)、润滑蛋白(smoothened,Smo)及GLi1蛋白表达;免疫组化法检测成骨细胞骨保护蛋白(osteoprotegerin,OPG)、核因子受体激活因子-κB配体(receptor activator for nuclear factor-κB ligand,RANKL)蛋白表达;对破骨细胞进行TRAP染色,计算TRAP+细胞数;ELISA检测共培养体系上清液中TRAP的含量。结果与假手术血清组比较,OVX血清组Ihh、Ptch、Smo、GLi1蛋白表达水平显著上调(P<0.01),RANKL/OPG比值增高;TRAP+细胞数和细胞上清液中TRAP含量亦明显增加(P<0.05)。与OVX血清组比较,左归丸含药血清组Ihh、Ptch、Smo、GLi1的蛋白表达明显下调,RANKL/OPG比值下降;TRAP+细胞数和细胞上清液中TRAP含量明显降低(P<0.05)。与激动剂组比较,加入左归丸含药血清后,Ihh、Ptch、Smo、GLi1的蛋白表达及RANKL/OPG比值均明显下降,TRAP+细胞个数及TRAP含量亦明显降低(P<0.05)。结论 左归丸含药血清可通过抑制异常活跃的Hedgehog-GLi信号通路,使Ihh、Ptch、Smo、GLi1的蛋白表达水平下调,降低RANKL/OPG比值,抑制破骨细胞的数量和活性,起到减少骨丢失作用。Objective Explore the effect of serum containing Zuogui pill on the expression of key proteins in the Hedgehog-GLi pathway of osteoblasts and the number of osteoclasts.Methods 50 female SD rats were randomly divided into control group,sham-operated group,and model group.The model group rats underwent bilateral ovariectomy.After 12 weeks,the rats in the model group were randomly divided into the ovariectomy group(OVX),positive control group,and Zuogui pill group.The positive control group rats were orally administered estradiol valerate suspension,the Zuogui pill group rats were administered with Zuogui pill water solution for 7 days.1.5 hours after the final administration,blood was collected to prepare corresponding drug containing serum.The co-culture system for osteoblasts,osteoclasts,and bone slices was established and divided into blank control serum group,sham-operated serum group,OVX serum group,positive control serum group,Zuogui pill containing serum group,agonist serum group,and agonist+Zuogui pill containing serum group.Corresponding serum was added to each group separately.The protein expression of Ihh,Ptch,Smo,and GLi1 in osteoblasts was detected by immunofluorescence.The protein expression of OPG and RANKL in osteoblasts was detected by immunohistochemistry.The number of TRAP-positive cells was calculated.ELISA was used to detect the content of TRAP in the supernatant of the co-culture system.Results Compared with sham-operated serum group,the protein expression level of Ihh,Ptch,Smo,and GLi1 were significantly upregulated(P<0.01),and the RANKL/OPG ratio increased,while the number of TRAP+cells and the TRAP content in the cellular supernatant also significantly increased in the OVX serum group(P<0.05).Compared with the OVX serum group,the protein expression of Ihh,Ptch,Smo,and GLi1 was significantly down-regulated and the RANKL/OPG ratio decreased,the number of TRAP+cells and the TRAP content in the cell supernatant also significantly decreased(P<0.05)in the Zuogui pill containing serum group.Compar

关 键 词：左归丸 骨质疏松症 Hedgehog-GLi信号通路 成骨细胞 破骨细胞 

分 类 号：R228[医药卫生—中医基础理论] R285[医药卫生—中医学]