左归丸含药血清对Hedgehog-GLi通路关键因子mRNA表达及骨吸收功能的影响
Effects of Serum Containing Zuogui Pill on mRNA expression of key factors of Hedgehog-GLi pathway and bone resorption function in osteoblast-osteoclast co-culture system
  
DOI:10.3969/j.issn.1006-7108.2024.06.005
中文关键词:  左归丸  骨质疏松症  Hedgehog-GLi信号通路  成骨细胞  破骨细胞  共培养体系
英文关键词:Zuogui pill  osteoporosis  Hedgehog signaling pathway  osteoblasts  osteoclasts  coculture system
基金项目: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)
作者单位
廖泽绮# 史婧儒# 王雨荷 赵宏艳 张元月 刘梅洁* 中国中医科学院医学实验中心北京 100700 
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中文摘要:
      目的 探讨左归丸含药血清对成骨-破骨细胞共培养体系中Hedgehog-GLi通路关键因子mRNA表达及骨吸收功能的影响。方法 建立成骨细胞、破骨细胞、骨磨片共培养体系,实验分为7组:空白对照组、假手术组、OVX组、阳性对照组、左归丸组、激动剂组和激动剂+左归丸组。选用50只雌性SD大鼠,分别制备相对应组含药血清,实验各组分别加入相对应组大鼠血清。通过实时荧光定量PCR检测成骨细胞Ihh、Ptch、Smo、GLi1、OPG及RANKL的mRNA表达;并采用甲苯胺蓝染色方法检测破骨细胞骨吸收功能。结果 与假手术组相比,OVX组Ihh、Ptch、Smo、GLi1mRNA表达显著上调,同时OPGmRNA表达水平显著降低,RANKL mRNA表达水平显著增加,RANKL/OPG比值明显增高。与OVX组相比,阳性对照组与左归丸组Ihh、Ptch、Smo、GLi1mRNA表达明显下调,同时OPG mRNA表达水平显著升高,RANKL mRNA表达水平显著降低,RANKL/OPG比值明显下降。OVX组加入激动剂Shh重组蛋白后,Hedgehog-GLi信号通路关键因子的mRNA表达较OVX组明显增加。与激动剂组比较,激动剂+左归丸组Ihh、Ptch、Smo、GLi1mRNA表达以及RANKL/OPG比值均明显下降。与假手术组相比,OVX组的骨吸收陷窝面积显著增加;与OVX组相比,阳性对照组和左归丸组的骨吸收陷窝面积显著下降。结论 左归丸含药血清可一定程度上抑制Hedgehog-GLi通路关键因子Ihh、Ptch、Smo、GLi1的活性,进而抑制OVX所致的骨吸收增强。
英文摘要:
      Objective To explore the effect of serum containing Zuogui Pill on the mRNA expression of key factors in the Hedgehog-GLi pathway and bone resorption function in an osteoblast-osteoclast-bone slice co-culture system. Methods An osteoblast-osteoclast-bone slice co-culture system was established. The experiment included blank control group, sham operation group, OVX group, positive control group, Zuogui Pill group, agonist group, and agonist + Zuogui Pill group. Serum containing drugs was prepared for the blank control group, sham operation group, OVX group, positive control group, and Zuogui Pill group using 50 female SD rats. Real-time PCR was used to measure the mRNA levels of Ihh, Ptch, Smo, GLi1, OPG, and RANKL in osteoblasts Tartrate-resistant acid phosphatase staining was employed to examine osteoc bone resorption function. Results Compared with the sham operation group, the OVX group exhibited a significant upregulation in mRNA expression levels of Ihh, Ptch, Smo, GLi1, while exhibiting significant decreases in OPG mRNA expression. In contrast, RANKL mRNA expression was significantly increased, resulting in a increase in the RANKL/OPG ratio. Compared with the OVX group, the mRNA expression levels of Ihh, Ptch, Smo, GLi1 in the positive control group and Zuogui Pill group were significantly downregulated, Meanwhile, there was a significant increase in OPG mRNA expression and a significant decrease in RANKL expression, which led to a significant decrease in the RANKL/OPG ratio. After adding Shh recombinant protein agonist to the OVX group, the mRNA expression of key HG pathway factors was significantly increased compared with the OVX group. In the agonist + Zuogui Pill group, the mRNA expression levels of Ihh, Ptch, Smo, GLi1 and RANKL/OPG ratio were significantly lowered compared to the agonist group. Moreover, the bone resorption pit area was significantly higher in the OVX group than in the sham group, compared with the OVX group, the positive control group and Zuogui Pill group showed a significant reduction. Conclusion Serum containing Zuogui Pill can inhibit the activity of key factors in the Hedgehog-GLi pathway, thereby suppressing the increase in bone resorption caused by OVX.
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