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二仙 Herbal Pair 通过调控 miRNA-34a-5p 促进感染性骨不连模型中成骨作用并减轻脂多糖诱导的成骨细胞抑制

Erxian herbal pair enhances bone formation in infected bone nonunion models and attenuates lipopolysaccharide-induced osteoblastinhibition by regulating miRNA-34a-5p.

机构信息

School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China.

Department of Pharmacy, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, China.

出版信息

Bioengineered. 2022 Jun;13(6):14339-14356. doi: 10.1080/21655979.2022.2085388.

DOI:10.1080/21655979.2022.2085388
PMID:36694425
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9995130/
Abstract

Bacterium-induced inflammatory responses cause bone nonunion. Although antibiotics suppress infection, bone loss after antibacterial treatment remains a critical challenge. Erxian herbal pair (EHP) has been proven effective in promoting bone formation. Our study aimed to investigate the effect of EHP on bone repair after anti-infection treatment, explore its effect on a lipopolysaccharide (LPS)-induced osteoblast. We evaluated effects of EHP on bone repair with Micro-CT, and morphology detecting. Chemical constituents of EHP and EHP-containing serum (EHP-CS) were identified by UHPLC-Q/TOF-MS. In addition, osteoblast induced by LPS was established and administrated with EHP-CS. Cell proliferationwas assessed by MTT. MiRNA mimic, inhibitor and siRNA were transiently transfected into osteoblasts. The mRNA levels and protein expressions of miRNA-34a-5p, BMP2, Runx2, SMAD2 were assessed. The results showed that the main biocactivity ingredients in EHP-CS were Baohuoside Ι and Orcinol Glucoside. EHP could promote bone remolding after anti-infection therapy and restore the activity of LPS-induced osteoblasts. Moreover, miRNA-34a-5p was dramatically downregulated and SMAD2 was upregulated after LPS stimulation, while EHP resisted the inhibition of LPS by promoting miRNA-34a-5p, ALP, and BMP2 expressions. Whereas downregulation of miRNA-34a-5p reversed these effects. Silencing endogenous SMAD2 expression markedly promoted BMP2 and ALP activity and enhanced osteogenesis. Taken together, EHP restored LPS-induced bone loss by regulating miRNA-34a-5p levels and repressing its target gene SMAD2. EHP might be a potential adjuvant herbal remedy for the treatment of bone nonunion, and miRNA-34a-5p is a novel target for controlling bone and metabolic diseases.

摘要

细菌诱导的炎症反应导致骨不连。尽管抗生素可以抑制感染，但抗菌治疗后骨丢失仍然是一个关键的挑战。二仙汤（EHP）已被证明能有效促进骨形成。本研究旨在探讨 EHP 对抗感染治疗后骨修复的影响，探索其对脂多糖（LPS）诱导的成骨细胞的作用。我们通过 Micro-CT 和形态学检测评估 EHP 对骨修复的影响。采用 UHPLC-Q/TOF-MS 鉴定 EHP 和含 EHP 血清（EHP-CS）的化学成分。此外，建立了 LPS 诱导的成骨细胞模型，并给予 EHP-CS 处理。通过 MTT 法评估细胞增殖。瞬时转染 miRNA 模拟物、抑制剂和 siRNA 至成骨细胞。检测 miRNA-34a-5p、BMP2、Runx2、SMAD2 的 mRNA 水平和蛋白表达。结果表明，EHP-CS 的主要生物活性成分是宝藿苷Ⅰ和紫菀苷。EHP 可促进抗感染治疗后的骨重塑，恢复 LPS 诱导的成骨细胞活性。此外，LPS 刺激后 miRNA-34a-5p 显著下调，SMAD2 上调，而 EHP 通过促进 miRNA-34a-5p、ALP 和 BMP2 的表达抵抗 LPS 的抑制作用。下调 miRNA-34a-5p 则逆转了这些作用。沉默内源性 SMAD2 表达显著促进 BMP2 和 ALP 活性，增强成骨作用。综上所述，EHP 通过调节 miRNA-34a-5p 水平并抑制其靶基因 SMAD2，恢复 LPS 诱导的骨丢失。EHP 可能是治疗骨不连的一种有潜力的辅助草药，miRNA-34a-5p 是控制骨代谢疾病的新靶点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fea/9995130/d9501605b158/KBIE_A_2085388_F0007_OC.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fea/9995130/ed8c775c411d/KBIE_A_2085388_UF0001_OC.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fea/9995130/d9501605b158/KBIE_A_2085388_F0007_OC.jpg

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