CircRNA-miRNA网络调控Ghrelin在艾灸“补益脾肾”法防治膝OA中的作用和机制

Knee osteoarthritis belongs to “Gu Bi” in traditional Chinese medicine。The chondrocytes reduction is the main pathophysiological process of OA. Ghrelin,the endogenous ligand of growth hormone secretagogue receptor，has been identified as an important role on participating in cartilage synthesis. In our previous study,we found Ghrelin was negatively related to disease severity in OA and may act as a protective factor of OA. However, what regulates ghrelin remains unclear.The deficiencies of “spleen” and “kidney” are important factors to cause OA. Epigenetic effect is associated with the function of “spleen” and “kidney”,and RNA regulation is one of the most important topics in epigenetics.Therefore,the current study is carried out to establish the SD rat OA model,based on the CircRNA-miRNA(ceRNA,competing endogenous RNAs )mechanism in epigenetics,to explore the distinction expression of miRNA-CircRNA during OA process, screening CircRNA-miRNA interaction pairs targeting Ghrelin.We further explore whether “Shen Shu””Zu San Li”moxibution serum “Tonifying spleen and kidney”method could promote ghrelin expression through our screened CircRNA-miRNA interaction pair,thus providing profounding theoretical basis for moxibution preventing and treating knee OA.

膝骨关节炎(OA)属中医“骨痹”范畴，软骨细胞减少是OA最主要的病理过程。最近研究表明生长激素释放促分泌素受体的内源性配体Ghrelin是参与软骨合成的重要因子，而我们前期发现Ghrelin与OA严重程度呈负相关，是OA进展的保护因子，但Ghrelin被何种因素调控尚不明确。脾肾亏虚是引发OA的重要原因，而表观遗传效应与“脾”“肾”功能密切相关，其中RNA调控是表观遗传的重要内容。本研究拟制造SD大鼠膝OA模型，从表观遗传中的CircRNA-miRNA(ceRNA，竞争性内源RNA)机制着眼，探讨OA发生时软骨CircRNA-miRNA 表达差异情况，筛选针对Ghrelin的CircRNA-miRNA，并进一步探讨“肾腧”“足三里”艾灸血清“补益脾肾”法是否可以通过调控筛选的CircRNA-miRNA促进Ghrelin表达，保护OA软骨细胞，为艾灸防治膝OA奠定深层次的理论基础。

膝骨关节炎(OA)属中医“骨痹”范畴，软骨细胞减少是OA最主要的病理过程。研究表明生长激素释放促分泌素受体的内源性配体Ghrelin是参与软骨合成的重要因子，而我们前期 发现Ghrelin与OA严重程度呈负相关，是OA进展的保护因子，但Ghrelin被何种因素调控尚不明确。脾肾亏虚是引发OA的重要原因，而表观遗传效应与“脾”“肾”功能密切相关，其中RNA 调控是表观遗传的重要内容。因此本项目采用SD 大鼠软骨细胞研究OA软骨细胞和大鼠正常软骨细胞miRNA/CircRNA array表达差异情况，qRT-PCR 检测证实 OA软骨细胞模型circSox6 和 circPan3 表达降低，生物信息学预测靶向结合 circPan3 的 miR-667-5p，qRT-PCR 验证其在 OA 软骨细胞中显著上调。双荧光素酶实验证实 miR-667-5p 可与Ghrelin 基因-3'UTR 端结合，circPan3 靶向作用于miR-667-5p，构建膝 OA 相关的circPan3-miR-667-5p-Ghrelin网络图。然后分别观察 SD 大鼠 OA 关节软骨细胞 转染miR-667-5p 和转染后 circPan3及共同过表达circPan3和miR-667-5p后，通过qRT-PCR 和 Western Blot 检测证明 circPan3 靶向作用于 miR-667-5p，促进 Ghrelin 合成和软骨细胞自噬，减少软骨细胞外基质的降解。通过制备不同浓度“肾腧”“足三里”艾灸血清，观察到艾灸血清促进circPan3、Ghrelin的表达和软骨基质合成，降低miR-667-5p的水平。艾灸血清可能通过circPan3- miR-667-5p - Ghrelin网络促进软骨基质合成和软骨自噬，发挥软骨保护作用。