Salvianolic acid B accelerates osteoporotic fracture healing via lncRNA-MALAT1/miR-155-5p/HIF1A axis
收藏资源简介：
Figure 1. SalB promotes osteogenic differentiation of hMSCs by activating MALAT1. (A) The hMSCs were subjected to osteogenic induction for 5 days, and different concentrations of SalB were added. The mRNA expression levels of MIAT, MALAT1, HOTAIR, MEG3 and H19 were evaluated by qPCR. (B-D) The hMSCs were transfected with siRNA targeting MALAT1, then the cells were treated with osteogenic induction medium with or without SalB (5 μM) for 7 days. The osteogenesis-related marker genes were evaluated by qPCR; (C&D) after 14 days of osteogenic induction, the cells were stained with Alizarin Red S, and quantified. Figure 2. MALAT1 sponges miR-155-5p and HIF1α is a bona fide target of miR-155-5p. (A) The HEK293T cells were transfected with MALAT1, empty vector, negative control (NC) or siMALAT1, then the level of mir-155-5p was quantified by qPCR. (B) The HEK293T cell lysate was incubated with biotin-labeled MALAT1. After pulldown, the miRNAs were extracted and measured by qPCR. (C) The HEK293 cells were transfected with NC, mimic or inhibitor of mir-155-5p, and the level of HIF1α was quantified by qPCR. (D) Wild type (WT) and mutant (MUT) 3′ UTR of HIF1α containing the binding site with miR-155-5p was inserted into pMIR-GLO vector, respectively. The HEK293 cells were co-transfected with miRNA oligos combined with luciferase reporter (WT or MUT). Then the relative luciferase activity was measured by luciferase reporter assay and calculated. (E) Immunofluorescent detection of HIF1α in hMSCs transfected with NC or mir-155-5p inhibitor. Figure 3. SalB promoted osteoporotic fracture healing in rats. (A-C) At 6 weeks after fracture surgery, the femurs were collected for micro-CT analysis. Representative 3D images and 2D sections generated from micro-CT analysis of femurs with fracture were shown, as well as the quantitative parameters including bone volume density (BMD), and BV/TV) (n = 5). (D-F) The sections were stained with Safranin O/Fast green or HE, and the relative area of bone and uncalcified region were calculated using Image J software. (G&H) The mechanical properties of femurs were tested using 3-point bending test. The ultimate load and stiffness of the fractured femur were normalized with the contralateral intact femur (n = 8). *P < 0.05. Figure 4. SalB increased osteogenesis and angiogenesis-related markers in the calluses. (A&B) The expressions of BMP2, OPN, HIF1α and Runx2 in the callus were detected by immunohistological staining. (C) The H-type vessels (CD31 and EMCN positive) in the calluses were detected by immunofluorescent staining.
图1 丹酚酸B（SalB）通过激活长链非编码RNA MALAT1促进人间充质干细胞（hMSCs）的成骨分化。(A) 将人间充质干细胞进行成骨诱导5天，同时添加不同浓度的丹酚酸B，采用实时荧光定量聚合酶链反应（qPCR）检测MIAT、MALAT1、HOTAIR、MEG3及H19的mRNA表达水平。 (B-D) 将人间充质干细胞用靶向MALAT1的小干扰RNA（siRNA）转染，随后分别用含或不含5 μM丹酚酸B的成骨诱导培养基处理细胞7天，通过qPCR检测成骨相关标记基因的表达；(C、D) 成骨诱导14天后，采用茜素红S染色对细胞进行染色并定量。 图2 MALAT1作为内源竞争RNA海绵吸附miR-155-5p，且缺氧诱导因子1α（HIF1α）是miR-155-5p的功能性靶基因。(A) 将HEK293T细胞分别转染MALAT1过表达质粒、空载体、阴性对照（NC）或siMALAT1，采用qPCR定量检测miR-155-5p的表达水平。 (B) 将生物素标记的MALAT1与HEK293T细胞裂解液共孵育，经下拉富集后提取microRNA（miRNA），通过qPCR进行检测。 (C) 将HEK293细胞分别转染miR-155-5p的阴性对照、模拟物（mimic）或抑制剂（inhibitor），采用qPCR定量检测HIF1α的表达水平。 (D) 将含有miR-155-5p结合位点的缺氧诱导因子1α（HIF1α）野生型（WT）及突变型（MUT）3'非翻译区（3' UTR）分别插入pMIR-GLO载体中。将HEK293细胞与miRNA寡核苷酸及荧光素酶报告质粒（野生型或突变型）共转染，随后通过荧光素酶报告基因实验检测并计算相对荧光素酶活性。 (E) 采用免疫荧光法检测转染阴性对照或miR-155-5p抑制剂的人间充质干细胞中HIF1α的表达。 图3 丹酚酸B可促进大鼠骨质疏松性骨折愈合。(A-C) 骨折术后6周，采集股骨进行显微计算机断层扫描（micro-CT）分析，展示骨折股骨的micro-CT重建代表性三维图像及二维切片，并定量分析骨密度（BMD）、骨体积分数（BV/TV）等参数（n=5）。 (D-F) 采用番红O/固绿染色或苏木精-伊红（HE）染色对组织切片进行染色，通过Image J软件计算骨组织与未钙化区域的相对面积。 (G、H) 采用三点弯曲实验检测股骨的力学性能，将骨折侧股骨的最大载荷及刚度与对侧完整股骨进行归一化处理（n=8）。*P < 0.05，即差异具有统计学意义。 图4 丹酚酸B可提高骨痂中成骨与血管生成相关标记物的表达。(A、B) 采用免疫组织化学染色检测骨痂中骨形态发生蛋白2（BMP2）、骨桥蛋白（OPN）、HIF1α及Runt相关转录因子2（Runx2）的表达。 (C) 采用免疫荧光染色检测骨痂中H型血管（CD31及EMCN阳性）的表达。
