Salvianolic acid B accelerates osteoporotic fracture healing via lncRNA-MALAT1/miR-155-5p/HIF1A axis

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.