Epimedium-Curculigo herb pair enhances bone repair with infected bone defects and regulates osteoblasts through LncRNA MALAT1/miR-34a-5p/SMAD2 axis

Abstract

Infected bone defects (IBDs) are the common condition in the clinical practice of orthopaedics. Although surgery and anti-infective medicine are the firstly chosen treatments, in many cases, patients experience a prolonged bone union process after anti-infective treatment. Epimedium-Curculigo herb pair (ECP) has been proved to be effective for bone repair. However, the mechanisms of ECP in IBDs are insufficiency. In this study, Effect of ECP in IBDs was verified by micro-CT and histological examination. Qualitative and quantitative analysis of the main components in ECP containing medicated serum (ECP-CS) were performed. The network pharmacological approaches were then applied to predict potential pathways for ECP associated with bone repair. In addition, the mechanism of ECP regulating LncRNA MALAT1/miRNA-34a-5p/SMAD2 signalling axis was evaluated by molecular biology experiments. In vivo experiments indicated that ECP could significantly promote bone repair. The results of the chemical components analysis and the pathway identification revealed that TGF-β signalling pathway was related to ECP. The results of in vitro experiments indicated that ECP-CS could reverse the damage caused by LPS through inhibiting the expressions of LncRNA MALAT1 and SMAD2, and improving the expressions of miR-34a-5p, ALP, RUNX2 and Collagen type І in osteoblasts significantly. This research showed that ECP could regulate the TGF-β/SMADs signalling pathway to promote bone repair. Meanwhile, ECP could alleviate LPS-induced bone loss by modulating the signalling axis of LncRNA MALAT1/miRNA-34a-5p/ SMAD2 in IBDs.

Keywords: Epimedium‐Curculigo herb pair; LncRNA MALAT1; SMAD2; infected bone defects; miR‐34a‐5p.

FIGURE 1

The bone repair effect of ECP in IBDs. (A) The appearance observation of tibia specimens in each group. (B) The micro‐CT scanning images of lesion sites in each group. (C) The HE staining images of tibia specimens (10×, scale bar = 1 mm, 40×, scale bar = 200 μm). (D) The IHC staining images of BMP‐2 in each group (200×, scale bar = 50 μm). (E) The BV/TV and BMD indexes (F) The protein degrees of RUNX2 in bone tissue. (*p < 0.05, **p < 0.01, vs. Control group; ^# p < 0.05, ^## p < 0.01, vs. Model group; n = 3).

FIGURE 2

Network pharmacology‐based prediction of the six absorbed constituents. (A) Intersection of the six absorbed constituents' targets with osteogenesis‐related genes involved in treating bone repair by Venn diagram. (B) Network diagram of osteogenic‐core targeted of the 6 absorbed constituents. (C) A circle diagram showed an analysis of the biological processes involved in the core targets of the six absorbed constituents by GO. (D) Analysis of the pathways involved in the core targets of the six absorbed constituents by the KEGG and bubble diagrams.

FIGURE 3

siMALAT1 and miR‐34a‐5p mimics drove the differentiation and mineralization of osteoblasts. (A) StarBase predicted the binding site between LncRNA MALAT1 and miR‐34a‐5p; Diagram of putative miR‐34a‐5p binding sequence in LncRNA MALAT1 3′UTR and its mutant in luciferase reporter assay. (B, G) qRT‐PCR was used to test the gene expression of LncRNA MALAT1, miR‐34a‐5p, SMAD2, ALP, RUNX2 and Collagen type І in osteoblasts. (C–F) Western Blot was used to test the protein expression of SMAD2, ALP, RUNX2 and Collagen type І. (H, J) The differentiation abilities of osteoblasts were observed by ALP staining (40×, scale bar = 500 μm). (I, K) The mineralization abilities of osteoblasts were observed by Alizarin red (40×, scale bar = 500 μm). (*p < 0.05, **p < 0.01, vs. NC or mimics NC; n = 3).

FIGURE 4

Verification of LncRNA MALAT1/miR‐34a‐5p/SMAD2 signalling pathway. (A, F) qRT‐PCR was adopted to detect the gene expressions of miR‐34a‐5p, SMAD2, ALP, RUNX2 and Collagen type І in osteoblasts. (B, E, I, J) Western Blot was adopted for testing the protein expressions of SMAD2, ALP, RUNX2 and Collagen type І. (C, G) The differentiation abilities of osteoblasts were discovered by ALP staining (40×, scale bar = 500 μm). (D, H) The mineralization abilities of osteoblasts were observed by Alizarin red staining (40×, scale bar = 500 μm). (*p < 0.05, **p < 0.01, vs. NC or inhibitor NC; n = 3).

FIGURE 5

LPS inhibited the differentiation and mineralization of osteoblasts. (A) Effect of LPS on differentiation of osteoblasts was detected by CCK‐8. (B) qRT‐PCR was used to detect the gene expressions of LncRNA MALAT1, miR‐34a‐5p, SMAD2, ALP, RUNX2 and Collagen type І in osteoblasts. (C, F) Western Blot was employed for detecting the protein expressions of total SMAD2, ALP, RUNX2 and Collagen type І. (D) The differentiation abilities of osteoblasts were observed by ALP staining (40×, scale bar = 500 μm). (E) The mineralization abilities of osteoblasts were observed by Alizarin red (40×, scale bar = 500 μm). (*p < 0.05, **p < 0.01, vs. Control, n = 3).

FIGURE 6

Verification of that LPS mediated LncRNA MALAT1/miR‐34a‐5p/SMAD2 signalling pathway on osteoblasts. (A) qRT‐PCR was employed for detecting the gene expression of miR‐34a‐5p, SMAD2, ALP, RUNX2 and Collagen type І in osteoblasts. (B, E) Western Blot was employed for detecting the protein expression of total SMAD2, ALP, RUNX2 and Collagen type І. (C) The differentiation abilities of osteoblasts were observed by ALP staining (40×, scale bar = 500 μm). (D) The mineralization abilities of osteoblasts were observed by Alizarin red (40×, scale bar = 500 μm). (*p < 0.05, **p < 0.01, vs. Control; ^# p < 0.05, ^## p < 0.01, vs. LPS; n = 3).

FIGURE 7

ECP‐CS reversed the inhibition of osteoblasts caused by LPS. (A) Effect of ECP‐CS on differentiation of osteoblasts was detected by CCK‐8. (B) qRT‐PCR was used to detect the gene expressions of LncRNA MALAT1, miR‐34a‐5p, SMAD2, ALP, RUNX2 and Collagen type І in osteoblasts. (C, F) Western Blot was used to test the protein expressions of SMAD2, ALP, RUNX2 and Collagen type І. (D) The differentiation abilities of osteoblasts were observed by ALP staining (40×, scale bar = 500 μm). (E) The mineralization abilities of osteoblasts were observed by Alizarin red (40×, scale bar = 500 μm). (*p < 0.05, **p < 0.01, vs. NC or ECP‐BS, n = 3).

FIGURE 8

Verification of that ECP‐CS mediated LncRNA MALAT1 /miR‐34a‐5p /SMAD2 signalling pathway on osteoblasts. (A) qRT‐PCR was employed for detecting the gene expression of miR‐34a‐5p, SMAD2, ALP, RUNX2 and Collagen type І in osteoblasts. (B, E) Western Blot was employed for detecting the protein expression of total SMAD2, ALP, RUNX2 and Collagen type І. (C) The differentiation abilities of osteoblasts were observed by ALP staining (40×, scale bar = 500 μm). (D) The mineralization abilities of osteoblasts were observed by Alizarin red (40×, scale bar = 500 μm). (*p < 0.05, vs. Control; **p < 0.01, vs. Control; ^# p < 0.05, vs. ECP‐CS; ^## p < 0.01, vs. ECP‐CS, n = 3).

FIGURE 9

The mechanism of Epimedium‐Curculigo herb pair on bone repair after IBDs.