Kartogenin Promotes the BMSCs Chondrogenic Differentiation in Osteoarthritis by Down-Regulation of miR-145-5p Targeting Smad4 Pathway

Abstract

Background: Transplantation of mesenchymal stem cells (MSCs) is a potential therapeutic strategy for cartilage degeneration of osteoarthritis (OA). But controlling chondrogenic differentiation of the implanted MSCs in the joints remains a challenge. The role of kartogenin (KGN) for chondrogenesis of MSCs has been widely reported, however, the mechanism of chondrogenesis has not been elucidated in OA.

Methods: In this study, we investigated the miR-145-5p, TGF-β, Samd4, and p-stat3/stat3 expression in cartilage of OA patients and bone marrow mesenchymal stem cells (BMSCs) treated with KGN or miR-145-5p inhibitor. In addition, the cell proliferation and chondrogenic differentiation in vitro and in vivo of BMSCs treated with KGN was also detected.

Results: In OA patients, the expression of miR-145-5p was up-regulated, and the expression of TGF-β, Samd4, and p-stat3/stat3 was inhibited. When the BMSCs treated with miR-145-5p inhibitor, the expression of TGF-β, Samd4, and p-stat3/stat3 was also significantly up-regulated. KGN-treated BMSCs had better proliferation and chondrogenic differentiation by up-regulating the expression of Sox 9, Col-2a1, aggrecan in vitro and in OA by down-regulation of miR-145-5p targeting Smad4 pathway. Moreover, intra-articular injection of KGN-treated BMSCs had a better pain relief effect in OA.

Conclusion: The double effect on cartilage protection and pain relief indicates a great potential of intra-articular injection of KGN-treated BMSCs for the treatment of OA.

Keywords: BMSCs; Kartogenin; Osteoarthritis; Pain; miR-145-5p.

Fig. 1

The expression of miR-145-5p was increased and Samd4 pathway was inhibited in OA patients. A The mRNA levels of miR-145-5p, TGF-β, and Samd4 in OA patients. B, C Protein expression of TGF-β, Samd4, stat3, and p-stat3 in OA patients (*p < 0.05 and **p < 0.01)

Fig. 2

Samd4 pathway was activated after BMSCs were treated with miR-145-5p inhibitor. A Predicted miR-145-5p target sequences. B The mRNA levels of miR-145-5p, Samd4, and TGF-β in BMSCs when treated with miR-145-5p inhibitor at 48 h was detected by RT-qPCR. C, D Protein expression of Samd4, stat3, p-stat3, and TGF-β in BMSCs when treated with miR-145-5p inhibitor at 72 h was detected by western blot (*p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 3

The expression of miR-145-5p was decreased and Samd4 pathway was activated in KGN-treated BMSCs. A The mRNA levels of miR-145-5p, TGF-β, and Samd4 in BMSCs when treated with KGN at 48 h was detected by RT-qPCR. B, C Protein expression of TGF-β, Samd4, stat3, and p-stat3 in BMSCs when treated with KGN at 72 h was detected by western blot (*p < 0.05 and **p < 0.01)

Fig. 4

Proliferation and chondrogenic differentiation of BMSCs treated with KGN. A The cell proliferation assay was studied after 1, 3, and 5 days by CCK-8 assay. B Representative fluorescence images of Calcein-AM/PI staining incubated for 3 days. C The survival rates of BMSCs. D The proliferation of BMSCs extracted after intra-articular injection by CCK-8 assay. E Safranine O and Alcain blue of BMSCs. F–I RT-qPCR analysis of chondrogenic genes Sox 9, Col-2a1, aggrecan, and Col-1 of BMSCs treated with KGN. (*p < 0.05, **p < 0.01)

Fig. 5

A, B H and E and Safranin O staining at 8 weeks after the IA injection. C OARSI scores in each group (* p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 6

KGN preconditioning BMSCs promoted cartilage regeneration in OA. A–D RT-qPCR analysis of Sox 9, Col-2a1, aggrecan, and Col-1 in OA rat model at 8 weeks post-injection. E Representative immunofluorescence images of Sox 9, Col-2a1, aggrecan, and Col-1 in cartilage at 8 weeks post-injection. (F–I) Quantitative statistics of Sox 9, Col-2a1, aggrecan, and Col-1 expression (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7

Paw withdrawal threshold (PWT) was measured to test mechanical allodynia (*p < 0.05, ***p < 0.001)

Fig. 8

KGN-pretreated BMSCs decreased miR-145-5p expression and activated Samd4 pathway in OA rats. A–C The mRNA expression of miR-145-5p, TGF-β, and Samd4 in cartilage after IA injection. D–I Protein expression of TGF-β, Samd4, stat3, and p-stat3 in cartilage after IA injection detected by western blot (*p < 0.05 and **p < 0.01)