ShK-modified UCMSCs Inhibit M1-Like Macrophage Polarization and Alleviate Osteoarthritis Progression via PI3K/Akt Axis

Abstract

The potassium channel Kv1.3 plays an important role in regulating immune cell functions in many inflammatory diseases whereas rarely in osteoarthritis (OA). Here, it is demonstrated that the Kv1.3 of macrophages is upregulated in response to LPS stimulation, as well as in human OA synovium samples than non-OA. Administration of Stichodactyla toxin (ShK), a Kv1.3 blocker, significantly inhibited cartilage degeneration and synovial inflammation in animal models of OA in vivo by inhibiting M1 macrophage polarization and reducing the production of inflammatory factors. In this study, a transgenically engineered human umbilical cord mesenchymal stem cell (UCMSC) delivery system is developed that secreted a peptide ShK, a Kv1.3 potassium blocker, into the knee articular cavity. Collectively, the results identified Kv1.3 as a potential therapeutic target for OA and demonstrated the efficacy of using ShK transgenic engineered UCMSCs as a delivery for the peptide in OA treatment.

Keywords: Kv1.3; Macrophage; Osteoarthritis; UCMSC.

Figure 1

Kv1.3 is highly expressed in M1 macrophage in the synovium of OA patients and mice experimental OA models. A,B) Representative images of H&E staining and immunohistochemistry staining of Kv1.3 from patients’ synovium with or without OA, and mice with or without DMM operation after 8 weeks. Scale bar, 50 µm. C,D) Immunofluorescence staining of Kv1.3 (red) and CD80 (green) for synovium from OA or non‐OA samples. Scale bar, 100 µm. E,F) Immunofluorescence staining of Kv1.3 (pink) and CD80 (green) for synovium 8 weeks after sham or DMM operation. Scale bar, 100 µm. G,H) Representative images of immunofluorescence co‐staining of M1 marker CD80 (green) and Kv1.3 (red) in RAW264.7 macrophages. Scale bar, 100 µm. Data are means ± SD. Student's two‐tailed t test, ns, not significant p ≥ 0.05; ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 2

Kv1.3 suppressor ShK effectively inhibits ROS and M1 polarization via the PI3K/Akt axis. A,B) Western blot and quantitative analysis of Kv 1.3, iNOS, COX2 and ARG1 proteins in RAW264.7 macrophages treated with LPS with or without ShK for 24 h. C,D) Representative images of ROS in RAW264.7 macrophages pretreated with ShK (100 nm) with or without LPS (50 ng mL⁻¹) in the 24 h. Scale bar, 100 µm. (E–G) ELISA analysis of IL‐1𝛽, IL‐6 and TNF‐𝛼 in supernatant of LPS treated RAW264.7 cells with or without ShK. (H‐I) Western blot analysis and quantification of p‐PI3K, PI3K, p‐AKT and AKT RAW264.7 in LPS treated macrophages with or without ShK. J,K) Representative immunofluorescence staining of macrophage marker F4/80 (green) and M1 polarization marker iNOS (red) in synovium 8 weeks after sham or DMM operation. Scale bar, 100 µm. Data are means ± SD. Student's two‐tailed t test, ns, not significant p ≥ 0.05; ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 3

Inhibition of Kv1.3 inhibits OA progression in vitro. A,B) Representative images of live‐dead staining for ATDC5 cells after coculture with RAW264.7 cells with or without ShK treated after LPS stimulation. Scale bar, 100 µm. C,D) Western blot analysis and quantification of MMP3, MMP13, COL2A1 and SOX9 in ADTC chondrocytes after added supernate from LPS‐treated RAW264.7 macrophages with or without ShK. E) RT‐qPCR for messenger RNA of Mmp13, Il1b, and Nlrp3 in chondrocytes in ADTC chondrocytes after added supernatant from LPS‐treated RAW264.7 macrophages with or without ShK. Data are means ± SD. Student's two‐tailed t test, ns, not significant p ≥ 0.05; ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 4

Kv1.3 suppressor ShK inhibits OA progression in vivo. A,B) Representative images of H&E staining of knee joint synovium and quantitative analysis of synovitis score. (n = 6). Scale bar, 50 µm. C,D) Representative images of safranin‐O/fast green staining of joint cartilage areas from sham, DMM and ShK‐treated DMM mice and OARSI scores were on the right side. (n = 6). Scale bar, 50 µm. E–H) Representative images of immunohistochemistry staining of COL2A1 and MMP13 for joint chondrocytes. (n = 6). Scale bar, 100 µm. I–K) 3D reconstructed images of knee joints and sagittal view of the medial joint compartment visualizing subchondral bone changes; a red line marks subchondral plate thickness. (n = 6) Scale bar, 1mm. Data presented as mean ± SD. ^** p < 0.01, ^* p < 0.05, ns, not significant, versus the indicated groups, Student's t‐test.

Figure 5

Preparation and characteristics of engineered UCMSCs. A) Experiment design diagram of transfection and screening of engineered UCMSCs. B) Representative images of Live‐dead staining staining of UCMSCs after screening by different concentrations of puromycin Scale bar, 100 µm. D) HPLC of supernatant from control or ShK‐UCMSCs after freeze‐drying and redissolved by the mobile phase. Detection is at 220 nm. E,F) Western blot analysis and quantification of COX2 and iNOS in LPS treated RAW264.7 macrophages added with supernate from with NC‐UCMSCs or ShK‐UCMSCs.Data are means ± SD. Student's two‐tailed t test, ns, not significant p ≥ 0.05; ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 6

Engineered UCMSCs attenuate OA phenotype by inhibiting M1 macrophage polarization. A) Representative images of H&E staining of knee joint synovium. Scale bar,100 µm. B) Representative images of safranin‐O/fast green staining of joint cartilage from sham, DMM, DMM+ShK, DMM+NC‐UCMSC and DMM+ShK‐UCMSC mice. Scale bar,50 µm. C,D) Representative images of immunohistochemistry staining of COL2A1 and MMP13 for joint chondrocytes. (n = 6). Scale bar, 100 µm. E) OARSI scores and quantification of intensity of IHC in (C) and (D) using ImageJ. (n = 6). F) Immunofluorescence staining of iNOS (red) and F4/80 (green) for synovium from sham or DMM group. (n = 6). Scale bar, 100 µm. G) Quantification of intensity of IF staining in (F) using ImageJ. Data presented as mean ± SD. ^** P < 0.01, ^* P < 0.05, ns, not significant, versus the indicated groups, Student's t‐test.

Figure 7

The scheme for ShK‐modified UCMSCs inhibit M1‐like macrophage polarization and alleviate osteoarthritis progression via PI3K/Akt axis.