Curcumin primed ADMSCs derived small extracellular vesicle exert enhanced protective effects on osteoarthritis by inhibiting oxidative stress and chondrocyte apoptosis

Abstract

Osteoarthritis (OA) is a common joint disease caused by progressive articular cartilage degeneration and destruction. Currently, there are no disease-modifying agents officially approved for OA patients. In this study, curcumin was loaded into adipose tissue-derived mesenchymal stem cells (ADMSCs)-derived small extracellular vesicle (ADMSCs-sEV) to synergistically exert chondro-protective effects in vitro and in vivo. We found curcumin primed ADMSCs derived sEV (sEV-CUR) exhibited an enhanced protective effect compared with free curcumin and ADMSCs-sEV in TBHP-induced chondrocytes. Moreover, our study demonstrated sEV-CUR more effectively down-regulated TBHP-induced oxidative stress and chondrocyte apoptosis in vitro. In OA mice model, our results indicated that sEV-CUR showed an improved cartilage protection, as biweekly intra-articular injection of sEV-CUR more efficaciously alleviated oxidative stress and chondrocyte apoptosis in OA cartilage. Overall, our findings showed sEV-CUR exhibited enhanced chondro-protective effects and holds great potential on the recovery of articular cartilage loss and destruction in OA patients.

Fig. 1

Construction and collection of sEV-CUR. a Chondrocyte proliferative ability after treated with different concentrations of curcumin (n = 6); b relative fluorescence units of curcumin were detected before and after ultracentrifugation (n = 3); c relative fluorescence units were detected after curcumin was isolated from sEV by ultracentrifugation (n = 3); d relative fluorescence intensity of sEV and CUR in PBS solution was detected for a 3-h period (n = 3); e relative fluorescence intensity of sEV-CUR and CUR in HA solution was detected for a 3-h period (n = 3). N.S., not significant, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Characterization of sEV-CUR. a Representative TEM images of sEV and sEV-CUR, scale bar: 100 nm; b average particle diameter distribution of sEV and sEV-CUR was analyzed by a nano-flow cytometry; c Western blot analysis of CD63, TSG101, and GM130 from ADMSCs, sEV, and sEV-CUR. CD63 and TSG101: sEV surface marker; GM130: the Golgi marker; d Zeta potential of sEV and sEV-CUR (n = 12). N.S., not significant

Fig. 3

Cellular uptake capacity of sEV-CUR. a Fluorescence images of cellular uptake assay was captured by a microscopy during a 24-h period after sEV-CUR treatment; b mean fluorescence intensity of curcumin was calculated and analyzed (n = 3); c mean fluorescence intensity of DiI was calculated and analyzed (n = 3). *P < 0.05, **P < 0.01, ***P < 0.01

Fig. 4

sEV-CUR exhibit enhanced chondro-protective effects in TBHP-induced chondrocytes. a Representative EdU staining fluorescence images of curcumin, sEV, or sEV-CUR treated chondrocytes, scale bar: 25 μm; b statistical evaluation of fluorescent positive rate after EdU staining (n = 3); c the chondrocyte proliferative ability in each group was detected and analyzed by CCK8 assay after 2 days treatment of curcumin, sEV, or sEV-CUR (n = 6); d, e relative mRNA expression of chondrocyte anabolism related markers aggrecan and collagen II (n = 3); f, g relative mRNA expression of chondrocyte catabolism related markers ADAMTS5 and MMP13 (n = 3); h, i relative mRNA expression of inflammatory related markers IL-1β and TNFα (n = 3). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

sEV-CUR exert an enhanced anti-oxidative and anti-apoptosis ability. a, b Immunofluorescence staining and statistical analysis of oxidative stress related marker 8-OHdG in each group (n = 3), scale bar: 50 μm; c MDA level was evaluated in each group (n = 3); d ROS level in each group was detected after curcumin, sEV, or sEV-CUR treatment for 2 days (n = 3); e, f representative images and quantitative analysis of TUNEL staining analysis to detect chondrocyte apoptosis (Red) different groups, scale bar, 50 μm; g, h chondrocyte apoptotic degree in each group was detected and analyzed by flow cytometry assay (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

sEV-CUR exhibit an enhanced protective function in ACLT-induced OA mice. a Representative images of H&E, Safranin O-Fast Green (S–O) staining, and Toluidine blue staining of articular cartilage in each group, scale bar: 100 μm; b statistic data of Mankin scores in each group (n = 6); c representative image of H&E staining of synovium in each group, scale bar: 100 μm; d statistical analysis of synovitis score in each group (n = 6); e representative image of immunohistochemistry staining for extracellular matrix anabolism biomarker aggrecan and collagen II in each group, scale bar: 100 μm; f, g quantitative analysis of immunohistochemical staining of aggrecan and collagen II (n = 6). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

sEV-CUR exert an improved anti-oxidative and anti-apoptosis ability in vivo. a, b Representative images and quantitative analysis of immunohistochemistry staining for a classical oxidative stress biomarker 8-OHdG in each group (n = 6), scale bar: 50 μm; c, d representative images and quantitative analysis of immunohistochemistry staining for a classical apoptosis biomarker cleaved caspase3 in each group (n = 6), scale bar: 50 μm; e, f representative images and quantitative analysis of TUNEL staining analysis to detect chondrocyte apoptosis (Red) in vivo in each group (n = 6), scale bar, 100 μm. NS, not significant, *P < 0.05, ***P < 0.001

Fig. 8

The therapeutic effects of sEV-CUR in ACLT-induced OA mouse