Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair

Abstract

Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10−6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks pos...

Icariin protects against iron overload‐induced bone loss via suppressing oxidative stress

“…However, there is a significant reduction of iron deposition in mice orally administrated with icariin, indicating icariin could reduce iron accumulation in vivo. Many herba flavonoids (e.g., quercetin and galangin) were reported to have the ability to chelate iron ions (Park, Bae, & Lee, ), studies by Wang et al () demonstrated that icariin could chelate cellular iron ions, leading to the inhibition of PHDs‐catalyzed HIF prolyl hydroxylation and subsequently activate HIF‐1α/2α. Our results indicated that besides iron chelation effect, icariin also could regulate iron metabolism, and promote excreting excess iron.…”

Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes

“…This study investigated effects of Sal B on passage 3/4 (P3/4) chondrocytes resembling those cultured cells for therapeutic applications. The P3/4 chondrocytes are successfully employed in clarification of regulatory role of factors on cellular functions in various cell models . However, accumulated evidence showed that passaged chondrocytes change their gene expression profiles, including matrix proteins, cytokines, matrix proteases and inhibitors, and signalling molecules , and dedifferentiate into a more fibroblastic state typically with decreased COL II and ACAN accompanied by increased COL I .…”

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1

“…Recent studies also demonstrated the involvement of hypoxia and HIF pathway for re-entry of cells in the cell cycle and for the cell contribution to organ regeneration (Ortmann et al, 2014;Brocato, Chervona, & Costa, 2014;Rios et al, 2016;Cho et al, 2015;Nakada et al, 2016;Zhang et al, 2015). Moreover, a relationship between HIF-1α induction in articular cartilage, cell proliferation, and cartilage repair has been reported (Wang et al, 2016). inhibiting DNA binding activity of HIF-1 or acriflavine, a direct inhibitor of HIF-1 because it prevents HIF-1 dimerization (Kong et al, 2005;Lee et al, 2009).…”

“…The cell cycle arrest of cardiomyocytes after birth was explained with the oxygen-rich postnatal environment (Puente et al, 2015). It was recently reported that induction of HIF-1α increased proliferation of cultured murine growth plate chondrocytes and promoted articular cartilage repair in an osteochondral defect mice model (Wang et al, 2016). The new finding that PL induces HIF-1α and that this factor is closely related to cell activation and proliferation opens a new perspective on the HIF-1α role during wound healing in different systems.…”