Ipriflavone attenuates the degeneration of cartilage by blocking the Indian hedgehog pathway

Zhiwei Zhang,Xiaojian Wang,Chunli Wang,Pengcui Li,Lei Wei,Chunfang Wang,Xiaochun Wei,Li Guo

doi:10.1186/s13075-019-1895-x

Abstract

BackgroundTo determine if ipriflavone, a novel and safe inhibitor of Indian hedgehog (Ihh) signaling, can attenuate cartilage degeneration by blocking the Ihh pathway.MethodsHuman chondrocytes were used to evaluate Ihh signaling, cell proliferation, apoptosis, gene, and protein expression of chondrocytes by cell proliferation and apoptosis assays, real-time qPCR, and Western blotting at 48 h after ipriflavone treatment. Human cartilage explants were further used to validate the cell culture results. The effects of ipriflavone on cartilage degeneration in vivo were assessed using the rat ACLT OA model. Two-month-old male SD rats were randomized into 3 groups (n = 75): (1) sham, (2) ACLT alone, and (3) ACLT+ ipriflavone. Ipriflavone was administered intragastrically at 24 h after ACLT for 6 weeks. The extent of OA progression was evaluated by the OARSI score and immunohistochemistry at 12 weeks after surgery. The Ihh signaling pathway and OA-related genes were quantified by real-time PCR.ResultsCell proliferation in the cells treated with ipriflavone was increased to 36.40% ± 1.32% (5 μM) and 28.54% ± 0.74% (10 μM) from 11.99% ± 0.35% (DMSO) (P < 0.001), and apoptosis was decreased to 12.64% ± 3.7% (5 μM) and 15.18% ± 3.13% (10 μM) from 25.76% ± 5.1% (DMSO) (P < 0.05). Ipriflavone blocked Runx-2 mainly through the Smo-Gli2 pathway. A similar result was found in the cartilage explant culture. Ihh signaling in vivo was inhibited in animals treated with ipriflavone. Safranin-O staining revealed a less cartilage damage with lower OARSI scores (P < 0.05) in the ipriflavone-treated animals compared with untreated animals. The gene expression of Smo and Gli2 was inhibited significantly by ipriflavone (P < 0.05). The OA-related gene and protein type X, MMP-13, and type II collagen-C fragment were reduced, while type II collagen and Agg were increased in the ipriflavone-treated animals (P < 0.05).ConclusionsCatabolic genes were disrupted by blocking the Ihh pathway. This finding suggests that disruption of Ihh signaling with ipriflavone provides chondral protection in rat posttraumatic OA.

Highlights

To determine if ipriflavone, a novel and safe inhibitor of Indian hedgehog (Ihh) signaling, can attenuate cartilage degeneration by blocking the Ihh pathway
The Cell Counting Kit-8 (CCK-8) assay results showed that the viability of chondrocytes was higher in the ipriflavone treatment groups than the dimethyl sulfoxide (DMSO) control group, and the viability gradually increased with a longer treatment time (Fig. 1B-c)
To further determine the effect of ipriflavone on chondrocyte apoptosis, we performed an annexin V-FITC/propidium iodide (PI) dual staining assay by flow cytometry, and the results showed that apoptosis was reduced in the ipriflavone treatment group than the DMSO control group after 48 h of treatment (Fig. 1C-a)

Summary

Introduction

A novel and safe inhibitor of Indian hedgehog (Ihh) signaling, can attenuate cartilage degeneration by blocking the Ihh pathway. OA is characterized by progressive degeneration of the articular cartilage and joint swelling, pain, stiffness, and loss of mobility. These characteristics significantly affect the quality of life and aggravate the financial burden of patients [1]. We further demonstrated that disrupting the Ihh signaling pathway in vivo attenuates surgically induced OA progression in Col2a1-CreERT2; Ihh fl/fl mice [8]. This evidence indicates that upregulation of the Ihh pathway plays a key role during OA progression and that inhibiting the Ihh pathway may attenuate the degeneration of cartilage. A safe inhibitor of the Hh pathway would be extremely important for human OA treatment

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