PPARγ inhibitors enhance the efficacy of statin therapy for steroid-induced osteonecrosis of the femoral head by directly inhibiting apoptosis and indirectly modulating lipoprotein subfractions

Abstract

Background: Steroid-induced osteonecrosis of the femoral head (SONFH) is a serious bone disease commonly seen in patients on long-term glucocorticoid therapy. Although statins have shown some efficacy in improving lipid metabolism, their efficacy in the treatment of SONFH remains limited. PPARγ inhibitors may enhance the efficacy of statins through several mechanisms. This study aims to investigate how PPARγ inhibitors may enhance the effects of statins in the treatment of SONFH by directly inhibiting apoptosis and indirectly modulating lipoprotein subfractions.

Methods: We first treated osteoblasts in vitro with high concentrations of hormones to simulate the SONFH environment. We then treated the cells with either the PPARγ inhibitor GW9662, the statin lovastatin, or a combination of both. We assessed cell proliferation and apoptosis using CCK-8, flow cytometry and Western blotting. We then established a SONFH rabbit model using high doses of methylprednisolone and lipopolysaccharide. The rabbits were randomly divided into four groups: control group, lovastatin group, GW9662 group and combination therapy group. We observed hip joint MRI before treatment, after 4 weeks of treatment, and 4 weeks after stopping treatment. We performed hematoxylin-eosin staining of the femoral head and analysed serum lipoprotein subfractions using VAP technology. In addition, we used quantitative polymerase chain reaction (qPCR) to analyse the expression of genes related to lipid metabolism at week 3.

Results: In vitro experiments showed that both GW9662 and lovastatin effectively inhibited hormone-induced apoptosis. In the animal studies, imaging and pathological results showed that the progression of SONFH was slower in the combination therapy group than in the other groups. VAP analysis showed that the lovastatin group had disturbed lipoprotein subfractions at the fourth week after stopping treatment, while the combination therapy group had more stable lipoprotein subfractions.

Conclusion: PPARγ inhibitors significantly enhance the efficacy of statins in the treatment of SONFH by directly inhibiting apoptosis and indirectly modulating lipoprotein subfractions. These findings provide new insights into the clinical management of SONFH and suggest that combination therapy may be an effective strategy.

Fig 1. The experimental results of osteoblast – related research.

(A). ARS and ALP- stained osteoblasts show brown-red color, indicating high mineralization with numerous, dense mineralized nodules, crucial for evaluating osteogenic differentiation. The cells have normal morphology with clear outlines and even cytoplasm. ALP – stained osteoblasts show strong activity, appearing dark blue – purple, reflecting high alkaline phosphatase expression during early osteogenic differentiation. (B). Cell proliferative activity after hormone and lovastatin treatment, revealing their impact on osteoblast growth. (C). The flow cytometry results showed the cell cycle and apoptosis status of cells in different treatment groups. (D). The WB detection results of PPAR – γ and apoptosis related protein expression changes elucidated the molecular mechanism.

Fig 2. Flow chart of animal experiment section (A) and hip T2WI results were detected by MRI (from left to right:Blank, Model, GW9662, Lovastain, GW9662

+ Lovastain) (B). Red arrows indicate changes in the bone marrow edema signal, and yellow arrows indicate changes in ischemia.

Fig 3. Lipoprotein subfraction plots for each experimental group in week two(A).

Lipoprotein subfraction plots for each experimental group in week three(B).Detection of gene expression levels in femoral head tissues of experimental groups by qPCR method(C).

Fig 4. HE staining was performed on the femoral head tissue after blood collection (A), and the rate of empty lacunae was calculated (B).

Additionally, the ultrastructure of the cells was observed by transmission electron microscopy (C).