Macrophage pyroptosis promotes cardiac fibroblast activation and myocardial fibrosis via the Hedgehog signaling pathway in radiation-induced heart damage

Radiotherapy is a crucial part of cancer treatment that applies to over 50 % of cancer patients. However, its administration could inadvertently damage healthy tissues, such as radiation-induced heart damage (RIHD), when thoracic radiation is implemented. Myocardial fibrosis is a key feature of RIHD that is characterized by excessive extracellular matrix (ECM) protein accumulation, resulting in cardiac stiffness and dysfunction. Macrophage pyroptosis, which is triggered by radiation, leads to the release of inflammatory mediators IL-1 beta and IL-18, which are crucial in inflammatory response and fibrosis. In this study, apoptosis-associated speck-like protein containing a CARD domain (ASC)-overexpressing RAW264.7 cells were exposed to 2 Gy, 4 Gy, and 8 Gy radiation to assess macrophage pyroptosis. Both IL-1 beta and IL-18 levels increased dose-dependently, peaking at 8 Gy. Similarly, LDH activity, which is a pyroptosis indicator, increases dose-dependently. Higher radiation dosages increased ASC specks. NLRP3, cleaved-caspase1 (P20), and GSDMD-N protein levels increased considerably in irradiation groups. Since macrophage pyroptosis promotes inflammation, it was investigated whether irradiated macrophages could cause cardiac fibroblast fibrosis. In co-culture with irradiated macrophages, cardiac fibroblasts showed dose-dependent elevation of fibrotic markers alpha-SMA and Collagen I. Blocking NLRP3-mediated pyroptosis by MCC950 in macrophages and found significant decreases in pyroptotic indicators, fibrosis markers, and Hh pathway activation in co-cultured fibroblasts. The activation of Hedgehog signaling in fibroblasts with Jervine successfully reverses fibrotic alterations caused by macrophage pyroptosis, as evidenced by decreased alpha-SMA, Collagen I, Shh, Smo, and Gli1 levels. These findings emphasize macrophage pyroptosis in radiation-induced cardiac fibrosis and identify NLRP3 and Hh pathway therapeutic targets. Collectively, targeting macrophage pyroptosis and the Hh pathway could offer new therapeutic avenues for preventing myocardial fibrosis in RIHD.