024 Antifibrotic effects of a calpain inhibitor ALLN on bleomycin-induced systemic sclerosis model via antagonizing TGF-β/Smad signaling pathway

Systemic sclerosis (SSc) is a connective tissue disorder representing fibrosis in the skin and internal organs such as lungs. An activated differentiation of local progenitor cells to myofibroblasts is likely a key mechanism underlying overproduction of extracellular matrix and resultant tissue fibrosis in SSc. Calpains are family members of Ca2+-dependent cysteine proteases for which the biological action may contribute to fibrosis in various organs. However, the precise mechanism of calpain-dependent fibrosis and therapeutic utility of their inhibitors in SSc remain unclear. This study aimed to investigate if a potent calpain inhibitor ALLN could possess the antifibrotic effects on a bleomycin-induced SSc model mice and human cultured cells. Normal human dermal fibroblasts pretreated with ALLN were stimulated with recombinant TGF-β1, followed by assessment for expression properties of TGF-β1/Smad signaling and fibrogenic molecules. ALLN (3mg/kg/day) was intraperitoneally administered 3 times a week in bleomycin-induced SSc model mice. ALLN treatment significantly inhibited over-phosphorylation and nuclear transport of Smad2/3 in TGF-β1-stimulated dermal fibroblasts. TGF-β1-dependent increase of α-smooth muscle actin, collagen type 1, fibronectin 1, and representative mesenchymal markers were attenuated in mRNA and protein expression by ALLN. Likewise, ALLN reverted a TGF-β1-dependent change of epithelial/mesenchymal markers in human lung epithelial cells. Consistent with these, ALLN remarkably suppressed the development of skin and lung fibrosis, following decrease of infiltrating CD3+T cells, in bleomycin-induced SSc model mice. No obvious side effects were observed. Our data provide evidence that calpains may be a primary contributor and novel therapeutic target for skin and lung fibrosis in SSc, with a treatment perspective of its inhibitor ALLN.