Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

Abstract

OBJECTIVE:To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice.METHODS:Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR.RESULTS:Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3).CONCLUSION:Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a reduction in ET-1.

The Mighty Fibroblast and Its Utility in Scleroderma Research

“…This conversion has been recapitulated with TGFβ treatment of in vitro cultures of fibroblasts (2, 24–25–26). Myofibroblasts usually undergo deactivation and apoptosis, dedifferentiation, or senescence in the latter stages of the wound healing process, the time when matrix reabsorption and TGFβ signaling terminate and redox homeostasis is restored (5, 7, 21, 23, 24, 27–28–29–30–31). The danger in fibroblast plasticity arises when the precipitating injury leads to excessive fibrogenic milieu activation wherein myofibroblast differentiation or persistence occurs, leading to wound strengthening through disproportionate collagen deposition, fiber alignment, and excessive force contraction, in part because myofibroblasts contribute to wound repair, not tissue regeneration (3, 7, 28).…”

“…Receptor modulation: wide study of the endothelin receptors has led to a myriad of clinical trials based upon basic research conducted with fibroblasts. Levels of the vasoactive peptide ET-1 and procontractile proteins are significantly increased in SSc fibroblasts, which enhance their ability to contract collagen matrix compared to normal fibroblasts (26, 31, 32). Treatment of normal and SSc human dermal fibroblasts with ET-1 promoted significant ECM production and matrix contraction through the endothelin A (ETA) receptor (32, 80).…”

Ethanol extract of gardenia fruit alleviates renal interstitial fibrosis induced by unilateral ureteral obstruction in rats

“…A recent study has discovered that crocetin treatment may protect against burn-induced small intestinal injury by inhibiting oxidative stress and inflammatory response ( 22 ). Furthermore, crocetin may alleviate cardiac, skin and lung fibrosis due to suppression of reactive oxygen species-dependent signaling pathways or a reduction in endothelin-1 ( 23 , 24 ). It has also been reported that geniposide is able to suppress the release of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6 and interleukin-1β in vitro and in vivo , and is also able to block the phosphorylation of nuclear factor-κB, p65 and p38, as well as inhibiting the expression of extracellular signal-related kinases (ERKs) and c-Jun N-terminal kinases in lipopolysaccharide-stimulated primary mouse macrophages ( 16 ).…”

Oxidative/nitrative stress in the pathogenesis of systemic sclerosis: are antioxidants beneficial?

“…Crocetin is an apocarotenoid dicarboxylic acid extracted from Crocus flowers and Gardenia jasminoides fruits. Crocetin inhibited the proliferation of normal and SSc fibroblasts and the trans-differentiation of SSc fibroblasts into myofibroblasts; in the bleomicin mouse model of SSc repeated intraperitoneal injection of crocetin alleviated J U S T A C C E P T E D skin and lung fibrosis in association with decreased levels of mRNAs for type 1 collagen and endothelin-1 in skin and lung [171].…”