Background: Systemic sclerosis (SSc) is an autoimmune rheumatic connective tissue disease. In normal wound healing process, fibroblasts are activated, proliferated and involved in tissue repair, and then removed by apoptosis. In systemic sclerosis, patient’s fibrosis occurs when fibroblasts become resistant to apoptosis and secrete a large amount of collagen and other extracellular matrixes. As the primary causes the disease are very complex and often unknown, it is necessary to consider or target the secondary causes of disease, such as the unresponsiveness of activated fibroblasts to apoptosis as the major factor in the creation and deployment of illness. In this study, we examined the expression levels of two key pro-apoptotic genes, Fas and Apaf-1, which are respectively involved in external and internal pathway of apoptosis.
Methods: In a case-control study skin biopsy samples were obtained from 19 patients with diffuse SSc, and 16 healthy controls. Dermal fibroblasts were cultured and total RNA was isolated from cell populations using High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany), followed by cDNA synthesis using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Massachusetts, USA). Real-time PCR was performed using SYBRGreen gene expression master mix (Takara Shuzo, Co., Ltd, Shiga, Japan) and specific primers for Fas and Apaf-1. Real-time data were analyzed using the (2-ΔCT)×1000 method. Statistical analysis was accomplished by using the SPSS software, v22 (IBM, Armonk, NY, USA). The P value less than 0.05 were recognized as a significant threshold. All data are represented as the mean ± SEM.
Results: Our results showed no significant difference in Fas (P=0.8) and Apaf-1 (P=0.17) mRNA expression levels between skin fibroblasts of systemic sclerosis patients and healthy controls.
Conclusion: In this study we observed no significant change in Apaf-1 and Fas mRNA levels in systemic sclerosis fibroblasts compared to control group. Hence, Apaf-1 and Fas are not transcriptionally activated in SSc fibroblasts. Further studies need to take place on protein levels and function of these proteins to confirm the mRNA transcription results.
