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To explore the effects of mechanical stretch on inflammatory cytokine expression and pro-fibrotic role in neonatal rat cardiac fibroblast.
Neonatal rat cardiac fibroblasts(CFs) were obtained through differential adhesion method. The 2nd passage cells were stimulated by FLEXCELL-5000T system. According to the stretch intensity and time applied,the cells were divided into following group: control, 5%,10%,15%, stimulation for 24 hours with the respective elongation. and 0h, 6h, 12h, 24h and 48h, stimulation with 10% elongation for respective time. The expression of α-SMA and Collagen I were detected by Q-PCR and Western blot. In the experiment of cytokine detection, The 2nd passage cells were stimulated through FLEXCELL-5000T system at 10% elongation, 10ˆ-6M pro-inflammatory media Angiotensin II(Ang II)as the positive control, the 4 groups were:control, Ang II, Stretch and Ang II+ Stretch. After 24h stimulation, the supernatant of cultural medium was collected, and the inflammatory factors were detected by antibody array(QAR-INF-1).
Instead of 15% elongation, both 5% and 10% stretch induced significant expression of α-SMA and Collagen I(P<0.05)and the effect of 10% elongation was more prominent. In contrast to 0h,the increased collagen I started to change at 6h and the increment of α-SMA at 12h(P<0.05), both reached the highest point at 24h and 48h(P<0.05). In vitro, CFs fundamentally secreted inflammatory factors with the most prominent MCP-1. The expression of pro-inflammatory cytokine (such as IL-2, IL-6, MCP-1 and TNF-α) increased in the groups of Ang II and Stretch (P<0.05),and these cytokines were lower in Ang II +Stretch compared to Ang II or Stretch stimulation alone (P<0.001). Besides, IFN-γ and IL-1α remained unchanged (P>0.05). In contrast to control, anti-inflammation cytokine (such as IL-13 and IL-4) decreased in the group of Ang II +Stretch, and stretch alone reduced the expression of IL-13 (P<0.05). IL-10 was not detected in all groups.
Mechanical stretch possesses a pro-fibrotic effect,CFs response to mechanical stretch through transdifferentiation to myofibroblast and increased expression of extracellular matrix. And cardiac fibroblasts were sensitive to mechanical stimulation with the secretion of pro-inflammatory factors and inhibition of anti-inflammatory cytokine expression, thus participated in mechanical stretch-induced inflammation.