本研究旨在验证钙敏感受体(CaSR)可以保护肠上皮屏障完整性并减少Ras相关C3肉毒毒素底物1(Rac1)/磷脂酶Cγ1(PLC-γ1)信号通路介导的炎症反应的假设。在不存在或存在CaSR拮抗剂(NPS2143)的情况下,不使用或使用TNF-α处理IPEC-J2单层,CaSR过表达,和Rac1沉默,PLCγ1沉默或精胺。结果显示精胺增加跨上皮电阻(TER),紧密连接蛋白水平,Rac1/PLC-γ1信号通路的蛋白浓度,在存在TNF-α的情况下,细胞旁通透性降低。NPS2143抑制精胺诱导的上述参数变化。CaSR过表达增加TER,紧密连接蛋白的水平和CaSR的蛋白质浓度,磷酸化PLCγ1,Rac1和IP3,并在TNF-α攻击后降低细胞旁通透性和白细胞介素8(IL-8)和TNF-α的含量。Rac1和PLCγ1沉默抑制了CaSR诱导的屏障功能和磷酸化PLCγ1,Rac1和IP3蛋白浓度的增加,并在TNF-α攻击后降低了IL-8和TNF-α的含量。这些结果表明,在TNF-α攻击后,CaSR激活通过激活Rac1和PLCγ1信号来保护肠道完整性并减轻炎症反应。精胺可通过CaSR/Rac1/PLC-γ1通路维持屏障功能。
This study aimed to test the hypothesis that the calcium-sensing receptor (CaSR) can protect intestinal epithelial barrier integrity and decrease inflammatory response mediated by the Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. IPEC-J2 monolayers were treated without or with TNF-α in the absence or presence of CaSR antagonist (NPS 2143), CaSR overexpression, and Rac1 silencing,
PLCγ1 silencing or spermine. Results showed that spermine increased transepithelial electrical resistance (TER), tight junction protein levels, the protein concentration of Rac1/PLC-γ1 signaling pathway, and decreased paracellular permeability in the presence of TNF-α. NPS2143 inhibited spermine-induced change in above-mentioned parameters. CaSR overexpression increased TER, the levels of tight junction proteins and the protein concentration of CaSR, phosphorylated
PLCγ1, Rac1, and IP3, and decreased paracellular permeability and contents of interleukin-8 (IL-8) and TNF-α after TNF-α challenge. Rac1 and
PLCγ1 silencing inhibited CaSR-induced increase in barrier function and the protein concentration of phosphorylated
PLCγ1, Rac1, and IP3, and decrease in contents of IL-8 and TNF-α after TNF-α challenge. These results suggest that CaSR activation protects intestinal integrity and alleviates the inflammatory response by activating Rac1 and
PLCγ1 signaling after TNF-α challenge, and spermine can maintain barrier function via CaSR/Rac1/PLC-γ1 pathway.