Liu Luqiong, Chen Tong, Xie Zhenrong, Zhang Yongjin, He Chenglu, Huang Yongkun
Kunming Medical University, Kunming, Yunnan, China.
Department of Pediatrics, the First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China.
PLoS One. 2024 Dec 26;19(12):e0316362. doi: 10.1371/journal.pone.0316362. eCollection 2024.
Butyric acid (BA) can potentially enhance the function of the intestinal barrier. However, the mechanisms by which BA protects the intestinal mucosal barrier remain to be elucidated. Given that the Ras homolog gene family, member A (RhoA)/Rho-associated kinase 2 (ROCK2)/Myosin light chain kinase (MLCK) signaling pathway is crucial for maintaining the permeability of the intestinal epithelium, we further investigated whether BA exerts a protective effect on epithelial barrier function by inhibiting this pathway in LPS-induced Caco2 cells. First, we aimed to identify the optimal treatment time and concentration for BA and Lipopolysaccharide (LPS) through a CCK-8 assay. We subsequently measured Trans-epithelial electrical resistance (TEER), FITC-Dextran 4 kDa (FD-4) flux, and the mRNA expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, along their protein expression levels, and average fluorescence intensity following immunofluorescence staining. We then applied the ROCK2 inhibitor Y-27632 and reevaluated the TEER, FD-4 flux, and mRNA, and protein expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, as well as their distribution in Caco2 cells. The optimal treatment conditions were determined to be 0.2 mmol/L BA and 5 μg/mL LPS for 24 hours. Compared with LPS treatment alone, BA significantly mitigated the reduction in the TEER, decreased FD-4 flux permeability, increased the mRNA expression of ZO-1 and Occludin, and normalized the distribution of ZO-1 and Occludin in Caco2 cells. Furthermore, BA inhibited the expression of RhoA, ROCK2, and MLCK, and normalized their localization within Caco2 cells. Following treatment with Y-27632, the epithelial barrier function, along with the mRNA and protein expression and distribution of ZO-1 and Occludin were further normalized upon inhibition of the pathway. These findings contribute to a deeper understanding of the potential mechanisms through which BA attenuates LPS-induced impairment of the intestinal epithelial barrier.
丁酸（BA）可能会增强肠道屏障的功能。然而，BA保护肠道黏膜屏障的机制仍有待阐明。鉴于Ras同源基因家族成员A（RhoA）/Rho相关激酶2（ROCK2）/肌球蛋白轻链激酶（MLCK）信号通路对于维持肠上皮的通透性至关重要，我们进一步研究了BA是否通过在脂多糖（LPS）诱导的Caco2细胞中抑制该信号通路来对上皮屏障功能发挥保护作用。首先，我们旨在通过CCK-8实验确定BA和LPS的最佳处理时间和浓度。随后，我们测量了跨上皮电阻（TEER）、4 kDa异硫氰酸荧光素葡聚糖（FD-4）通量，以及紧密连接蛋白1（ZO-1）、闭合蛋白（Occludin）、RhoA、ROCK2和MLCK的mRNA表达、蛋白表达水平，以及免疫荧光染色后的平均荧光强度。然后，我们应用ROCK2抑制剂Y-27632，并重新评估TEER、FD-4通量，以及ZO-1、Occludin、RhoA、ROCK2和MLCK的mRNA和蛋白表达，以及它们在Caco2细胞中的分布。确定最佳处理条件为0.2 mmol/L BA和5 μg/mL LPS处理24小时。与单独LPS处理相比，BA显著减轻了TEER的降低，降低了FD-4通量通透性，增加了ZO-1和Occludin的mRNA表达，并使ZO-1和Occludin在Caco2细胞中的分布正常化。此外，BA抑制了RhoA、ROCK2和MLCK的表达，并使它们在Caco2细胞中的定位正常化。在用Y-27632处理后，该信号通路被抑制后，上皮屏障功能以及ZO-1和Occludin的mRNA和蛋白表达及分布进一步恢复正常。这些发现有助于更深入地了解BA减轻LPS诱导的肠上皮屏障损伤的潜在机制。
