Division of Pediatric Surgery, Department of Surgery, St. Louis Children's Hospital, Washington University School of Medicine, St. Louis, Missouri.
Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.
J Surg Res. 2021 Feb;258:73-81. doi: 10.1016/j.jss.2020.08.049. Epub 2020 Sep 28.
Short bowel syndrome resulting from small bowel resection (SBR) is associated with significant morbidity and mortality. Many adverse sequelae including steatohepatitis and bacterial overgrowth are thought to be related to increased bacterial translocation, suggesting alterations in gut permeability. We hypothesized that after intestinal resection, the intestinal barrier is altered via toll-like receptor 4 (TLR4) signaling at the intestinal level.
B6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice underwent 50% SBR or sham operation. Transcellular permeability was evaluated by measuring goblet cell associated antigen passages via two-photon microscopy. Fluorimetry and electron microscopy evaluation of tight junctions (TJ) were used to assess paracellular permeability. In parallel experiments, single-cell RNA sequencing measured expression of intestinal integral TJ proteins. Western blot and immunohistochemistry confirmed the results of the single-cell RNA sequencing.
There were similar number of goblet cell associated antigen passages after both SBR and sham operation (4.5 versus 5.0, P > 0.05). Fluorescein isothiocyanate-dextran uptake into the serum after massive SBR was significantly increased compared with sham mice (2.13 ± 0.39 ng/μL versus 1.62 ± 0.23 ng/μL, P < 0.001). SBR mice demonstrated obscured TJ complexes on electron microscopy. Single-cell RNA sequencing revealed a decrease in TJ protein occludin (21%) after SBR (P < 0.05), confirmed with immunostaining and western blot analysis. The KO of iTLR4 mitigated the alterations in permeability after SBR.
Permeability after SBR is increased via changes at the paracellular level. However, these alterations were prevented in iTLR4 mice. These findings suggest potential protein targets for restoring the intestinal barrier and obviating the adverse sequelae of short bowel syndrome.
小肠切除（SBR）引起的短肠综合征与显著的发病率和死亡率相关。许多不良后果，包括脂肪性肝炎和细菌过度生长，被认为与细菌易位增加有关，这表明肠道通透性发生改变。我们假设，在肠切除后，肠道屏障通过肠道水平的 toll 样受体 4（TLR4）信号发生改变。
B6 和肠道特异性 TLR4 敲除（iTLR4 KO）小鼠接受 50%的 SBR 或假手术。通过双光子显微镜测量杯状细胞相关抗原的通过来评估跨细胞通透性。使用荧光法和电子显微镜评估紧密连接（TJ）来评估旁细胞通透性。在平行实验中，单细胞 RNA 测序测量了肠道完整 TJ 蛋白的表达。Western blot 和免疫组织化学证实了单细胞 RNA 测序的结果。
SBR 和假手术组的杯状细胞相关抗原通过量相似（4.5 对 5.0，P>0.05）。与假手术组相比，大量 SBR 后荧光素异硫氰酸酯-葡聚糖进入血清的摄取明显增加（2.13±0.39ng/μL 对 1.62±0.23ng/μL，P<0.001）。SBR 小鼠在电子显微镜下显示 TJ 复合物模糊。单细胞 RNA 测序显示 SBR 后 TJ 蛋白紧密连接蛋白（occludin）减少（21%，P<0.05），免疫染色和 Western blot 分析证实了这一点。iTLR4 的 KO 减轻了 SBR 后通透性的改变。
SBR 后通透性的增加是通过旁细胞水平的变化引起的。然而，这些改变在 iTLR4 小鼠中被预防。这些发现为恢复肠道屏障和避免短肠综合征的不良后果提供了潜在的蛋白质靶标。
