抑制组蛋白去乙酰化酶6可恢复失血性休克时的肠道紧密连接。
Inhibition of histone deacetylase 6 restores intestinal tight junction in hemorrhagic shock.
作者信息
Chang Zhigang, Li Yongqing, He Wei, Liu Baoling, Duan Xiuzhen, Halaweish Ihab, Bambakidis Ted, Pan Baihong, Liang Yingjian, Nikolian Vahagn C, Georgoff Patrick, Alam Hasan B
机构信息
From the Department of Surgical ICU (Z.C.), Beijing Hospital, Beijing, China; Department of Cardiothoracic Surgery (W.H.), Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China; Department of Surgery (Z.C., Y.L., W.H., B.L., I.H., T.B., B.P., Y.L., V.C.N., P.G., H.B.A.), University of Michigan Hospital, Ann Arbor, Michigan; The First Hospital (Y.L.), China Medical University, Shengyang, China; and Department of Pathology (X.D.), Loyola University Medical Center, Maywood, Illinois.
出版信息
J Trauma Acute Care Surg. 2016 Sep;81(3):512-9. doi: 10.1097/TA.0000000000001137.
BACKGROUND
We recently discovered that Tubastatin-A, a histone deacetylase (HDAC6) inhibitor, can improve survival in a rodent model of hemorrhagic shock (HS), but mechanisms remain poorly defined. In this study, we investigated whether Tubastatin-A could protect intestinal tight junction (TJ) in HS.
METHODS
In an in-vivo study with Wistar-Kyoto rats, the rats underwent HS (40% blood loss) followed by Tubastatin-A (70 mg/kg) treatment, without fluid resuscitation. The experimental groups were (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, without treatment), and (3) treatment (hemorrhage with Tubastatin-A administration). Six hours after hemorrhage, ileum was harvested. Whole cell lysate were analyzed for acetylated α-tubulin (Ac-tubulin), total tubulin, acetylated histone 3 at lysine 9 (Ac-H3K9), β-actin, claudin-3 and zonula occludens 1 (ZO-1) proteins by Western blot. Histological effects of Tubastatin-A on small bowel were examined. In an in-vitro study, human intestinal epithelial cells (Caco-2) were divided into three groups: (1) sham (normoxia), (2) control (anoxia, no treatment), and (3) treatment (anoxia, treatment with Tubastatin-A). After 12 hours in an anoxia chamber, the cells were examined for Ac-tubulin and Ac-H3K9, cellular viability, cytotoxicity, claudin-3 and ZO-1 protein expression, and transwell permeability study.
RESULTS
Tubastatin-A treatment significantly attenuated HS-induced decreases of Ac-tubulin, Ac-H3K9, ZO-1 and claudin-3 proteins in small bowel in-vivo (p < 0.05). In cultured Caco-2 cells, anoxia significantly decreased cellular viability (p < 0.001) and increased cytotoxicity (p < 0.001) compared to the sham group, while Tubastatin-A treatment offered significant protection (p < 0.0001). Moreover, expression of claudin-3 was markedly decreased in vitro compared to the sham group, whereas this was significantly attenuated by Tubastatin-A (p < 0.05). Finally, anoxia markedly increased the permeability of Caco-2 monolayer cells (p < 0.05), while Tubastatin-A significantly attenuated the alteration (p < 0.05).
CONCLUSION
Inhibition of HDAC6 can induce Ac-tubulin and Ac-H3K9, promote cellular viability, and prevent the loss of intestinal tight junction proteins during HS and anoxia.
背景
我们最近发现，组蛋白去乙酰化酶（HDAC6）抑制剂Tubastatin-A可提高失血性休克（HS）啮齿动物模型的存活率，但其机制仍不清楚。在本研究中，我们调查了Tubastatin-A是否能在HS中保护肠道紧密连接（TJ）。
方法
在一项针对Wistar-Kyoto大鼠的体内研究中，大鼠经历HS（失血40%），随后进行Tubastatin-A（70mg/kg）治疗，不进行液体复苏。实验组为：（1）假手术组（无出血，未治疗），（2）对照组（出血，未治疗），（3）治疗组（出血并给予Tubastatin-A）。出血6小时后，采集回肠。通过蛋白质印迹法分析全细胞裂解物中的乙酰化α-微管蛋白（Ac-微管蛋白）、总微管蛋白、赖氨酸9位乙酰化组蛋白3（Ac-H3K9）、β-肌动蛋白、闭合蛋白-3和闭锁小带蛋白1（ZO-1）。检查Tubastatin-A对小肠的组织学影响。在一项体外研究中，将人肠上皮细胞（Caco-2）分为三组：（1）假手术组（常氧），（2）对照组（缺氧，未治疗），（3）治疗组（缺氧，用Tubastatin-A治疗）。在缺氧箱中放置12小时后，检查细胞的Ac-微管蛋白和Ac-H3K9、细胞活力、细胞毒性、闭合蛋白-3和ZO-1蛋白表达，并进行跨膜通透性研究。
结果
Tubastatin-A治疗显著减轻了HS诱导的体内小肠中Ac-微管蛋白、Ac-H3K9、ZO-1和闭合蛋白-3蛋白的减少（p<0.05）。在培养的Caco-2细胞中，与假手术组相比，缺氧显著降低了细胞活力（p<0.001）并增加了细胞毒性（p<0.001），而Tubastatin-A治疗提供了显著的保护作用（p<0.0001）。此外，与假手术组相比，体外闭合蛋白-3的表达明显降低，而Tubastatin-A显著减轻了这种降低（p<0.05）。最后，缺氧显著增加了Caco-2单层细胞的通透性（p<0.05），而Tubastatin-A显著减轻了这种改变（p<0.05）。
结论
抑制HDAC6可诱导Ac-微管蛋白和Ac-H3K9，促进细胞活力，并防止HS和缺氧期间肠道紧密连接蛋白的丢失。
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