Background

Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn’s disease (CD). Rho kinases (ROCKs) play multiple roles in TGFβ-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis.

Methods

Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures.

Results

ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFβ1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis.

Conclusions

Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

中文翻译：

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局部Rho激酶抑制剂的多效作用治疗实验性肠炎的肠道纤维化

背景

导致（亚）阻塞的肠纤维化是克罗恩病（CD）的常见并发症。Rho激酶（ROCKs）在TGFβ诱导的成纤维细胞活化中起多种作用，这可能是治疗靶标。由于全身性ROCK抑制会引起心血管副作用，因此我们评估了局部作用的ROCK抑制剂（AMA0825）对肠道纤维化的影响。

方法

使用硫酸葡聚糖钠（DSS）和过继性T细胞转移在小鼠模型中评估纤维化。在不同的细胞类型和CD活检培养物中研究了AMA0825的体外和离体作用。

结果

ROCK在人肠道的成纤维细胞，上皮，内皮和肌肉细胞中表达，并在发炎和纤维化组织中被激活。在两种纤维化模型中，AMA0825的预防性治疗可抑制肌纤维母细胞的积累，促纤维化因子的表达以及纤维化组织的积累，而不会影响临床疾病活动和组织学炎症。ROCK抑制作用可逆转慢性DSS模型中已建立的纤维化，并阻止狭窄CD活检组织中离体前纤维化蛋白的分泌。AMA0825减少了TGFβ1诱导的心肌相关转录因子（MRTF）和p38丝裂原活化蛋白激酶（MAPK）的激活，下调了基质金属蛋白酶，胶原蛋白和成纤维细胞IL6的分泌。在这些细胞中，ROCK抑制增强了自噬，这是观察到的胶原蛋白和IL6生成减少所必需的。AMA0825不会影响其他ROCK阳性细胞类型促炎性细胞因子的分泌，从而证实了对纤维化的选择性体内作用。

结论

局部ROCK抑制通过减少MRTF和p38 MAPK激活并增加成纤维细胞自噬来预防和逆转肠道纤维化。总的来说，我们的结果表明，局部ROCK抑制作为CD的附加疗法有望抵消纤维化。