The Effects And Mechanisms Of Rho Kinase And Its Isoforms During The Intestinal Inflammation And Epithelial Barrier Dysfunction

Background and Aims:The intestinal epithelial barrier dysfunction takes part in the entire progress of inflammatory bowel disease, with the anatomy basis of tight junction structure damage. Rho kinase (ROCK) can alter the cell cytoskeleton structure by regulating the phosphorylation status of the myosin II regulation light chain (MLC), which has an important impact on the intestinal epithelial barrier function. NF-κB, another downstream molecule of ROCK, is also one of the most important translocation factors which regulate varies of inflammatory cytokines. Thus it might be possible that ROCK can promote the intestinal inflammation through activating NF-κB. The objection of this study is to investigate the effect and mechanism of Rho/ROCK during the intestinal inflammation and intestinal epithelial barrier dysfunction.Methods:Balb/c mice were randomly divided into three groups:control group (n=10), TNBS group (n=30) and Y-27632group (n=20). On day1, mice were challenged with trinitrobenzesulfonic acid (TNBS) to induce colitis. Mice of Y-27632group were exposed to10mg/kg Y-27632by intraperitoneal injection q.d from day1to day7. All mice were sacrificed on day8. Colon inflammation was quantified by mice common appearance and mortality; transmission electron microscope (TEM) was used to evaluate the morph of the tight junctions in the intestinal mucosa; the intestinal permeability were evaluated using fluorescein labeling method; both the protein and gene expression of the tight junction proteins (ZO-1and Occludin) were assessed using immunohistochemistry and Realtime-PCR; the colonic expression of ROCK1, ROCK2and their downstream molecules such as phospho-MYPT-1Thr696, phospho-MLC Ser19, IκBα, phospho-IκBα Ser32/36, NF-κB p65and NF-κB p65Ser536were also assessed using Western blot. Results:Mice of TNBS group present significant bloody stool and have a higher mortality compared with the other two groups. Histological damage and intestinal permeability were much more severe in the TNBS group than the other two groups. No significant difference in the morphology of colonic tight junctions was found by TEM. A highly increased expression of tight junction proteins ZO-1and Occludin in the colon was detected in the TNBS group. Y-27632interfering produced a dramatic reduction in Occludin expression compared with that of TNBS group. The expressions of P-MYPT-1Thr696were similar in the TNBS group and control group while there was a significant increase in the expression of P-MLC Serl9in the TNBS group compared to that of the control group. After challenged with Y-27632, both P-MYPT-1Thr696and P-MLC Ser19expressions were significantly inhibited. The colonic expressions of IκBα were reduced in the TNBS group compared with control group, and there was an obvious reduction in the expression of P-IκBα Ser32/36, while the downstream NF-κB p65expression together with its phosphorylated form P-NF-κB p65Ser536was elevated compared to the control group; Y-27632could increase the overall and phosphorylated expression of IκBα, thus decreased the expression of downstream NF-κB p65and P-NF-κB p65Ser536.Conclusions:1. ROCK inhibitor can relieve the intestinal inflammation and improve the intestinal epithelial permeability during the TNBS-induced colitis in mice.2. ROCK can alter the tight junction functions and thus affect the intestinal epithelial barrier function by facilitating the phosphorylation of their downstream molecules like MYPT-1and MLC.3. Once being activated, ROCK can promote the overall expressions of IκBα and activate it by facilitate its phosphorylation, meanwhile the expressions of the downstream NF-κB p65were also increased, which might be activated through its Ser536phosphorylation.4. It is probable that ROCK activation might result in a huge secretion of inflammation cytokines through the NF-κB pathway, and thus promote the intestinal inflammation. Background and Aims:There are two isoforms of ROCK in mammals:ROCK1and ROCK2. Despite their high affinity in structure, data shows that there still remains differences in the functions. Our aims are to select the effective interfering fragments targeting ROCK1and ROCK2; approach their differences in activating MLC and NF-κB signaling pathway. Thus we can drill deeper into the mechanism of ROCK in colitis and find out the key isoform of ROCK which plays a critical role in the intestinal epithelial barrier dysfunction.Methods:We designed the interfering fragments targeting ROCK1and ROCK2and produced LV3-shROCKl and LV3-shROCK2by cloning them to a lentiviral vector of pGLV-H1-GFP+Puro (LV3). After the identification using gene sequencing, LV3-shROCKl and LV3-shROCK2were transacted into293T cells together with a helper vehicle and a packaging vehicle. When successfully packaged, the recombinated vectors were transacted into Caco-2cells. Realtime-PCR and Western blot were used to detect the interfering efficiency on72h and96h after infestation and the effective fragments were chose out for the follow-up experiments.Caco-2cells were randomly divided into7groups:NC group, NC+TNF-a group, LV3-shROCK1group, LV3-shROCK2group, and LV3-shROCKl-shROCK2group. Realtime-PCR and Western blot were used to detect the gene and protein expressions of ROCK1, ROCK2and tight junction proteins like ZO-1and Occludin. Using Western blot, we also assessed NF-κB p65and phospho-NF-κB p65Ser536to evaluate the NF-κB pathway, and phospho-MYPT-1Thr696, phosphor-MLC Serl9to evaluate the MLC pathway.Results:According to the results of Realtime-PCR and Western blot, we picked out the most efficient interfering fragments of ROCK1(ROCK1-homo-1197and ROCK1-homo-1479) and ROCK2(ROCK2-homo-797and ROCK2-homo-3051).After24h’s stimulation by TNF-a, the expression of ROCK1and ROCK2were significantly upregulated, and were downregulated to variant extent when ROCKs were silenced. The expressions of NF-κB p65were inhibited in a similar extent in group LV3-shROCKl and LV3-shROCK2, and were obvious downregulated when ROCK1and ROCK2were blocked simultaneously. The expression of phospho-NF-κB p65Ser536were downregulated primarily in groups in which ROCK2were blocked. Both phospho-MYPT-1Thr696and phospho-MLC Ser19were elevated after stimulated with TNF-a. Expressions of phospho-MYPT-1Thr696were extremely inhibited when ROCK1were blocked, while there were no apparent changes in LV3-shROCK2group. Expressions of phospho-MLC Ser19were also downregulated when ROCKs were blocked regardless of its isoform types.Conclusions1. The expressions of tight junction proteins are independent with TNF-a stimulations and ROCKs interfering.2. ROCK1and ROCK2have similar NF-κB p65activation effects, while ROCK2takes more part in the phosphorylation of NF-κB p65Ser536.3. ROCK1and ROCK2have similar phosphylation effect of MLC Ser19, while MYPT-1Thr696was phosphorylated primarily by ROCK1, which suggest that MLC Ser19was activated by ROCK2in an independent manner of MYPT-1Thr696.