Myosin Light Chain Kinase Inhibitor Inhibits Dextran Sulfate Sodium-Induced Colitis in Mice

Background and Aims

Myosin light chain kinase (MLCK) plays a central role in the mechanisms of barrier dysfunction, and intestinal epithelial MLCK protein expression is upregulated in active ulcerative colitis (UC). ML-7, a MLCK inhibitor, has been used in many MLCK studies. However, the effect of ML-7 has never been estimated in colitis models. The aim of this study was to determine whether ML-7 can treat UC.

Methods

Experimental colitis was induced and ML-7 was administered by intraperitoneal injection. The disease activity index (DAI) scores were evaluated and colon tissue was collected for the assessment of histological changes, myeloperoxidase (MPO) activity, and tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-13 and interleukin (IL)-17 levels. The small intestinal mucosa was ultrastructurally examined, epithelial MLCK protein expression and enzymatic activity were determined, and intestinal permeability was assayed using FITC-dextran 4000 (FD-4) and Evans blue (EB).

Results

ML-7 was found to be significantly effective in reducing the DAI scores and histological index scores, and decreasing MPO activity and TNF-α, IFN-γ, IL-13 and IL-17 levels. The small intestinal epithelial MLCK protein expression and enzymatic activity were downregulated by ML-7. The epithelial cells and intercellular tight junctions were ameliorated, and the amount of FD-4 in blood and EB permeating into the intestine were decreased by ML-7 in colitis mice.

Conclusions

ML-7 has a significant anti-colitis effect in colitis mice. It is mainly associated with the inhibition of the epithelial MLCK protein expression, resulting in ameliorated intestinal mucosal permeability.     

Myosin Light Chain Phosphorylation Correlates with Contractile Force in Guinea Pig Gallbladder Muscle

Acetylcholine (ACh) -induced gallbladder smooth muscle contraction involves myosin light chain phosphorylation (MLCP). ACh-induced contraction is dose dependent. Whether MLCP by ACh is also dose dependent and how it correlates with contractile force have not been carefully evaluated. This study investigated the correlation between gallbladder muscle contraction and MLCP. Guinea pig gallbladder muscle strips were studied isometrically and frozen after different doses of ACh (0, 0.1, 5, 100 M) for different periods of incubation (0.5, 1, 2, 3, 4 min). MLCP was determined using gel electrophoresis. Both contraction and MLCP in response to ACh were concentration dependent. Peak MLCP to ACh 100 M occurred at 30 sec. There was a high correlation between active force and MLCP (r = 0.991; P = 0.009 at 30 sec stimulation). Nifedipine 1 M reduced ACh-induced contraction and MLCP by a similar degree (31% and 33%, respectively). In conclusion, gallbladder contractile force significantly correlates with MLCP. This is consistent with the hypothesis that initiation of gallbladder cholinergic contraction is dependent on phosphorylation of myosin light chains.     

糖基化终末产物和磷酸化肌球蛋白轻链在糖尿病结肠动力障碍中的作用

背景:结肠动力障碍是糖尿病(DM)的常见并发症。糖基化终末产物(AGEs)在DM患者体内显著升高,然而其在DM结肠动力障碍中的作用尚未完全明确。目的:探讨AGEs和磷酸化肌球蛋白轻链(p-MLC)在DM结肠动力障碍中的作用。方法:将Sprague-Dawley大鼠随机分为DM组和正常对照组,以链脲菌素腹腔注射建立DM模型,8周后处死所有大鼠,取远端结肠组织。检测离体结肠肌条肌张力;以免疫组化染色检测结肠组织p-MLC表达;以蛋白质印迹法检测结肠组织p-MLC、总肌球蛋白轻链(t-MLC)表达。分离培养结肠平滑肌细胞(SMCs),以不同浓度、不同作用时间AGEs作用于SMCs,以蛋白质印迹法检测SMCs的p-MLC、t-MLC表达。结果:与正常对照组相比,DM组大鼠结肠平滑肌张力显著减弱[(0.89±0.09)g对(1.98±0.12)g,P0.05],DM组大鼠结肠组织pMLC/t-MLC/β-actin水平显著降低(0.98±0.10对1.61±0.12,P0.05)。SMCs经不同浓度、不同作用时间AGEs干预后,p-MLC/t-MLC/β-actin水平显著降低。结论:DM结肠动力障碍可能由AGEs抑制SMCs中MLC磷酸化所致。     

Myosin Light Chain Kinase Is Involved in the Mechanism of Gastrointestinal Dysfunction in Diabetic Rats

Background  

It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca²⁺ via myosin light chain phosphorylation, which is activated by myosin light chain kinase (MLCK). Recently, MLCK has been demonstrated to play an important role in smooth muscle contraction and normal gastrointestinal motility.     

Contribution of Myosin Light Chain Kinase to Neurotransmitter Release from Peripheral Sympathetic Nerve Endings of Spontaneously Hypertensive Rats

A dissociation between changes in blood pressure (BP) and plasma renin activity (PRA) has been noted after administration of renin inhibitors. In the present study, the renin inhibitor PD 132002 was given to salt-deplete, anesthetized dogs. PRA was measured at pH 6.0 by a conventional angiotensin I (ANG I) RIA method (PRA-C) and by an ANG I antibody-trapping RIA method (PRA-AT) performed at pH 7.4. PD 132002 at 0.01, 0.1, 1, and 10 mg/kg IV, reduced BP by 3 ±2, 9 ± 2, 24 ± 4, and 39 ± 4 mm Hg, respectively, (baseline of 136 ± 8 mm Hg, N = 5)     

Adiponectin Modulates DCA-Induced Inflammation via the ROS/NF-Kappa B Signaling Pathway in Esophageal Adenocarcinoma Cells

Background

Deoxycholic acid (DCA) promotes the development and progression of esophageal adenocarcinoma (EAC) by inducing inflammation. Adiponectin is reported to have anti-inflammatory and anti-tumor effects.

Purpose

This study investigated the effects of two types of adiponectin, full-length adiponectin (f-Ad) and globular adiponectin (g-Ad), on DCA-induced inflammation, and investigated the involvement of the reactive oxygen species (ROS)/NF-κB signaling pathway in inflammation in EAC.

Methods

OE19 cells were treated with DCA (50–300 μM) and/or f-Ad/g-Ad (10.0 μg/ml) or N-acetylcysteine (NAC). The viability of cells exposed to DCA was measured by use of the MTT assay. mRNA and protein levels of the inflammatory factors were examined by real-time PCR and ELISA. Intra-cellular ROS levels were determined by use of flow cytometry. Protein levels of total and p-NF-κB p65 were measured by western blot.

Results

DCA induced dose and time-dependent cytotoxicity. mRNA and protein expression of TNF-α, IL-8, and IL-6 in cells treated with DCA alone were up-regulated, and intra-cellular ROS and p-NF-κB p65 protein levels were also increased. g-Ad promoted inflammatory factor production, ROS levels, and p-NF-κB p65 protein expression whereas f-Ad had a suppressive effect. When combined with DCA, g-Ad enhanced the pro-inflammatory effect of DCA whereas f-Ad, similar to NAC, suppressed the effect.

Conclusion

DCA has a pro-inflammatory effect in EAC. f-Ad has an anti-inflammatory effect whereas g-Ad seems to have a pro-inflammatory effect in an ROS/NF-κB p65-dependent manner. This indicates that f-Ad could be a potential anti-inflammatory reagent for cancer therapy.     

XRCC2 Promotes Colorectal Cancer Cell Growth,Regulates Cell Cycle Progression,and Apoptosis

X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2) and poly(ADP-ribose) polymerase 1 (PARP1) both play important roles in homologous recombination DNA repair. According to the theory of synthetic lethality, XRCC2-deficient cells are more sensitive to PARP1 inhibitors compared to XRCC2-expressing cells. We investigated XRCC2 expression and function in colorectal cancer (CRC), and the characteristics of sensitivity to PARP1 inhibitor in CRC cells with different XRCC2 levels.We enrolled 153 patients with CRC who had undergone surgery in this study. XRCC2 expression was assessed using immunohistochemistry. Stable CRC SW480 cell lines with low or high XRCC2 expression were constructed. Following treatment with the PARP1 inhibitor olaparib, the viability of cells with different XRCC2 levels was determined; cell cycle distribution and apoptosis were analyzed using flow cytometry. B-cell lymphoma-2 (Bcl-2) protein expression was measured by Western blotting.The positive rates of XRCC2 in primary CRC tissue were significantly higher than that in the matched adjacent noncancerous tissue, and XRCC2 expression status in primary CRC was related to tumor site, Dukes’ stage, and tumor-nodes-metastasis (TNM) stage. XRCC2 overexpression inhibited CRC cell apoptosis and promoted proliferation by enriching cells in the G0/G1 phase. Moreover, olaparib suppressed proliferation, and olaparib sensitivity in CRC cells with high XRCC2 expression was greater.High XRCC2 expression promotes CRC cell proliferation and enriches cells in the G0/G1 phase but inhibits apoptosis. High XRCC2 expression cells are more sensitive to olaparib, which inhibits their viability.     

Decreased Pulmonary Inflammation Following Ethanol and Burn Injury in Mice Deficient in TLR4 but not TLR2 Signaling

Background: Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro‐inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll‐like receptors 2 and 4 (TLR2 and 4) pathways. Methods: Wild‐type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty‐four hours postinjury, systemic and pulmonary levels of pro‐inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination. Results: Higher numbers of neutrophils were observed in the lungs of wild‐type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL‐6 and tumor necrosis factor‐α were also elevated in wild‐type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL‐6 were increased in wild‐type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild‐type mice, of neutrophil infiltration as well as IL‐6 and KC in the lung. Conclusions: These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.     

Tumor Buds Show Reduced Expression of Laminin-5 Gamma 2 Chain in DNA Mismatch Repair Deficient Colorectal Cancer

Purpose Tumor budding at the invasive margin of colorectal cancer is an important adverse prognostic factor. The subset of colorectal cancer that is deficient in DNA mismatch repair has been associated with a good prognosis. It is hypothesized that tumor budding in this subset may lack biologic aggressiveness because it is not associated with aberrant expression of the independent prognostic factor, laminin-5 gamma 2. Methods Eighty colorectal cancers with high-grade tumor budding were studied, including nine sporadic colorectal cancers with immunohistochemical loss of expression of MLH1 (MLH1(−)), seven colorectal cancers from patients with hereditary nonpolyposis colorectal cancer, and 64 sporadic colorectal cancers expressing both MLH1 and MSH2 (MLH1(+)). Two regulatory mechanisms for laminin-5 gamma 2 expression were explored, including aberrant nuclear expression of β-catenin by immunohistochemistry and promoter methylation of laminin-5 gamma 2 by methylation-specific polymerase chain reaction. Results Only three of nine MLH1(−) colorectal cancers showed expression of laminin-5 gamma 2 compared with 46 of 64 MLH1(+) colorectal cancers (P = 0.05). Only two of seven hereditary nonpolyposis colorectal cancers expressed laminin-5 gamma2 compared with MLH1(+) colorectal cancers (P= 0.03). Expression of nuclear β-catenin was more frequent (58 percent) in MLH1(+) colorectal cancers compared with MLH1(−) colorectal cancers (11 percent, P = 0.01). Methylation of laminin-5 gamma 2 was found in 5 of 38 (13 percent) cases but did not differ among colorectal cancer subsets. Four of five colorectal cancers with methylation of laminin-5 gamma 2 were scored as negative for laminin-5 gamma 2 by immunohistochemistry. Conclusions The reduced expression of laminin-5 gamma 2in colorectal cancers with deficient DNA mismatch repair may underlie a variant of tumor budding that is relatively nonaggressive. Supported by a grant from the Canadian Institutes of Health Research (grant no. MOP-67206).