肌球蛋白轻链激酶介导的肌球蛋白轻链磷酸化在严重烧伤早期肠粘膜屏障损害中的作用
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摘要
一、背景与目的
肠粘膜上皮屏障是体内重要的生物防御屏障,在抵御肠腔内细菌和/或内毒素的移位过程中起着中坚作用。过去的研究表明,严重烧伤后肠道粘膜上皮组织明显受损,屏障功能紊乱,通透性增加,肠道细菌和/或内毒素移位,从而引发或加重烧伤后一系列病理生理变化,但严重烧伤后肠粘膜屏障功能损害的分子机制仍不完全清楚。肠粘膜屏障完整性得以维持的结构基础是细胞间的紧密连接(TJ)及其相关蛋白。
TJ是由多种蛋白质相互作用而形成的复合体,在调节肠粘膜通透性和维持上皮细胞极性中发挥着重要作用。TJ由特异的连接蛋白使两个相邻细胞之间的间歇完全闭锁,与其密切相关的蛋白主要包括ZO-1、occludin、claudin等,其中ZO-1又与胞内细胞骨架肌动蛋白相连,使胞内细胞骨架与细胞外连接蛋白实现信号互为沟通。在相关信号刺激下,由于骨架蛋白活动和细胞回缩,引起连接蛋白结构和功能失调,细胞间隙形成,屏障通透性增加。
研究表明,屏障通透性变化与调节涉及细胞骨架重组、紧密连接、粘附连接结构等不同的机制。细胞收缩是各种原因引起屏障通透性增加的共同通路,主要受肌动蛋白(actin)和肌球蛋白(myosin)的影响,并依赖于肌球蛋白轻链(myosin light chain,MLC)的磷酸化。
鉴于MLC磷酸化在生物屏障功能的调控中起着重要作用,而既往有关研究资料又缺乏对MLC磷酸化与烧伤后肠粘膜屏障功能损害关系的探讨,本研究围绕MLC磷酸化与烧伤早期肠粘膜屏障功能损害,重点探讨肌球蛋白轻链激酶(myosin light chain kinase,MLCK)所介导的MLC磷酸化与严重烧伤早期肠粘膜上皮屏障功能损害、细胞骨架F-肌动蛋白、细胞紧密连接蛋白等变化的关系,初步明确MLCK所介导的MLC磷酸化在严重烧伤早期肠粘膜上皮屏障功能损害中的作用,以进一步完善烧伤后肠粘膜上皮屏障损害的发生机制。
二、方法
1.健康成年Sprague Dawley大鼠随机分为正常对照组(伤前)及伤后1、2、6、12、24h组,制备30% TBSAⅢ度烧伤模型。用异硫氰酸荧光素-葡聚糖(FITC-Dextran)示踪法检测大鼠肠粘膜通透性,免疫荧光法检测肠粘膜组织紧密连接蛋白ZO-1、F-肌动蛋白及p-MLC表达。
2.健康成年BALB/c小鼠随机分为4个组:①正常对照组:正常小鼠行假致伤;②ML-9对照组:于假致伤的正常对照小鼠腹腔注射ML-9 (2 mg/kg);③烧伤组:小鼠接受30% TBSAⅢ度烧伤;④烧伤治疗组:于30% TBSAⅢ度烧伤小鼠腹腔注射ML-9(2mg/kg)。FITC-Dextran示踪法检测肠粘膜通透性,HE染色及光镜观察肠粘膜组织结构,透射电镜观察肠粘膜超微结构,免疫荧光法检测肠粘膜ZO-1及F-肌动蛋白,Western blot法检测肠粘膜MLCK及磷酸化型MLC(p-MLC)蛋白表达。
3.以培养的单层肠上皮细胞(Caco-2)为模型,Caco-2细胞分为正常对照组(缺氧0h)、缺氧2、6、8、12和24 h组,另设ML-9对照组及缺氧组(ML-9终浓度为100μmol/L,不缺氧或缺氧6h),细胞缺氧是在1% O2条件下进行培养。电阻测定仪检测单层Caco-2细胞的跨细胞电阻(TER),Western blot法检测Caco-2细胞MLCK、MLC及磷酸化型MLC(p-MLC)蛋白表达。
三、主要结果
1.烧伤后大鼠肠粘膜通透性明显增加,伤后6h达高峰,为正常对照的2.41倍。烧伤后肠上皮通透性的增加伴随有细胞骨架F-actin及紧密连接蛋白ZO-1的明显重分布,肠上皮细胞p-MLC表达明显增加。
2.与正常对照组相比较,烧伤后6h小鼠肠粘膜通透性明显增加,光镜下肠粘膜组织结构受损明显,电镜下肠粘膜上皮细胞间连接增宽。同时,紧密连接蛋白ZO-1明显重分布、细胞骨架F-actin损害,肠粘膜组织p-MLC及MLCK蛋白表达明显增加。给予MLCK特异性抑制剂ML-9(2mg/mL)治疗后上述变化程度减轻。
3.与正常对照相比较,缺氧后单层肠上皮细胞TER呈下降趋势,肠上皮细胞p-MLC和MLCK蛋白表达增加,但MLC蛋白表达无明显变化。用MLCK特异性抑制剂ML-9(100μmol/L)预处理能有效防止缺氧引起的单层肠上皮细胞TER降低及p-MLC蛋白表达增加,但对肠上皮细胞MLCK蛋白表达无明显影响。
四、结论
1.严重烧伤早期肠粘膜屏障通透性显著增加,以伤后6h最重。肠粘膜上皮细胞骨架F-actin及紧密连接蛋白ZO-1的重分布或重排参与了严重烧伤早期肠粘膜屏障通透性增加的发生机制。
2.严重烧伤早期肠粘膜屏障通透性的增加伴随有MLCK蛋白表达及MLC磷酸化增加;特异性抑制MLCK介导的MLC磷酸化能明显降低烧伤引起的肠粘膜通透性增加,减轻组织结构受损、F-actin及紧密连接蛋白ZO-1的改变,表明MLCK介导的MLC磷酸化参与了严重烧伤早期肠粘膜屏障功能损害的发生。
3.缺氧引起肠上皮细胞屏障功能损害,并伴随有MLCK蛋白表达及MLC磷酸化增加。特异性抑制MLCK活性能抑制缺氧引起的肠上皮细胞MLC磷酸化,减轻缺氧后肠上皮屏障功能损害,MLCK介导的MLC磷酸化参与了缺氧后肠上皮屏障功能损害的发生。
Background & Aims
An intact intestinal barrier is critical to normal physiological function and prevention of disease, and plays an important role in defending the translocation of opportunistic bacteria and toxins from the gut. It is well recognized that severe burn injury often induces the loss of intestinal barrier function, increased intestinal permeability, and the translocation of bacteria and/or endotoxin from the gut, which can lead to the development of distant organ damages and multiple organ failure. However, the mechanism and the signalling cascade of the intestinal barrier dysfunction after severe burn injury are not fully elucidated.
The structure fundament of maintaining the intestinal barrier function is tight junction (TJ) and its associated proteins. TJ is the most apical component of the intercellular junctional complex, which is composed of multiple proteins interactionally, and plays an important role in regulating the intestinal permeability and cell polarity. There are at least three different types of transmembrane proteins, i.e., ZO-1, occludin and claudin. ZO-1 protein, an actin-and-myosin-binding protein, can communicate with the exterior signals of the cell. The relevant stimulating signal can alter the structure of linking proteins and impair the barrier function via skeleton protein activity and cell retraction, which forms intercellular space and increases the permeability.
It has been shown that the changes and regulation of barrier permeability involve cytoskeleton rearrangement or redistribution, tight junction, adheren junction structure and so on. Cell retraction is the common pathway for the increase of barrier permeability, and mainly regulated by actin and myosin via a myosin light chain (MLC) phosphorylation-dependent mechanism.
Although MLC phosphorylation is involved in the regulation of biological barrier function, the role of MLC phosphorylation in the pathogenesis of intestinal barrier dysfunction induced by severe burn injury is poorly understood. The aim of this study was to determine the role of MLC kinase (MLCK)-mediated MLC phosphorylation in intestinal epithelial barrier dysfunction after severe burn injury, hoping to better understand the mechanism of postburn intestinal barrier dysfunction.
Methods
1. Healthy adult Sprague Dawley rats were randomly divided into control, 1, 2, 6, 12 and 24h postburn groups. The intestinal permeability of rats was measured by fluorescein-isothiocyanate-dextran (FITC-Dextran) method. The changes of tight junction protein ZO-1, cytoskeleton F-actin, and the protein expression of phosphorylated MLC (p-MLC) in intestinal epithelia of the rats were analyzed by immunofluorescence.
2. Healthy adult BALB/c mice were randomly divided into four groups, i.e., (1) sham burn mice given vehicle, (2) sham burn mice given ML-9 (2mg/kg peritoneal injection, immediately after sham burn), (3) mice given a 30% total body surface area (TBSA) full-thickness burn, and (4) burn rats given ML-9 (2mg/kg peritoneal injection, immediately after burn injury). The intestinal permeability of mice was measured by FITC-dextran method. The histological changes of the intestinal mucosa were observed by microscope and electromicroscope. The changes of F-actin and tight junction protein ZO-1 in intestinal mucosa were analyzed by immunofluorescence. The expressions of MLCK and p-MLC in intestinal mucosa were analyzed by Western blot.
3. The cultured Caco-2 cell monolayers were randomly divided into control, 2, 6, 8, 12 and 24h posthypoxia groups. In some monolayers, the Caco-2 cells were treated with ML-9 (100μmol/L) prior to normoxia or hypoxia. Hypoxia was induced by 1%O2. The transepithelial electrical resistance (TER) of the monolayers was monitored with a volt/ohmmeter. The protein expressions of MLCK, MLC and p-MLC in Caco-2 cells were detected by Western blot analysis.
Results
1. After severe burn injury, the intestinal permeability of the rats increased significantly when compared with that of control, and peaked at 6h with 2.41 times higher than that of control. The increased intestinal permeability was accompanied by the striking reorganization of F-actin and the tight junction protein ZO-1, and the significant increase of p-MLC in intestinal epithelia of the burned rats.
2. When compared with that of the control mice, the intestinal permeability of the burned mice increased significantly at 6h. Meanwhile, the intestinal mucosa was damaged, and the intercellular space of intestinal mucosa was increased in burned mice. The protein expressions of both MLCK and p-MLC were increased significantly. However, treatment of the mice with ML-9, a specific inhibitor of MLCK, can lessen the burn-induced changes mentioned above.
3. Hypoxia treatment caused a significant decrease of TER in Caco-2 cell monolayers. Hypoxia also induced the increased expressions of MLCK and p-MLC protein in Caco-2 cells, whereas the expression of MLC protein was not significantly affected by hypoxia treatment. However, treatment the Caco-2 cell monolayers with ML-9, a specific inhibitor of MLCK, prevented the TER decrease and p-MLC increase, but not the MLCK increase induced by hypoxia.
Conclusions
1. The intestinal epithelial permeability increase significantly following severe brun injury, and peaks at 6h postburn. The rearragement or redistribution of F-actin and tight junction protein ZO-1 may contribute to the intestinal epithelial barrier dysfunction following severe burn injury.
2. The increased intestinal epithelial permeability induced by severe burn injury is accompanied by the increased protein expressions of both MLCK and p-MLC. Specific inhibition of MLC phosphorylation mediated by MLCK can attenuate the permeability increase, the mucosa damage and the changes of F-actin and tight junction protein ZO-1, suggesting that the MLCK-mediated MLC phosphorylation is involved in the pathogenesis of intestinal epithelial barrier dysfunction following severe burn injury.
3. The intestinal barrier dysfunction induced by hypoxia is also accompanied by the increase of both MLCK protein expression and MLC phosphorylation. Inhibiting MLCK activity prevents both the increased MLC phosphorylation and the damaged barrier function induced by hypoxia. It is suggested that the MLCK-mediated MLC phosphorylation may also play an critical role in the intestinal barrier dysfunction induced by hypoxia.
肠粘膜上皮屏障是体内重要的生物防御屏障,在抵御肠腔内细菌和/或内毒素的移位过程中起着中坚作用。过去的研究表明,严重烧伤后肠道粘膜上皮组织明显受损,屏障功能紊乱,通透性增加,肠道细菌和/或内毒素移位,从而引发或加重烧伤后一系列病理生理变化,但严重烧伤后肠粘膜屏障功能损害的分子机制仍不完全清楚。肠粘膜屏障完整性得以维持的结构基础是细胞间的紧密连接(TJ)及其相关蛋白。
TJ是由多种蛋白质相互作用而形成的复合体,在调节肠粘膜通透性和维持上皮细胞极性中发挥着重要作用。TJ由特异的连接蛋白使两个相邻细胞之间的间歇完全闭锁,与其密切相关的蛋白主要包括ZO-1、occludin、claudin等,其中ZO-1又与胞内细胞骨架肌动蛋白相连,使胞内细胞骨架与细胞外连接蛋白实现信号互为沟通。在相关信号刺激下,由于骨架蛋白活动和细胞回缩,引起连接蛋白结构和功能失调,细胞间隙形成,屏障通透性增加。
研究表明,屏障通透性变化与调节涉及细胞骨架重组、紧密连接、粘附连接结构等不同的机制。细胞收缩是各种原因引起屏障通透性增加的共同通路,主要受肌动蛋白(actin)和肌球蛋白(myosin)的影响,并依赖于肌球蛋白轻链(myosin light chain,MLC)的磷酸化。
鉴于MLC磷酸化在生物屏障功能的调控中起着重要作用,而既往有关研究资料又缺乏对MLC磷酸化与烧伤后肠粘膜屏障功能损害关系的探讨,本研究围绕MLC磷酸化与烧伤早期肠粘膜屏障功能损害,重点探讨肌球蛋白轻链激酶(myosin light chain kinase,MLCK)所介导的MLC磷酸化与严重烧伤早期肠粘膜上皮屏障功能损害、细胞骨架F-肌动蛋白、细胞紧密连接蛋白等变化的关系,初步明确MLCK所介导的MLC磷酸化在严重烧伤早期肠粘膜上皮屏障功能损害中的作用,以进一步完善烧伤后肠粘膜上皮屏障损害的发生机制。
二、方法
1.健康成年Sprague Dawley大鼠随机分为正常对照组(伤前)及伤后1、2、6、12、24h组,制备30% TBSAⅢ度烧伤模型。用异硫氰酸荧光素-葡聚糖(FITC-Dextran)示踪法检测大鼠肠粘膜通透性,免疫荧光法检测肠粘膜组织紧密连接蛋白ZO-1、F-肌动蛋白及p-MLC表达。
2.健康成年BALB/c小鼠随机分为4个组:①正常对照组:正常小鼠行假致伤;②ML-9对照组:于假致伤的正常对照小鼠腹腔注射ML-9 (2 mg/kg);③烧伤组:小鼠接受30% TBSAⅢ度烧伤;④烧伤治疗组:于30% TBSAⅢ度烧伤小鼠腹腔注射ML-9(2mg/kg)。FITC-Dextran示踪法检测肠粘膜通透性,HE染色及光镜观察肠粘膜组织结构,透射电镜观察肠粘膜超微结构,免疫荧光法检测肠粘膜ZO-1及F-肌动蛋白,Western blot法检测肠粘膜MLCK及磷酸化型MLC(p-MLC)蛋白表达。
3.以培养的单层肠上皮细胞(Caco-2)为模型,Caco-2细胞分为正常对照组(缺氧0h)、缺氧2、6、8、12和24 h组,另设ML-9对照组及缺氧组(ML-9终浓度为100μmol/L,不缺氧或缺氧6h),细胞缺氧是在1% O2条件下进行培养。电阻测定仪检测单层Caco-2细胞的跨细胞电阻(TER),Western blot法检测Caco-2细胞MLCK、MLC及磷酸化型MLC(p-MLC)蛋白表达。
三、主要结果
1.烧伤后大鼠肠粘膜通透性明显增加,伤后6h达高峰,为正常对照的2.41倍。烧伤后肠上皮通透性的增加伴随有细胞骨架F-actin及紧密连接蛋白ZO-1的明显重分布,肠上皮细胞p-MLC表达明显增加。
2.与正常对照组相比较,烧伤后6h小鼠肠粘膜通透性明显增加,光镜下肠粘膜组织结构受损明显,电镜下肠粘膜上皮细胞间连接增宽。同时,紧密连接蛋白ZO-1明显重分布、细胞骨架F-actin损害,肠粘膜组织p-MLC及MLCK蛋白表达明显增加。给予MLCK特异性抑制剂ML-9(2mg/mL)治疗后上述变化程度减轻。
3.与正常对照相比较,缺氧后单层肠上皮细胞TER呈下降趋势,肠上皮细胞p-MLC和MLCK蛋白表达增加,但MLC蛋白表达无明显变化。用MLCK特异性抑制剂ML-9(100μmol/L)预处理能有效防止缺氧引起的单层肠上皮细胞TER降低及p-MLC蛋白表达增加,但对肠上皮细胞MLCK蛋白表达无明显影响。
四、结论
1.严重烧伤早期肠粘膜屏障通透性显著增加,以伤后6h最重。肠粘膜上皮细胞骨架F-actin及紧密连接蛋白ZO-1的重分布或重排参与了严重烧伤早期肠粘膜屏障通透性增加的发生机制。
2.严重烧伤早期肠粘膜屏障通透性的增加伴随有MLCK蛋白表达及MLC磷酸化增加;特异性抑制MLCK介导的MLC磷酸化能明显降低烧伤引起的肠粘膜通透性增加,减轻组织结构受损、F-actin及紧密连接蛋白ZO-1的改变,表明MLCK介导的MLC磷酸化参与了严重烧伤早期肠粘膜屏障功能损害的发生。
3.缺氧引起肠上皮细胞屏障功能损害,并伴随有MLCK蛋白表达及MLC磷酸化增加。特异性抑制MLCK活性能抑制缺氧引起的肠上皮细胞MLC磷酸化,减轻缺氧后肠上皮屏障功能损害,MLCK介导的MLC磷酸化参与了缺氧后肠上皮屏障功能损害的发生。
Background & Aims
An intact intestinal barrier is critical to normal physiological function and prevention of disease, and plays an important role in defending the translocation of opportunistic bacteria and toxins from the gut. It is well recognized that severe burn injury often induces the loss of intestinal barrier function, increased intestinal permeability, and the translocation of bacteria and/or endotoxin from the gut, which can lead to the development of distant organ damages and multiple organ failure. However, the mechanism and the signalling cascade of the intestinal barrier dysfunction after severe burn injury are not fully elucidated.
The structure fundament of maintaining the intestinal barrier function is tight junction (TJ) and its associated proteins. TJ is the most apical component of the intercellular junctional complex, which is composed of multiple proteins interactionally, and plays an important role in regulating the intestinal permeability and cell polarity. There are at least three different types of transmembrane proteins, i.e., ZO-1, occludin and claudin. ZO-1 protein, an actin-and-myosin-binding protein, can communicate with the exterior signals of the cell. The relevant stimulating signal can alter the structure of linking proteins and impair the barrier function via skeleton protein activity and cell retraction, which forms intercellular space and increases the permeability.
It has been shown that the changes and regulation of barrier permeability involve cytoskeleton rearrangement or redistribution, tight junction, adheren junction structure and so on. Cell retraction is the common pathway for the increase of barrier permeability, and mainly regulated by actin and myosin via a myosin light chain (MLC) phosphorylation-dependent mechanism.
Although MLC phosphorylation is involved in the regulation of biological barrier function, the role of MLC phosphorylation in the pathogenesis of intestinal barrier dysfunction induced by severe burn injury is poorly understood. The aim of this study was to determine the role of MLC kinase (MLCK)-mediated MLC phosphorylation in intestinal epithelial barrier dysfunction after severe burn injury, hoping to better understand the mechanism of postburn intestinal barrier dysfunction.
Methods
1. Healthy adult Sprague Dawley rats were randomly divided into control, 1, 2, 6, 12 and 24h postburn groups. The intestinal permeability of rats was measured by fluorescein-isothiocyanate-dextran (FITC-Dextran) method. The changes of tight junction protein ZO-1, cytoskeleton F-actin, and the protein expression of phosphorylated MLC (p-MLC) in intestinal epithelia of the rats were analyzed by immunofluorescence.
2. Healthy adult BALB/c mice were randomly divided into four groups, i.e., (1) sham burn mice given vehicle, (2) sham burn mice given ML-9 (2mg/kg peritoneal injection, immediately after sham burn), (3) mice given a 30% total body surface area (TBSA) full-thickness burn, and (4) burn rats given ML-9 (2mg/kg peritoneal injection, immediately after burn injury). The intestinal permeability of mice was measured by FITC-dextran method. The histological changes of the intestinal mucosa were observed by microscope and electromicroscope. The changes of F-actin and tight junction protein ZO-1 in intestinal mucosa were analyzed by immunofluorescence. The expressions of MLCK and p-MLC in intestinal mucosa were analyzed by Western blot.
3. The cultured Caco-2 cell monolayers were randomly divided into control, 2, 6, 8, 12 and 24h posthypoxia groups. In some monolayers, the Caco-2 cells were treated with ML-9 (100μmol/L) prior to normoxia or hypoxia. Hypoxia was induced by 1%O2. The transepithelial electrical resistance (TER) of the monolayers was monitored with a volt/ohmmeter. The protein expressions of MLCK, MLC and p-MLC in Caco-2 cells were detected by Western blot analysis.
Results
1. After severe burn injury, the intestinal permeability of the rats increased significantly when compared with that of control, and peaked at 6h with 2.41 times higher than that of control. The increased intestinal permeability was accompanied by the striking reorganization of F-actin and the tight junction protein ZO-1, and the significant increase of p-MLC in intestinal epithelia of the burned rats.
2. When compared with that of the control mice, the intestinal permeability of the burned mice increased significantly at 6h. Meanwhile, the intestinal mucosa was damaged, and the intercellular space of intestinal mucosa was increased in burned mice. The protein expressions of both MLCK and p-MLC were increased significantly. However, treatment of the mice with ML-9, a specific inhibitor of MLCK, can lessen the burn-induced changes mentioned above.
3. Hypoxia treatment caused a significant decrease of TER in Caco-2 cell monolayers. Hypoxia also induced the increased expressions of MLCK and p-MLC protein in Caco-2 cells, whereas the expression of MLC protein was not significantly affected by hypoxia treatment. However, treatment the Caco-2 cell monolayers with ML-9, a specific inhibitor of MLCK, prevented the TER decrease and p-MLC increase, but not the MLCK increase induced by hypoxia.
Conclusions
1. The intestinal epithelial permeability increase significantly following severe brun injury, and peaks at 6h postburn. The rearragement or redistribution of F-actin and tight junction protein ZO-1 may contribute to the intestinal epithelial barrier dysfunction following severe burn injury.
2. The increased intestinal epithelial permeability induced by severe burn injury is accompanied by the increased protein expressions of both MLCK and p-MLC. Specific inhibition of MLC phosphorylation mediated by MLCK can attenuate the permeability increase, the mucosa damage and the changes of F-actin and tight junction protein ZO-1, suggesting that the MLCK-mediated MLC phosphorylation is involved in the pathogenesis of intestinal epithelial barrier dysfunction following severe burn injury.
3. The intestinal barrier dysfunction induced by hypoxia is also accompanied by the increase of both MLCK protein expression and MLC phosphorylation. Inhibiting MLCK activity prevents both the increased MLC phosphorylation and the damaged barrier function induced by hypoxia. It is suggested that the MLCK-mediated MLC phosphorylation may also play an critical role in the intestinal barrier dysfunction induced by hypoxia.
引文
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