脉络宁局部灌注对挤压伤综合征模型猪Wnt/β-catenin和Notch信号通路的影响

Effect of Mailuoning local perfusion on Wnt/β-catenin and Notch signaling pathways in crush injury syndrome model pigs

目的:探讨脉络宁局部灌注对挤压伤综合征模型猪Wnt/β-连接蛋白(β-catenin)和Notch通路的影响。方法:24只巴马小型猪随机分为正常对照组、模型组、常规灌注组和脉络宁灌注组,每组6只。除正常对照组外,其余各组建立挤压伤模型,建模结束后模型组即刻恢复血供,常规灌注组和脉络宁灌注组建模前处理,建模后分别给予常规灌注和脉络宁灌注1 h后恢复自身血供。各组恢复自身血供4 h后,采用苏木素-伊红(HE)染色检测骨骼肌组织形态;透射电镜观察骨骼肌超微结构;ELISA法检测血清中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平;RT-qPCR检测骨骼肌组织中血管内皮生长因子(VEGF)和基质金属蛋白酶2(MMP-2)mRNA水平;Western blot检测骨骼肌组织中β-catenin、Delta样配体4(Dll4)和Notch的蛋白水平。结果:HE结果显示模型组骨骼肌肌纤维排列紊乱,肌核肿胀或固缩明显,肌膜皱缩,血管破裂、肿胀,肌间质水肿,炎症细胞浸润明显;电镜结果显示模型组肌丝排列紊乱、出现溶解,肌纤维I带、A线和Z线部分丧失,内质网、线粒体和肌核肿胀明显,出现大量的核聚集和核内移。常规灌注组略轻于模型组。脉络宁灌注组HE显示骨骼肌形态基本正常,肌纤维较常规灌溉组排列整齐,肌膜皱缩或肿胀现象不明显,血管基本恢复正常;电镜结果显示肌纤维I带、A线和Z线清晰可见且规则,细胞核排列整齐。与正常对照组相比,模型组组织肿胀、血管破损比例和Z线异常百分率,血清中IL-1β和TNF-α水平,骨骼肌组织中VEGF和MMP-2的mRNA水平,β-catenin核蛋白/β-catenin总蛋白、Dll4和Notch蛋白水平升高(P<0.05);与模型组相比,常规灌注组组织肿胀、血管破损比例和Z线异常百分率,血清中IL-1β和TNF-α水平,骨骼肌组织中MMP-2的mRNA水平,核β-catenin/β-catenin总蛋白、Dll4、Notch的蛋白水平及脉络宁灌注组血清中的IL-1β和TNF-α水平,骨骼肌组织中VEGF和MMP-2的mRNA水平,β-catenin核蛋白/β-catenin总蛋白、Dll4、Notch的蛋白水平均降低(P<0.05);与常规灌注组相比,脉络宁灌注组组织肿胀、血管破损比例和Z线异常百分率,血清中的TNF-α水平,骨骼肌组织中VEGF和MMP-2的mRNA水平,核β-catenin/β-catenin总蛋白、Dll4、Notch蛋白水平降低(P<0.05)。结论:脉络宁局部灌注可抑制Wnt/β-catenin和Notch通路,缓解炎症和血管损伤,从而实现对挤压伤综合征的保护。

AIM:To investigate the effects of local perfusion of Mailuoning on Wnt/β-catenin and Notch signaling pathway in crush injury syndrome model pigs.METHODS:A total of 24 Bama mini pigs were randomly divided into normal control group,model group,normal perfusion group and Mailuoning perfusion group,with 6 in each group.Except for normal control group,the other groups were established crush injury model.After modeling,the blood supply of the pigs in model group was immediately restored.The normal perfusion group and Mailuoning perfusion group were preprocessed for modeling,and were given normal perfusion and Mailuoning perfusion for 1 h to restore their blood supply.After the recovery of blood supply for 4 h,the skeletal muscle morphology was detected by hematoxylin-eosin(HE)staining.The ultrastructure of the skeletal muscle was observed under transmission electron microscope.The serum levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were measured by ELISA.The mRNA levels of vascular endothelial growth factor(VEGF)and matrix metalloproteinase-2(MMP-2)were detected by RT-qPCR,and the protein levels ofβ-catenin,Delta-like ligand 4(Dll4)and Notch in skeletal muscular tissue were observed by Western blot.RESULTS:The HE results of model group showed disordered arrangement of skeletal muscle fibers,swollen or obviously constricted nucleus,shrunken sarcolemma,ruptured and swollen blood vessels,edematous interstitium,obvious infiltrating inflammatory cells.The results of electron microscopy in model group showed disorderly arranged and dissolved myofilaments,partial loss of I-band,A-line and Z-line muscle fibers,obviously swollen endoplasmic reticulum,mitochondria and nuclei,and a large number of aggregated and internally shifted nuclei.The above indexes in normal perfusion group were slightly lighter than those in model group.In Mailuoning perfusion group,the results of HE staining observation showed almost normal skeletal muscle,neatly arranged muscle fibers,almost invisible shrinkage or swelling muscle membrane,and basically returned to normal blood vessels;the results of electron microscopy showed clearly visible and regular muscle fiber I-band,A-line,Z-line and neatly arranged nuclei.Compared with normal control group,the proportion of tissue swelling and vascular damage,the percentage of abnormal Z-line,the serum levels of IL-1βand TNF-α,the mRNA expression of VEGF and MMP-2,the protein levels of nucleoprotein/totalβ-catenin,Dll4 and Notch in skeletal muscular tissue in model group were increased(P<0.05).Compared with model group,the proportion of tissue swelling and vascular damage,the percentage of abnormal Z-line,the serum levels of IL-1βand TNF-α,the mRNA expression of MMP-2,the protein levels of nuclear/totalβ-catenin,Dll4 and Notch in skeletal muscular tissue in normal perfusion group,and the serum levels of IL-1βand TNF-α,the mRNA expression of VEGF and MMP-2,the protein levels of nuclear/totalβ-catenin,Dll4 and Notch in skeletal muscular tissue in Mailuoning perfusion group were decreased(P<0.05).Compared with normal perfusion group,the proportion of tissue swelling and vascular damage,the percentage of abnormal Z-line,the serum levels of TNF-α,the mRNA expression of MMP-2,the protein levels of nuclear/totalβ-catenin,Dll4 and Notch in skeletal muscular tissue in Mailuoning perfusion group were decreased(P<0.05).CONCLUSION:Mailuoning local perfusion suppresses Wnt/β-catenin and Notch signaling pathways,attenuates inflammation and vascular damage,thus realizing the protection of crush injury syndrome.