脂氧素A4对大鼠皮瓣和肌肉组织缺血再灌注损伤保护作用及机制研究

发布时间：2018-06-19 22:37

本文选题：缺血再灌注损伤 + 皮瓣　；参考：《第二军医大学》2017年博士论文

【摘要】：第一部分:脂氧素A4对大鼠带蒂皮瓣缺血再灌注损伤保护作用研究目的:研究脂氧素A4对大鼠带蒂皮瓣缺血再灌注损伤的保护作用。方法:建立大鼠腹壁上动脉带蒂皮瓣缺血再灌注损伤模型,静脉注射脂氧素A4,在术后72小时,通过大体形态计算皮瓣成活面积,激光多普勒血流成像检测皮瓣血流灌注。HE染色观察皮瓣组织学改变,ELISA检测皮瓣中IL-1β,IL-6,TNF-α炎症因子含量。同时检测皮瓣中MDA含量及SOD活性。TUNEL染色观察皮瓣细胞凋亡比例,通过western blotting检测抗氧化应激相关蛋白Nrf2、HO-1及凋亡相关蛋白Bax,Bcl2含量变化。通过免疫组织化学染色和western blotting检测LC3蛋白含量。结果:LA4显著增加缺血再灌注损伤皮瓣成活面积,增加皮瓣血流灌注量。LA4减轻缺血再灌注导致的组织损伤,降低缺血再灌注损伤皮瓣中MDA含量,升高SOD活性,同时抑制炎症因子IL-1β,IL-6,TNF-α释放。LA4抑制皮瓣缺血再灌注损伤所致细胞凋亡,同时降低Bax表达,促进Bcl2表达。LA4促进Nrf2向细胞核内转位并促进HO-1表达。LA4还促进缺血再灌注损伤皮瓣组织中LC3Ⅰ向LC3Ⅱ转变,提高细胞自噬水平。结论:LA4通过抑制氧化应激、炎症反应、凋亡,调节自噬水平保护皮瓣组织缺血再灌注损伤。第二部分:脂氧素A4对大鼠骨骼肌缺血再灌注损伤保护作用研究目的:研究LA4对大鼠骨骼肌缺血再灌注损伤的保护作用及相关机制。方法:建立大鼠下肢肌肉缺血再灌注损伤模型,通过静脉注射不同剂量的LA4。再灌注后3小时,观察下肢肌肉组织水肿,免疫荧光染色法标记中性粒细胞浸润,HE染色观察肌肉组织损伤状态,ELISA检测肌肉组织中炎症因子TNF-α、IL-Iβ及IL-6含量。同时检测抗氧化应激相关酶SOD、CAT、GSH-Px活性,TUNEL染色观察肌细胞凋亡比例。Western blotting检测抗氧化应激相关蛋白Nrf2、HO-1和凋亡相关蛋白Bax、Bcl2表达变化。透射电镜观察肌肉组织自噬体数量。免疫荧光染色和western blotting检测自噬相关蛋白LC3和Beclin1含量变化。结果:大鼠肌肉缺血再灌注损伤导致肌肉水肿,中性粒细胞浸润、肌肉组织学损伤,炎症因子水平增高,抗氧化应激相关酶活性下降,细胞凋亡比例增高。LA4能减轻肌肉组织水肿,减少中性粒细胞浸润。LA4还能减轻肌肉组织学损伤程度。LA4降低缺血再灌注组织中炎症因子含量,增加抗氧化应激相关酶的活性。LA4还增加抗凋亡蛋白bcl2表达并降低促凋亡蛋白bax表达。la4对肌肉缺血再灌注损伤的保护作用与其激活nrf2/ho-1信号通路相关,当加入ho-1抑制剂(znppⅨ)时,la4对肌肉组织缺血再灌注损伤的保护作用明显被抑制。la4还促进缺血再灌注损伤肌肉组织中自噬体的形成,促进lc3Ⅰ向lc3Ⅱ转变,增加beclin1表达。结论:la4通过抑制炎症反应、氧化应激、细胞凋亡、调节自噬保护肌肉组织缺血再灌注损伤,其保护作用与激活nrf2/ho-1信号通路相关。第三部分:脂氧素a4对骨骼肌细胞氧化应激损伤的保护作用研究目的:通过体外培养的骨骼肌细胞氧化应激损伤模型,观察la4对骨骼肌细胞氧化应激损伤的保护作用。方法:使用不同浓度的h2o2(0,25,50,100,200,400μmol/l)刺激大鼠骨骼肌成肌细胞(l6细胞),模拟骨骼肌缺血再灌注损伤,确定h2o2损伤骨骼肌细胞的最佳时间和浓度。在h2o2损伤细胞前,不同浓度(0,0.1,1,10,100nmol/l)la4预处理细胞12小时,确定la4对l6细胞氧化应激损伤的最佳保护浓度。mtt实验观察细胞活性变化,检测ros含量判断细胞氧化应激损伤程度。光镜下形态学观察细胞氧化应激损伤所致细胞形态学改变,并检测培养液中ck、ldh活性判断细胞损伤严重程度。测定细胞内gsh含量判断细胞抗氧化应激能力。tunel染色观察骨骼肌细胞凋亡比例,westernblotting检测凋亡相关蛋白bcl2,bax含量变化。结果:h2o2氧化应激损伤骨骼肌细胞的最佳浓度为200μmol/l,最佳作用时间为4小时,可造成细胞活性下降至对照组55%。la4(10nmol/l)显著增加h2o2损伤的骨骼肌细胞活力。la4还能够抑制h2o2损伤所致ros的生成,抑制ck、ldh向培养液中释放,促进细胞内gsh的消耗。la4促进bcl2表达,抑制bax表达,降低h2o2损伤所致细胞凋亡比例。结论:la4在体外培养的骨骼肌细胞氧化应激损伤中,显著增加细胞活力、抑制ros生成、增加细胞抗氧化应激能力,抑制氧化应激损伤所致细胞凋亡,对骨骼肌氧化应激损伤有保护作用。第四部分:脂氧素a4对骨骼肌细胞氧化应激损伤保护机制研究目的:在骨骼肌细胞氧化应激损伤模型中,研究la4保护作用的相关机制。方法:在体外骨骼肌细胞氧化应激损伤模型中,通过免疫荧光染色,westernblotting检测la4对nrf2/ho-1信号通路及自噬水平的影响。同时通过westernblotting检测la4对erk、p38、jnk、pi3k/akt信号通路的作用,并研究erk、p38、jnk、pi3k/akt信号通路与nrf2/ho-1信号通路、自噬的关系。结果:LA4在体外骨骼肌细胞氧化应激损伤时激活Nrf2/HO-1信号通路,促进Nrf2向细胞核内转移,同时增加HO-1蛋白的表达。LA4提高骨骼肌细胞氧化应激损伤时自噬水平。LA4通过激活ERK通路而激活Nrf2/HO-1信号通路。LA4对P38和JNK、PI3K/AKT信号通路无明显调节作用。LA4通过激活ERK/Nrf2信号通路而提高骨骼肌氧化应激损伤时自噬水平,抑制ERK/Nrf2信号通路后,自噬水平降低。结论:LA4通过激活ERK/Nrf2信号通路提高氧化应激损伤的骨骼肌细胞自噬水平,增强抗氧化应激蛋白HO-1的表达,从而保护骨骼肌细胞氧化应激损伤。
[Abstract]:The first part: the protective effect of lipoxin A4 on ischemic reperfusion injury in rats with pedicle flap: To study the protective effect of lipoxygenin A4 on ischemia reperfusion injury in rats with pedicle flap. Method: establish the model of ischemia reperfusion injury in the upper abdominal wall flap of the abdominal wall of rats, intravenous lipoxygenase A4, through the large body shape after 72 hours of operation. The survival area of the skin flap was calculated. The skin flap was stained by laser Doppler flow imaging to observe the histological changes of the skin flap. The content of IL-1 beta, IL-6, and TNF- alpha in the skin flap was detected by ELISA. The MDA content in the flap and the proportion of the apoptosis of the skin flap were detected by SOD activity.TUNEL, and the proportion of apoptosis in the skin flap was observed by SOD activity.TUNEL, and the antioxidant should be detected by Western blotting. Changes in the content of Nrf2, HO-1 and apoptosis related protein Bax and Bcl2. The content of LC3 protein was detected by immunohistochemical staining and Western blotting. Results: LA4 significantly increased the survival area of the skin flap with ischemia-reperfusion injury, increased the volume of blood flow perfusion of the flap to reduce the tissue damage caused by ischemia-reperfusion, and reduced the ischemia-reperfusion injury. The content of MDA in the wound skin flap, the increase of SOD activity, and the inhibition of the inflammatory factor IL-1 beta, IL-6, and TNF- alpha release.LA4 inhibit the cell apoptosis induced by the ischemia-reperfusion injury of the flap, and reduce the expression of Bax, promote the expression of.LA4 to promote the transposition of Nrf2 to the nucleus and promote HO-1 expression.LA4 to promote the ischemia-reperfusion injury in the skin flap. Change, improve the level of autophagy. Conclusion: LA4 protects the ischemic reperfusion injury of skin flap by inhibiting oxidative stress, inflammatory response, apoptosis and regulating autophagy level. The second part: the protective effect of lipoxygenin A4 on skeletal muscle ischemia reperfusion injury in rats: To study the protective effect of LA4 on skeletal muscle ischemia reperfusion injury in rats and the protective effect of lipoxygenin on the protection of ischemia reperfusion injury in rat skeletal muscle and Methods: to establish a rat model of lower limb muscle ischemia reperfusion injury, the edema of the muscle tissue of the lower extremities was observed 3 hours after intravenous injection of different doses of LA4.. Immunofluorescence staining was used to mark neutrophils infiltration, HE staining was used to observe the injury state of the muscle tissue. ELISA was used to detect the inflammatory factor TNF- a, IL-I beta in the muscle tissue. And IL-6 content. Detection of antioxidant stress related enzymes SOD, CAT, GSH-Px activity. TUNEL staining was used to observe the apoptosis ratio of muscle cells,.Western blotting was used to detect antioxidant stress related protein Nrf2, HO-1 and apoptosis related proteins Bax, Bcl2 expression. Transmission electron microscopy was used to observe the number of autophago in muscle tissue. Immunofluorescence staining and Western assay Changes in the content of autophagy related proteins LC3 and Beclin1. Results: muscle edema, neutrophil infiltration, muscle tissue injury, increased inflammatory factors, decreased activity of antioxidant stress related enzymes, and increased apoptosis ratio.LA4 could reduce muscle edema and decrease neutrophils infiltration.LA4 in rats. .LA4 can reduce the degree of muscle tissue injury, reduce the content of inflammatory factors in ischemia reperfusion tissue, increase the activity of antioxidant stress related enzymes, and increase the expression of anti apoptotic protein BCL2 and reduce the protective effect of.La4 on myocardial ischemia reperfusion injury, which is related to the activation of nrf2/ho-1 signaling pathway, when ho is added to ho. When -1 inhibitor (ZnPP IX), the protective effect of LA4 on ischemia-reperfusion injury of muscle tissue is obviously inhibited by.La4, which also promotes the formation of autophagic in the muscle tissue of ischemia reperfusion injury, promotes the transformation of LC3 I to LC3 II, and increases the expression of Beclin1. Conclusion: LA4 can regulate autophagy by inhibiting the inflammatory response, oxidative stress, and apoptosis. The protective effect of ischemic reperfusion injury is related to the activation of nrf2/ho-1 signaling pathway. Third part: the protective effect of lipoxygenin A4 on oxidative stress injury in skeletal muscle cells: the protective effect of LA4 on oxidative stress damage in skeletal muscle cells is observed by the oxidative stress damage model of skeletal muscle cells in vitro. The rat skeletal myoblasts (L6 cells) were stimulated with different concentrations of H2O2 (0,25,50100200400 mol/l) to simulate the injury of skeletal muscle ischemia-reperfusion injury, and the optimal time and concentration of H2O2 damaged skeletal muscle cells were determined. Before H2O2 damaged cells, 0,0.1,1,10100nmol/l LA4 pretreated cells for 12 hours to determine LA4 on the oxidation of L6 cells. The best protective concentration of stress injury.Mtt test observed cell activity changes, detect ROS content to judge the degree of oxidative stress injury. Morphological observation of cell morphology changes caused by oxidative stress injury under light microscope, and detection of CK, LDH activity to judge the severity of cell damage in the culture medium. The determination of GSH content in cells to determine cell resistance The apoptosis ratio of skeletal muscle cells was observed by oxidative stress.Tunel staining, and westernblotting was used to detect the changes of BCL2 and Bax content of apoptosis related proteins. Results: the optimum concentration of skeletal muscle cells by H2O2 oxidative stress was 200 mu mol/l and the best time of action was 4 hours, which could cause the cell viability to decrease to 55%.la4 (10nmol/l) in the control group. The damage of skeletal muscle cell vitality.La4 can also inhibit the formation of ROS caused by H2O2 damage, inhibit the release of CK, LDH into the culture medium, promote the consumption of GSH in the cell.La4 to promote BCL2 expression, inhibit Bax expression and reduce the percentage of apoptosis induced by H2O2 damage. Conclusion: LA4 in the oxidative stress injury of skeletal muscle cells cultured in vitro, significantly increased Cell vitality, inhibiting ROS formation, increasing cell antioxidant stress ability, inhibiting apoptosis induced by oxidative stress injury and protecting skeletal muscle oxidative stress damage. The fourth part: Study on the protective mechanism of lipoxygenin A4 on oxidative stress injury in skeletal muscle cells: the study of LA4 protection in the oxidative stress damage model of skeletal muscle cells Methods: in the model of oxidative stress injury in skeletal muscle cells in vitro, the effects of LA4 on nrf2/ho-1 signaling pathway and autophagy were detected by immunofluorescence staining, and the effects of LA4 on ERK, p38, JNK, pi3k/akt signaling pathway were detected by westernblotting, and ERK, p38, JNK, and signaling pathways were studied. The relationship with autophagy with nrf2/ho-1 signaling pathway. Results: LA4 activates Nrf2/HO-1 signaling pathway during oxidative stress injury in skeletal muscle cells in vitro, promotes the transfer of Nrf2 into the nucleus, and increases the expression of HO-1 protein expression.LA4 to increase the autophagy level in skeletal muscle cell oxidative stress,.LA4 activates the Nrf2/HO-1 signal through the activation of the ERK pathway. Road.LA4 has no obvious regulating effect on P38 and JNK, PI3K/AKT signaling pathway..LA4 increases autophagy level in oxidative stress injury of skeletal muscle by activating ERK/Nrf2 signaling pathway and reduces autophagy level after ERK/Nrf2 signaling pathway. Conclusion: LA4 enhances autophagy level in skeletal muscle cells damaged by oxidative stress by activating ERK/Nrf2 signaling pathway. The expression of strong antioxidant stress protein HO-1 can protect skeletal muscle cells from oxidative stress injury.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R658

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