中华急诊医学杂志  2022, Vol. 31 Issue (5): 636-643   DOI: 10.3760/cma.j.issn.1671-0282.2022.05.013
间充质干细胞上清液调控腺苷酸活化蛋白激酶保护脂多糖诱导的急性肺损伤
陈海洋1 , 杨宏锋1 , 沈嘉琪2 , 朱伟2 , 虞志新1     
1. 江苏大学附属人民医院重症医学科,镇江 212002;
2. 江苏大学医学院,镇江 212013
摘要: 目的 研究脐带间充质干细胞上清液对脂多糖(lipopolysaccharide, LPS)诱导的急性肺损伤(acute lung injury, ALI)的保护作用及机制。方法 选择6周龄C57BL/6雄性小鼠共40只,随机数字法分为假手术(sham)组,LPS模型组,LPS+人脐带间充质干细胞上清液(HucMSC-cm)(LPS+cm)治疗组,LPS+HucMSC-cm+Compound C(LPS+cm+cc)干预组,每组10只。通过气管内注射LPS 5 mg/kg构建ALI模型,4 h后予以气管内注射HucMSC-cm 50 μL/只构建治疗组;干预组在LPS及HucMSC-cm前30 min予腹腔注射Compound C 15 mg/kg;72 h后留取小鼠外周血检测中性粒细胞比例,并处死小鼠留取肺组织。通过苏木精-伊红染色检测小鼠肺组织病理学改变,Western Blot及免疫组化法分析肺组织中IL-6、ICAM-1、VCAM-1和磷酸化腺苷酸活化蛋白激酶(phosphate AMP-activated protein kinase, p-AMPK)的表达。体外培养人肺微血管内皮细胞(HuLEC-5a),将细胞分为三组:control组、LPS组(10 μg/mL)、LPS+HucMSC-cm组。处理24 h后,Western Blot检测p-AMPK及AMPK蛋白表达水平,RT-PCR检测IL-6及IL-8的mRNA表达。多组间比较采用单因素方差分析,两组比较采用Tukey法检验。结果 与sham组相比,LPS组小鼠肺组织充血水肿加重,肺间隔增厚和炎性细胞浸润增多,肺组织IL-6(P=0.003)、ICAM-1(P < 0.001)及VCAM-1(P=0.001)蛋白水平明显上升,p-AMPK蛋白水平表达下降(P=0.013),外周血中性粒细胞比例上升(P < 0.001),LPS+HucMSC-cm组肺组织充血水肿及病理学损伤较LPS组得到改善,肺组织中IL-6(P=0.003)、ICAM-1(P=0.001)及VCAM-1(P=0.006)蛋白水平下降,p-AMPK蛋白水平表达上升(P=0.002),外周血中性粒细胞比例下降(P < 0.001),而LPS+cm+cc组肺组织病理学损伤较LPS+HucMSC-cm组再次加重,肺组织IL-6、ICAM-1及VCAM-1蛋白水平明显上升,p-AMPK蛋白水平表达下降,免疫组化法得到与蛋白相一致的结果。体外实验中,LPS处理后,IL-6(P < 0.001)及IL-8(P=0.027)的mRNA表达较control组上升,且p-AMPK蛋白水平下降(P=0.005);LPS+HucMSC-cm组较LPS组IL-6(P=0.003)及IL-8(P=0.002)的mRNA表达下降,p-AMPK蛋白水平上升(P=0.003)。结论 人脐带间充质干细胞上清液通过激活AMPK活性改善LPS诱导的ALI。
关键词: 人脐带间充质干细胞上清液    腺苷酸活化蛋白激酶    急性肺损伤    粘附分子    肺微血管内皮细胞    
HucMSC-cm protects lipopolysaccharide-induced acute lung injury by activating AMP-activated protein kinase
Chen Haiyang1 , Yang Hongfeng1 , Shen Jiaqi2 , Zhu Wei2 , Yu Zhixin1     
1. Department of Intensive Care Medicine, the Affiliated People's Hospital, Jiangsu University, Zhenjiang 212002, China;
2. School of Medicine, Jiangsu University, Zhenjiang 212013, China
Abstract: Objective To investigate the protective effect of human umbilical cord mesenchymal stem cell conditioned medium (HucMSC-cm) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and relevant mechanism of action. Methods Forty 6-week-old male C57BL/6 mice were selected and randomized (random number) into the sham group, LPS group, LPS + HucMSC-cm (LPS+cm) group, and LPS+HucMSC-cm+Compound C (LPS+cm+cc) group, with 10 mice in each group. Mice were intratracheally injected with LPS (5 mg/kg) to establish ALI model, and intratracheally injected with hucMSC-CM (50 μL) 4 h after LPS treatment. Mice in the LPS+cm+cc group were intraperitoneally treated with Compound C (15 mg/kg) prior to LPS treatment. Neutrophils in peripheral blood were counted with the automated hematology analyzer 72 h after LPS administration. After that, mice were sacrificed, and the lung tissue pathology was observed using hematoxylin eosin (HE) staining. Besides, the expressions of IL-6, ICAM-1, VCAM-1 and P-AMP-activated protein kinase (P-AMPK) in the lung tissues were analyzed by Western blot and immunohistochemical assay. In vitro, human lung microvascular endothelial cells (HuLEC-5a) were cultured and divided into three groups: control group, LPS group (10 μg/ mL), and LPS + HucMSC-cm group. After 24 h of treatment, the expressions of p-AMPK and AMPK were detected by Western blot, and the expressions of IL-6 and IL-8 were detected by real-time fluorescence quantitative PCR. Oneway analysis of variance was used to compare the mean values of normally distributed measurement data between groups. Comparisons between two groups were performed using the Tukey's multiple comparison test. Results Compared with the sham group, the LPS group showed lungs with congestion and swelling, thickened pulmonary septum, and inflammatory cell infiltration. Moreover, in the LPS group, the protein expressions of IL-6 (P=0.003), ICAM-1 (P < 0.001) and VCAM-1 (P=0.001) were increased significantly, while the expression of p-AMPK was decreased (P=0.013), accompanied by an increase in the proportion of neutrophils in peripheral blood (P < 0.001). Compared with the LPS group, the LPS+HucMSC-cm group demonstrated eased congestion, edema and pathological injury of lung tissue, reversed protein expressions of IL-6 (P=0.003), ICAM-1 (P=0.002), VCAM-1 (P=0.006) and P-AMPK (P=0.002), as well as decreased proportion of neutrophils in peripheral blood (P < 0.005). Compared with the LPS+HucMSC-cm group, the LPS+cm+cc group exhibited more severe lung histopathological injury, significantly increased protein expressions of IL-6, ICAM-1 and VCAM-1 in lung tissues, as well as decreased expression of P-AMPK protein. The results of immunohistochemistry were consistent with those of protein. In vitro experiment, after LPS treatment, the mRNA expressions of IL-6 (P < 0.001) and IL-8 (P=0.027) were increased and p-AMPK protein expression (P=0.005) was decreased as compared with the control group. In comparison with the LPS group, the LPS+HucMSC-cm group showed decreased mRNA expression levels of IL-6 (P=0.003) and IL-8 (P=0.002), but increased protein level of p-AMPK (P=0.003). Conclusions HucMSC-cm has a protective effect against LPS-induced acute lung injury, which is mainly attributed to the inhibited expression of adhesion molecules and inflammatory factors under the activation of AMPK.
Key words: Human umbilical cord mesenchymal stem cell conditioned medium    AMP-activated protein kinase    Acute lung injury    Adhesion molecules    Lung microvascular endothelial cell    

急性肺损伤(acute lung injury, ALI)以及其严重形式急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)是重症监护病房常见的一种疾病,ARDS的病死率可高达30%~45%[1]。ARDS发病早期会有大量炎症因子涌入肺组织,而中性粒细胞是早期炎症的核心因素[2]。细胞间粘附分子1(intercellular adhesion molecule-1, ICAM-1)和血管细胞粘附分子1(vascular cell adhesion molecule-1, VCAM-1)均属于免疫球蛋白超家族的成员,越来越多的证据表明,两者在ALI的中性粒细胞跨内皮细胞迁移起关键作用[3-4]。因此抑制这两者在内皮细胞上的表达成为控制炎症的关键,而腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)作为炎症过程中的调控因子,可以降低粘附分子的表达进而抑制炎症反应[5]

AMPK是生物能量代谢调节的关键分子,近年研究发现其是炎症过程中的关键调控因子[6-7]。有研究证明乌司他汀可以通过调节AMPK/NF-κB通路来抑制炎症因子减轻脂多糖(lipopolysaccharide, LPS)诱导的ALI[8]

间充质干细胞(mesenchymal stem cell, MSC)是具有自我更新能力的多能干细胞[9],可以调节免疫和炎症反应并减轻感染[10],而人脐带间充质干细胞上清液(human umbilical cord mesenchymal stem cell conditioned medium, HucMSC-cm)在LPS诱导ALI中的作用机制仍不明确。本研究旨在阐明HucMSC-cm逆转LPS导致的ALI的机制,并为临床治疗ALI提供新的治疗靶点。

1 材料与方法 1.1 动物实验模型构建

动物:6周龄SPF级雄性C57BL/6小鼠购自江苏大学实验动物中心,许可证号为SYXK(苏)2018-0053,合格证号为202112223,所有动物实验均经江苏大学动物实验中心批准,并按照《实验动物护理使用指南》进行。

动物模型构建及分组:40只C57BL/6小鼠随机分成4组(每组10只):sham组、LPS组、LPS+HucMSC-cm组、LPS+HucMSC-cm+Compound C组。根据既往文献报道构建ALI模型[11],气管内缓慢滴注LPS,剂量5 mg/kg,50 μL/只;HucMSC-cm于LPS注射4 h后气管内注射,剂量50 μL/只;Compound C溶于生理盐水,剂量为15 mg/kg,下一药物给药前30 min腹腔注射。

1.2 人肺微血管内皮细胞分组及处理

人肺微血管内皮细胞株(HuLEC-5a)购自上海宾穗生物科技有限公司,用含10%胎牛血清(BI,以色列)的DMEM(BI,以色列)培养细胞,置于37 ℃,5% CO2培养箱中孵育。将1×105个细胞种于六孔板,细胞分组:对照组(control组)、LPS组、LPS+HucMSC-cm组,细胞融合度达约70%时加LPS(10 μg/mL)及HucMSC-cm,24 h后提取蛋白或RNA。

1.3 脐带间充质干细胞培养及上清液收集

本研究中,脐带标本来源于江苏大学附属医院,通过江苏大学附属人民医院伦理委员会批准,(批件编号:SQK-20210108-W),所有受试者均提供知情同意。根据以前发表的研究[12],从健康新生儿脐带中分离培养HucMSCs。细胞扩增至第3~5代进行实验。

当细胞培养达到70%的融合度,换用6 mL人间充质干细胞无血清培养基(灏洋生物,天津)培养72 h,然后收集上清液。上清液在1 000 r/min下离心5 min去除细胞碎片,然后将其转移到分子截量为3 000 Da的高速离心管(Millipore,美国)中离心,浓缩50倍。最后,上清液通过0.22 μm过滤器(Corning,美国)过滤,保存在-80 ℃冰箱,用于每次实验。

1.4 Western Blot分析

用RIPA裂解液(Vazyme公司,中国)裂解细胞及肺组织。蛋白质样品用SDS-PAGE分离,然后转移到PVDF膜(Millipore,美国)。封闭1 h后,与一抗在4 ℃孵育过夜。使用抗体包括抗IL-6(1∶1 000,武汉三鹰,中国)、抗p-AMPK(1∶1 000,CST,美国)、抗AMPK(1∶1 000,CST,美国)、抗VCAM-1(1∶3 000,abcam,英国)、抗ICAM-1(1∶1 000,abcam,英国)、抗GAPDH(1∶3 000,CWBIO,中国)、抗β-actin(1∶2 000,CST,美国)。第2天,用辣根过氧化物酶标记的山羊抗兔或鼠抗体(1∶3 000,ABM,加拿大)在37 ℃下孵育1 h。

1.5 肺组织切片及免疫组织化学法

肺组织用4%多聚甲醛固定,石蜡包埋,切成5 μm切片。经过一系列脱蜡脱水后,用3%H2O2封闭内源过氧化物酶活性,然后用新鲜制备的0.01 mol/L柠檬酸缓冲液进行抗原修复。在用PBS洗涤3次后,用5%牛血清白蛋白封闭切片30 min,然后与下列一抗共同孵育4 ℃过夜:抗p-AMPK(1:800,CST,美国)、VCAM-1(1:800,abcam,英国)、IL-6(1:200,武汉三鹰,中国)。次日将切片与生物素标记的山羊抗鼠/兔二抗在37 ℃孵育30 min。DAB显色液用于显色,苏木精用于复染。免疫组织化学图像用全景扫描仪MIDI(3DHISTECH,匈牙利)捕获。

1.6 苏木精-伊红(HE)染色法观察肺损伤程度

将肺组织置于4%多聚甲醛中固定,按浓度梯度酒精脱水,二甲苯透明,石蜡包埋,行5 μm切片,置于载玻片上,贴片后进行HE染色,在光镜下观察肺损伤程度。

1.7 肺湿重(W)/干重(D)比值检测肺水肿

肺组织置于滤纸上吸干组织表面液体后称重,记为W,之后将肺组织置于70 ℃烘箱72 h至恒重,称重记为D,计算肺组织W/D。

1.8 逆转录聚合酶链反应(RT-PCR)

用TRIzol试剂(Invitrogen,美国)裂解细胞及肺组织,提取RNA。利用反转录试剂盒(CWBIO,中国)获得cDNA。采用UltraSYBR mix(CWBIO,中国)进行RT-PCR。细胞以GAPDH为内参基因,动物组织以ACTB为内参基因。PCR引物如表 1表 2所示。

表 1 扩增靶基因的引物序列(人) Table 1 Primer sequences for the amplification of target genes (human)
基因 引物序列(5’-3’)
IL-6 forward: 5’-GAGGAGACTTGCCTGGTGAA-3’
reverse: 5’-GCGCAGAATGAGATGAGTTG-3’
IL-8 forward: 5’-ACCGGAAGGAACCATCTCAC-3’
reverse: 5’-GTGGATCCTGGCTAGCAGAC-3’
GAPDH forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’
reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’

表 2 扩增靶基因的引物序列(小鼠) Table 2 Primer sequences for the amplification of target genes (mice)
基因 引物序列(5’-3’)
ICAM-1 forward: 5’-CTGAAAGATGAGCTCGAGAGTG-3’
reverse: 5’-AAACGAATACACGGTGATGGTA-3’
VCAM-1 forward: 5’-GACATTTACCCAGTTTACAGGC-3’
reverse: 5’-TGACGGGAGTAAAGGTTACTTC-3’
ACTB forward: 5-GTGCTATGTTGCTCTAGACTTCG- 3’
reverse: 5- ATGCCACAGGATTCCATACC- 3’
1.9 统计学方法

采用GraphPad Prism 8.0软件进行统计分析,服从正态分布的计量资料以均值±标准差(x±s)表示;多组间比较采用单因素方差分析,组间两两比较采用Tukey法检验,以P < 0.05为差异有统计学意义。

2 结果 2.1 HucMSC-cm减轻LPS诱导的ALI

为明确脐带间充质干细胞上清液对ALI的作用。首先用LPS及HucMSC-cm构建ALI及治疗组模型(图 1A),发现LPS模型组小鼠肺组织充血水肿明显,而治疗组较LPS组减轻(图 1B);通过HE染色发现,相较于sham组,LPS组肺泡结构破坏,肺泡间隔明显增厚,充血、水肿明显,并伴大量炎性细胞浸润;当给予HucMSC-cm处理后,肺组织损伤得到明显改善(图 1C)。此外,通过比较外周血中性粒细胞比例,发现HucMSC-cm能有效抑制LPS诱导升高的中性粒细胞比例(P < 0.001,图 1D)。综上说明脐带间充质干细胞上清液可以改善LPS诱导的小鼠ALI。

A:体内实验设计模式图;B:小鼠肺组织;C:小鼠肺组织病理切片HE染色(放大倍数,×400);D:小鼠外周血中性粒细胞比例;aP < 0.001 图 1 HucMSC-cm减轻LPS诱导的ALI Fig 1 HucMSC-cm alleviated LPS-induced ALI
2.2 HucMSC-cm减少LPS诱导的ALI中炎症因子

在体内试验中,通过Western Blot检测小鼠肺组织中IL-6蛋白水平,发现LPS气管内注射72 h后能引起肺组织中IL-6蛋白水平明显升高(P=0.003),而LPS+HucMSC-cm能抑制IL-6的升高(P=0.003)(图 2A)。同样地,通过免疫组织化学法检测各组小鼠肺组织中IL-6的表达,得到了与上述一致的结果(图 2B)。在体外试验中,当给予LPS刺激肺微血管内皮细胞24 h后,IL-6(P < 0.001)及IL-8(P=0.027)的mRNA水平均明显升高,而给予HucMSC-cm预处理,则可抑制IL-6(P=0.003)及IL-8(P=0.002)的mRNA水平(图 2CD)。

A:Western Blot检测小鼠肺组织IL-6蛋白表达水平(左)及量化图(右);B:免疫组化检测小鼠肺组织IL-6的表达(放大倍数,×400);C和D:RT-PCR检测肺微血管内皮血细胞IL-6及IL-8 mRNA表达水平;aP < 0.01,bP < 0.001,cP < 0.05 图 2 HucMSC-cm能减少LPS诱导的ALI肺组织中IL-6 Fig 2 HucMSC-cm reduced IL-6 in LPS-induced ALI lung tissue
2.3 HucMSC-cm抑制LPS诱导的肺血管内皮细胞表面粘附分子的表达

进一步探究HucMSC-cm在体内对肺微血管内皮细胞粘附分子的影响。结果发现LPS组ICAM-1(P < 0.001)及VCAM-1(P=0.001)蛋白水平较sham组高,而在HucMSC-cm预处理组,ICAM-1(P=0.001)及VCAM-1(P=0.006)蛋白水平明显下降(图 3A);mRNA水平上结果相同(P < 0.001)(图 3B)。此外,免疫组织化学也证明LPS组VCAM-1的阳性表达较sham组高,并且主要聚集于肺血管周围,而LPS+HucMSC-cm组能有效抑制VCAM-1的表达(图 3C)。

A:Western Blot检测小鼠肺组织ICAM-1及VCAM-1蛋白水平及量化图;B:RT-PCR检测肺组织中ICAM-1及VCAM-1 mRNA表达水平;C:免疫组化检测小鼠肺组织VCAM-1的表达(放大倍数,×400);aP < 0.01,bP < 0.001 图 3 HucMSC-cm能减少LPS诱导的ALI肺组织中粘附分子 Fig 3 HucMSC-cm reduced the adhesion molecules in LPS-induced ALI lung tissue
2.4 HucMSC-cm通过激活AMPK减轻LPS诱导的ALI

既往研究发现AMPK的活化可以改善ALI[13],本研究检测ALI模型中p-AMPK及AMPK蛋白水平,发现LPS组中p-AMPK蛋白水平较sham组降低(P=0.013),而AMPK蛋白水平未见明显改变;在治疗组中,HucMSC-cm可以改善p-AMPK的表达(P=0.002),而并未改变AMPK蛋白水平(图 4A)。此外,通过免疫组化方法亦得到与前述相似的结果,并且p-AMPK的表达主要分布于血管周围(图 4B)。在体外实验中,用HucMSC-cm及LPS处理肺微血管内皮细胞,得到了与体内一致的结果,即LPS组中p-AMPK蛋白水平降低(P=0.005),而HucMSC-cm可以改善p-AMPK的表达(P=0.003)(图 4C)。因此,HucMSC-cm可以通过激活肺微血管内皮细胞AMPK磷酸化水平缓解LPS诱导的ALI。

A:Western Blot分析小鼠肺组织中p-AMPK,AMPK蛋白水平及量化图;B:免疫组化检测小鼠肺组织中p-AMPK表达(放大倍数,400×);C:Western Blot分析肺微血管内皮细胞中p-AMPK及AMPK蛋白表达水平及量化图;aP < 0.05,bP < 0.01 图 4 HucMSC-cm上调肺微血管内皮细胞p-AMPK表达 Fig 4 HucMSC-cm up-regulated the expression of P-AMPK in pulmonary microvascular
2.5 AMPK抑制剂逆转HucMSC-cm对LPS诱导的ALI的保护作用

为明确HucMSC-cm对于ALI的保护作用是否通过AMPK途径,本实验通过给予Compound C预处理,发现小鼠肺组织LPS+cm+cc组较LPS+cm组充血水肿更加明显(图 5A);HE染色发现LPS+cm+cc组中肺组织病理学变化更加严重(图 5B)。同时Compound C能抑制体内HucMSC-cm诱导的p-AMPK水平,而不影响AMPK总蛋白水平(图 5CD)。进一步分析肺组织中炎症因子及粘附分子的变化,发现Compound C处理后能进一步增加IL-6及VCAM-1在肺组织中的表达(图 5CE)。检测该过程中对于肺血管通透性的影响发现LPS组肺W/D比值小鼠较sham组高(P=0.006),LPS+cm组下降(P=0.004),而AMPK抑制剂组干预后再次上升(P=0.040)(图 5F)。

A:小鼠肺组织;B:小鼠肺组织HE染色(放大倍数,×400);C:免疫组化检测小鼠肺组织p-AMPK、VCAM-1及IL-6表达(放大倍数,×400);D:Western Blot分析小鼠肺组织中p-AMPK蛋白表达;E:Western Blot检测小鼠肺组织ICAM-1、VCAM-1及IL-6蛋白水平;F:小鼠肺组织湿/干重比例;aP < 0.05,bP < 0.01 图 5 Compound C抑制HucMSC-cm对LPS诱导的ALI的保护作用 Fig 5 Compound C attenuated the protective effect of HucMSC-cm on LPS-induced ALI
3 讨论

ALI和ARDS是重症监护病房常见的疾病,目前对该疾病认识尚不充分,以至于治疗仍存在缺陷,导致该疾病始终保持较高的发病率和病死率[14]。在临床上,ALI常继发于脓毒血症患者,其中革兰阴性杆菌所致感染较常见[15]。而作为革兰阴性细菌壁的组成成分,LPS由脂质和多糖构成,完全能够模拟构建脓毒症和ALI模型,并且已在大量研究中得到证实[16-17]。在本研究中通过给予气管内注射LPS,足以引起肺部炎症反应,这也与已有的研究相一致[18]。本研究发现,在LPS处理72 h后的小鼠肺组织充血水肿,HE染色可见正常的肺结构破坏,肺泡塌陷,肺泡及肺间质中大量炎性细胞浸润,Western Blot及免疫组化法检测发现肺组织中炎症因子IL-6明显上升。

在近几年的研究中,间充质干细胞已成为在炎症疾病中候选的治疗方法[19], 并且此前已有研究证明间充质干细胞可以减轻LPS诱导的炎症因子[20];而与细胞相比,细胞培养基更容易保存及运输,且没有细胞给药造成的不良反应,如排斥反应、致瘤性、致血栓性等[21-22]。在不同来源的间充质干细胞中,脐带间充质干细胞来源丰富,收集方便,生长较快,能分泌更多类型的因子来构建免疫抑制环境[23-24]。同时脐带间充质干细胞及其条件培养基也都被证明具有抗炎作用[25-26]。然而,脐带间充质干细胞上清液在LPS诱导的ALI中的作用机制尚未明确,本研究使用HucMSC-cm处理LPS构建的ALI的小鼠,发现小鼠肺组织充血水肿有所改善,病理学检查同样发现小鼠肺组织结构有所恢复,肺泡及肺间质中炎性细胞减少,Western Blot及免疫组化检测发现肺组织中的IL-6表达下降。从而有效证明了脐带间充质干细胞上清液能保护由LPS诱导的ALI,进一步验证发挥作用的机制。

ALI和ARDS发病早期大量炎症因子浸润在肺组织,这与本研究动物模型病理结果一致,并且中性粒细胞是早期炎症的核心因素[2]。在发生ALI后,中性粒细胞会迅速向肺损伤部位迁移,而这一过程主要是由于肺血管内皮细胞被激活,内皮细胞上粘附分子ICAM-1和VCAM-1表达增加,与血管中的中性粒细胞表面的选择素结合,中性粒细胞牢固地粘附在炎症部位的毛细血管内皮上,随后中性粒细胞在粘附处发生扁平化和极化,在内皮管腔表面爬行并从血管渗出,沿着趋化因子的浓度梯度移动到炎症部位。因此,造成ALI炎症聚集的前提是肺血管内皮细胞激活,粘附分子表达增高。既往研究发现在ARDS患者死亡后的肺组织样本中,ICAM-1、VCAM-1和E-选择素的表达明显升高[27]。同样在本实验中,当气管内注射LPS后,小鼠肺组织中粘附分子ICAM-1及VCAM-1表达较sham组升高,但是HucMSC-cm对于LPS诱导的ALI中的粘附分子的影响并不清楚,因此在本研究中发现使用脐带间充质干细胞上清液治疗后,无论在蛋白水平还是mRNA水平,这两种粘附分子都能被明显抑制。另外,本研究通过免疫组化法更直观地观察到,HucMSC-cm对于LPS诱导的VCAM-1的抑制主要体现在肺微血管内细胞上,从而证明了HucMSC-cm主要是通过下调肺组织中的粘附分子、减少中性粒细胞与内皮细胞粘附减少炎症反应。

AMPK是一种丝氨酸/苏氨酸蛋白激酶,近期对其的研究逐渐扩展到炎症反应及氧化应激。既往研究发现,通过5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷激活动脉内皮细胞AMPK时,可以抑制ICAM-1及VCAM-1表达,从而减少白细胞粘附在内皮细胞抑制炎症[28];亦有研究证实,二甲双胍通过激活巨噬细胞AMPK表达从而抑制LPS诱导的炎症因子及趋化因子表达,达到抑制炎症的作用[29]。基于AMPK活化可抑制粘附分子及炎症因子的表达,笔者猜测HucMSC-cm是否通过激活AMPK活性达到抑制粘附分子的表达。因此本研究首先检测各组小鼠肺组织中p-AMPK及AMPK的表达,发现在LPS刺激后,p-AMPK表达明显受到抑制,而在给予脐带间充质干细胞上清液处理后,p-AMPK表达重新上调,这一过程并未对AMPK总蛋白水平产生影响,在体外实验中得到一致的结果。接下来为进一步证明AMPK的活化在ALI中的作用,给予AMPK抑制剂Compound C预处理,发现HucMSC-cm所诱导的AMPK活性被逆转;并且肺组织中粘附分子ICAM-1、VCAM-1及炎症因子IL-6水平再次上调,说明AMPK活化在脐带间充质干细胞上清液治疗LPS诱导的ALI中起关键作用。

本研究利用LPS诱导的小鼠ALI模型证明HucMSC-cm在ALI中的保护作用,发现HucMSC-cm可以减少LPS诱导的肺组织中ICAM-1、VCAM-1及IL-6的表达,并且证实AMPK在该过程中的重要作用。

利益冲突  所有作者声明无利益冲突

作者贡献声明  陈海洋、杨宏锋、虞志新:酝酿和设计实验;陈海洋、沈嘉琪:实施研究;陈海洋:采集数据;陈海洋、沈嘉琪、朱伟:分析/解释数据;陈海洋:起草文章;杨宏锋、朱伟、虞志新:对文章的知识性内容作批评性审阅,支持性贡献

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