摘要
目的探讨巨噬细胞中1-磷酸鞘胺醇受体(sphingosine 1-phosphate receptor,S1PR)的表达及其作用,观察干预S1PR3(S1P3)对脂多糖诱导的心肌损伤的影响。方法传代培养小鼠Ana-1巨噬细胞,给予脂多糖(lipopolysaccharide,LPS,100 ng/ml)刺激或S1P3特异性抑制剂CAY-10444(10μmol/L)干预,细胞随机分为对照组、LPS组、CAY-10444组、CAY-10444预处理2h+LPS组,Transwell小室观测巨噬细胞迁移,蛋白免疫印迹检测巨噬细胞S1PR的表达,并检测p-Akt/Akt蛋白水平。在体实验,6~8周龄雄性C57/B6小鼠,随机分为对照组、LPS组、CAY-10444组、CAY-10444干预+LPS组,每组12只,LPS(10 mg/kg)腹腔注射,或CAY-10444 1 mg/kg于LPS诱导后30 min腹腔注射干预,24 h后取心脏组织HE染色观察病理改变,免疫组化染色观察巨噬细胞浸润程度以及炎症因子的表达情况,实时荧光定量PCR检测心肌损伤标记分子BNP、巨噬细胞表面分子F4/80、炎症因子TNF-α、IL-1β、IL-6的mRNA水平。结果与对照组比较,LPS诱导巨噬细胞大量迁移S1P3蛋白表达增加(P<0.01),p-Akt Ser473/Akt表达上调(P<0.01);与LPS组相比,S1P3抑制剂CAY-10444干预后再给予LPS刺激,巨噬细胞迁移被抑制(P<0.01),p-Akt Ser473/Akt表达也降低(P<0.01);在体实验,LPS诱导小鼠后BNP mRNA水平明显上调(P<0.01),同时F4/80以及炎症因子TNF-α、IL-1β、IL-6的mRNA水平上调(P<0.01),HE染色可见心肌损伤及炎细胞浸润,免疫组化染色法显示F4/80及炎症因子的大量阳性表达(P<0.01);使用S1P3抑制剂后,与LPS组比较,心肌损伤减轻免疫组化中巨噬细胞减少(P<0.01),炎症因子表达降低(P<0.01),BNP mRNA水平降低(P<0.01),F4/80以及TNF-α、IL-1β、IL-6的mRNA水平也明显降低(P<0.01)。结论抑制巨噬细胞S1P3表达可抑制巨噬细胞的迁移并提示p-Akt/Akt与了这一过程,此外,S1P3抑制剂的干预可有效减轻LPS诱导的心肌损伤。
AIM To investigate the effect and the potential mechanism of Sphingosine 1-phosphate （S1P） receptors in lipopolysaccharide （LPS）-induced macrophages migration and to observe the effect of S1P3 inhibitor on LPS-induced cardiac injury. METHODS Macrophages （murine Ana-1 cells） were cultured and exposed LPS and the expression of S1P receptors was detected by Western blotting. Cultured Ana-1 cells were then incubated with LPS or with or without pretreatment with S1P3 inhibitor （CAY-10444）. Transwell assay was used to observe the migration of macrophages and Western blot assay was used to confirm that CAY-10444 could effectively inhibit S1P3 expression in Ana-1 cells and to detect Akt and p-Akt Ser473 protein. In vivo, LPS-induced heart injury mouse model was established to demonstrate the cardioprotective properties of S1P3 inhibitor. Mice in each experiment were randomly assigned to four groups： Control group （n=12）, which was intraperitoneal with saline, LPS group （n=12）, which was intraperitoneally injected with LPS （10 mg/kg）, CAY-10444 group （n=12）, which was intraperitoneally injected with CAY （1mg/kg） and LPS＋CAY-10444 group （n=12）, which was intraperitoneally injected with CAY 30min after LPS challenge. Tissue samples from the myocardium were collected and the pathological changes of myocardium were observed by hematoxylin-eosin （HE） staining. Immunohistochemistry was used to observe the expressions of mature macrophage-specific marker F4/80 and proinflammatory cytokines, including TNF-α, IL-1β and IL-6. Quantitative real-time PCR assay was used to detect mRNA level of B-type natriuretic peptide （BNP）, F4/80 and proinflammatory cytokines. RESULTS Compared with those in the NC group, the expression of S1P3 in Ana-1 cells was up-regulated by LPS （P〈0.01） and the amount of phosphorylated Akt （p-Akt Ser473/Akt） was significantly increased in LPS group （P〈0.01）. When pretreated with S1P3 inhibitor CAY-10444, the migration of macrophages increased when incubated with LPS （P〈0.01）, but CAY-10444 inhibited such effect （P〈0.01）. Compared with that in LPS group, S1P3 inhibitor also reduced the level of phosphorylation of Akt （Ser 473） （P〈0.01）. In vivo study, LPS treated mice exhibited severe heart injury characterized by the prominent cardiac inflammation in HE staining and higher mRNA level of BNP （P〈0.01）. The expressions of F4/80, TNF-α, IL-1β and IL-6 in myocardial tissue were markedly up-regulated after LPS challenge. However, treatment with CAY-10444 dramatically attenuated the cardiac histopathological changes in comparison with those in LPS group. The level of BNP was also decreased significantly in LPS＋CAY group （P〈0.01）. Macrophage-specific marker and proinflammatory cytokines were down-regulated in LPS＋CAY group （P〈0.01）. CONCLUSION Suppressed expression of S1P3 in macrophages could inhibit LPS-induced migration and the changes of phosphorylated Akt level might be involved in this process. The intervention of S1P3 inhibitor effectively ameliorates LPS-induced cardiac injury.
出处
《心脏杂志》
CAS
2017年第5期522-528,共7页
Chinese Heart Journal
