探讨脓毒症相关性急性肺损伤（ALI）中粒细胞集落刺激因子受体（G-CSFR）对多配体蛋白聚糖4（SDC4）表达的影响及调控机制。

选取30只C57bl/6小鼠，采用随机数字表法分为空白对照组（sham组，等量等渗NaCl溶液腹腔注射），脓毒症相关性ALI组[脂多糖（LPS）组，20 mg/kg的LPS腹腔注射]，LPS联合靶向G-CSFR的短发夹RNA腺相关病毒（shG-CSFR）干预组[LPS + shG-CSFR组，气管注射shG-CSFR后进行LPS腹腔注射]，每组10只。利用苏木素-伊红染色、免疫荧光以及实时荧光定量PCR检测肺组织炎症因子的表达水平、炎症损伤及G-CSFR、SDC4的荧光表达。对体外肺血管内皮细胞分别进行LPS刺激、慢病毒过表达以及PP2[非受体酪氨酸激酶（SRC）抑制剂]干预，利用转录组测序（RNA-seq）、实时荧光定量PCR及western-blotting技术分析对肺血管内皮细胞进行G-CSFR过表达产生的差异基因，研究其不同表达水平对下游分子SDC4的表达影响以及磷酸化SRC（pSRC）/核因子κB（NF-κB）通路的表达改变。

与sham组相比，LPS组小鼠肺组织炎症因子肿瘤坏死因子α、白细胞介素6（IL-6）、IL-1β基因表达以及肺组织炎症损伤评分均显著升高，但是shG-CSFR的进一步基因干扰能够明显抑制以上表型改变（P均< 0.05）。RNA-seq结果显示，在LPS刺激的基础上，G-CSFR过表达可进一步提升SDC4的基因表达量[（66 ± 12）比（152 ± 19），t = 43.575，q = 0.001]，且SDC4参与的京都基因与基因组百科全书（KEGG）信号通路富集于细胞外基质受体相互作用。在LPS刺激下，体外肺血管内皮细胞可以产生显著增高的SDC4表达，且相比于LPS组，LPS + G-CSFR组细胞经过G-CSFR慢病毒过表达干预后SDC4信使RNA和蛋白表达水平均显著增高，且伴随着pSRC和磷酸化pNF-κB/NF-κB蛋白的增高；而随着PP2的抑制作用，pNF-κB/NF-κB和SDC4的蛋白表达均被显著抑制（P均< 0.05）。

脓毒症相关性ALI背景下，内皮细胞膜受体G-CSFR活化并且过表达后引起细胞内pSRC/NF-κB通路的激活，产生炎症协调因子SDC4的高表达，从而加重炎症反应。

To investigate the effect of granulocyte colony-stimulating factor receptor (G-CSFR) on the expression of syndecan-4 (SDC4) in endothelial cells during sepsis-associated acute lung injury (ALI) and its regulatory mechanisms.

A total of 30 C57bl/6 mice were randomly divided into three groups using a random number table: a negative control group (sham group, intraperitoneal injection of an isotonic NaCl solution of equal concentration), a sepsis-associated ALI group [lipopolysaccharide (LPS) group, intraperitoneal injection of 20 mg/kg LPS], and a LPS combined with short hairpin RNA targeting G-CSFR (shG-CSFR) intervention group [LPS + shG-CSFR group, intratracheal injection of shG-CSFR followed by intraperitoneal injection of LPS], with 10 mice in each group. Hematoxylin-eosin staining, immunofluorescence staining and real-time quantitative PCR were used to detect the expression levels of inflammatory factors, the degree of inflammatory damage, and the fluorescence intensities of G-CSFR and SDC4 in lung tissue. For in vitro experiments, pulmonary microvascular endothelial cells were subjected to LPS stimulation, lentivirus overexpression, and PP2 [a non-receptor tyrosine kinase (SRC) inhibitor] treatment. RNA sequencing (RNA-seq), real-time quantitative PCR, and western-blotting were used to identify differentially expressed genes following G-CSFR overexpression in pulmonary microvascular endothelial cells and to investigate the effects of different G-CSFR expression levels on downstream SDC4 expression, as well as the activation status of the phosphorylation SRC (pSRC)/nuclear factor-kappaB (NF-κB) pathway.

Compared with the sham group, the LPS group exhibited significantly higher expression levels of tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-1β in lung tissue, as well as higher inflammatory damage scores; meanwhile, these changes were significantly attenuated by shG-CSFR-mediated gene interference (all P < 0.05). RNA-seq showed that G-CSFR overexpression further increased SDC4 expression [(66 ± 12) in the LPS group vs. (152 ± 19) in the LPS + shG-CSFR group, t = 43.575, q < 0.001], and the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway involving SDC4 was enriched in the extracellular matrix receptor interaction. LPS stimulation significantly increased SDC4 expression in pulmonary microvascular endothelial cells, and G-CSFR overexpression further enhanced SDC4 messenger RNA and protein in the LPS + shG-CSFR group as compared with the LPS group, accompanied by increased levels of pSRC and phosphorylation pNF-κB/NF-κB (all P < 0.05). Moreover, treatment with PP2 significantly suppressed the expression of pNF-κB/NF-κB and SDC4.

In the context of sepsis-associated ALI, activation and overexpression of G-CSFR in endothelial cells lead to activation of the intracellular pSRC/NF-κB pathway, resulting in overexpression of the inflammatory coordinating factor SDC4, which aggravates inflammation.