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.