The Sonic hedgehog signaling pathway involved in the expression of RACK1 in the pulmonary microvascular endothelial cells induced by lipopolysaccharide

Abstract

Objective To explore the effect of expression of protein kinase C receptor 1(RACK1) induced by lipopolysaccharide (LPS) on Sonic hedgehog(SHH) signaling pathway in rat puhnonary microvascular endothelial cells (RPMVEC). Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Anhui province. Using immunocytochemistry method, the expression of RACK1 protein in RPMVECs was detected, cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group, SAG(smoothened Agonist, a SHH signaling pathway specific agonist) dose-dependent group, LPS time-dependent group, SAG time-dependent group and LPS+SAG group. In LPS dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 mg/L LPS for 8 h. In LPS time-dependent groups, RPMVECs were cultured with 10 mg/L LPS for 0, 2, 4, 8, 12, 24 h. In SAG dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 μmol/L for 8 h. In SAG time-dependent groups, RPMVECs were cultured with 1 μmol/L SAG for 0, 2, 4, 8, 12, 24 h. In LPS+SAG group, RPMVECs were cultured with 1 μmol/L SAG 8 h after 10 mg/L LPS treatment for 1 h. In addition, blank group, LPS group and SAG group were set for control. Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention. Results Immunocytochemistry revealed that RACK1 were present in RPMVEC. 1. In LPS dose-dependent groups (0, 0.1, 1, 10 mg/L), the level of RACK1 elevated as LPS dose increased correspondingly with inter-group difference (P 0.05; the rest P 0.05). The relative expression levels of GLI-1 mRNA were (1.109 ± 0.063), (1.169 ± 0.052), (3.468 ± 0.128), (3.434 ± 0.054), (0 μmol/L vs. 0.1 μmol/L and 1 μmol/L vs. 10 μmol/L, P>0.05, the rest P 0.05). The relative expression level of GLI-1 mRNA increased at 2 h (3.027 ± 0.065), and compared with 0 h (2.651 ± 0.123), there was significant differences (F=132.841, P<0.05). 3. In LPS+SAG intervention groups, the expression of RACK1 was lower than that in LPS group (0.831 ± 0.040 vs. 1.189 ± 0.149, P<0.05), and the expression of GLI-1 mRNA was higher than that in LPS group (2.720 ± 0.130 vs. 0.796 ± 0.082, P<0.05). Conclusions The LPS up-regulates the expression of RACK1 in RPMVECs, and the activated SHH signaling pathway can down-regulate the expression of RACK1 induced by LPS in RPMVECs. Key words: Lipopolysaccharide; Pulmonary microvascular endothelial cells; Receptor for activated C kinase 1; Sonic hedgehog signaling pathways; Acute respiratory distress syndrome