The SHH Signaling Pathway Involved In The Expression Of RACK1 In The Pulmonary Microvascular Endothelial Cells Induced By Lipopolysaccharide

Objective To explore whether the expression of protein kinase C receptor 1(RACK1)can be induced by lipopolysaccharide(LPS)and it interacts with SHH signaling pathway in rat puhnonary microvascular endothelial cells(RPMVEC).Methods The healthy male SPF grade SD rats with 200-250 g body weight were purchased from the laboratory animal center of Anhui province.Using immunocytochemistry method to detect the expression of RACK1 protein in RPMVEC,Cultured RPMVEC were randomly divided into different groups of LPS dose-dependent group,SAG dose-dependent group,LPS time-dependent group,SAG time-dependent group and LPS+SAG group.1.For LPS dose-dependent treatment,RPMVEC were cultured with 0.1,1,10 mg/L LPS for 8h.For LPS time-dependent treatment,RPMVEC were cultured with 10 mg/L LPS for 0,2,4,8,12,24 h.2.For SAG dose-dependent treatment,RPMVEC were cultured with 0.1,1,10 ?mol/L for 8 h.For SAG time-dependent treatment,RPMVEC were cultured with 1 ?mol/L SAG for 0,2,4,8,12,24 h.For LPS+SAG treatment,RPMVEC were cultured with 1 ?mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h,in addition,blank,LPS and SAG groups were set as references.Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the level of GLI-1 m RNA after intervention.Results Immunocytochemistry revealed that RACK1 were present in RPMVEC.1.In LPS dose-dependent manner(0,0.1,1,10 mg/L)the concentration dependence of relative expression levels of RACK1 for LPS(P < 0.05);the relative expression levels of GLI-1 m RNA were(1.109 ± 0.063),(1.039 ± 0.135),(0.813 ± 0.066),(0.770 ± 0.105),(1 mg/L vs 10 mg/L,P > 0.05;the rest P < 0.05).In LPS time-dependent manner the relative expression level of RACK1 at 2 h(0.370 ± 0.010)was higher than 0 h(0.329 ± 0.008),peaked at 12 h(1.296 ± 0.048).When compared with 0 h,there were significant differences(F = 1272.204,P < 0.05);the relative expression level of GLI-1 m RNA was decreased at 2 h(0.929 ± 0.007.When compared with 0 h(1.089 ± 0.042),there were significant differences(F = 306.609,P < 0.05).2.In SAG dose-dependent manner the relative expression levels of RACK1 were no significant,(all P > 0.05);the relative expression levels of GLI-1 m RNA were(1.109 ± 0.063),(1.169 ± 0.052),(3.468 ± 0.128),(3.434 ± 0.054),(0 ?mol/L vs 0.1 ?mol/L and 1 ?mol/L vs 10 ?mol/L,P > 0.05,the rest P < 0.05).In SAG time-dependent manner the relative expression levels of RACK1 protein were no significant(P > 0.05);the relative expression level of GLI-1 m RNA rose at 2 h(3.027 ± 0.065),When compared with 0 h(2.651 ± 0.123),there were significant differences(F = 132.841,P < 0.05).3.In LPS+SAG intervention manner the expression of RACK1 was lower than the LPS group(0.831 ± 0.040 vs 1.189 ± 0.149,P < 0.05),the expression of GLI-1 m RNA is higher than the LPS group(2.720 ± 0.130 vs 0.796 ± 0.082,P < 0.05).Conclusions The LPS up-regulates the expression of RACK1 in RPMVEC,and the activated SHH signaling pathway can down-regulate the expression of RACK1 induced by LPS in RPMVEC.