Heme oxygenase-1 in cholecystokinin-octapeptipe attenuated injury of pulmonary artery smooth muscle cells induced by lipopolysaccharide and its signal transduction mechanism

Abstract

Aim: To study the effect of cholecystokinin-octapeptide (CCK-8) on lipopolysaccharide (LPS) -induced pulmonary artery smooth muscle cell (PASMCs) injury and the role of heme oxygenase-1 (HO-1), and to explore the regulation mechanism of c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1) signal transduction pathway in inducing HO-1 expression further.

Methods: Cultured PASMCs were randomly divided into 4 or 6 groups: normal culture group, LPS (10 mg/L), CCK-8 (10(-6) mol/L) plus LPS (10 mg/L) group, CCK-8 (10(-6) mol/L) group, zinc protoporphyrin 9 (ZnPPIX) (10(-6) mol/L) plus LPS (10 mg/L) group, CCK-8 (10(-6) mol/L) plus ZnPPIX and LPS (10 mg/L) group. Seven hours after LPS administration, ultrastructural changes and content of malondialdehyde (MDA) of PASMCs in each group were investigated by electron microscopy and biochemical assay respectively. HO-1 mRNA and protein of PASMCs in the former 4 groups were examined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry staining. Changes of c-fos expression and activation of JNK of PASMCs in the former 4 groups were detected with immunocytochemistry staining and Western blot 30 min after LPS administration.

Results: The injuries of PASMCs and the increases of MDA content induced by LPS were alleviated and significantly reduced by CCK-8 (P<0.05). The specific HO-1 inhibitor- ZnPPIX could worsen LPS-induced injuries and weaken the protective effect of CCK-8. The expressions of c-fos, p-JNK protein and HO-1 mRNA and protein were all slightly increased in LPS group, and significantly enhanced by CCK-8 further (P<0.05).

Conclusion: HO-1 may be a key factor in CCK-8 attenuated injuries of PASMCs induced by LPS, and HO-1 expression may be related to the activation of JNK and activator protein (AP-1).

Figure 1

Mitochondria ultrastructural changes shown in EM photograph of PASMCs (×25 000). A: Control group, B: LPS group, C: CCK-8+LPS group, D: CCK-8 group, E: LPS+ZnPP group, F: CCK-8+LPS+ZnPP group.

Figure 2

Effect of CCK-8 on LPS-induced c-fos (upper, ×400) and HO-1 (lower, ×200) protein expression in PASMCs. A: Control group, B: LPS group, C: CCK-8+LPS group, D: CCK-8 group.

Figure 3

RT-PCR product gel electrophoresis. M: DNA marker, 1: Control, 2: LPS, 3: CCK-8 + LPS, 4: CCK-8.

Figure 4

Effect of CCK-8 on LPS-induced HO-1 mRNA ex-pression and JNK activation in PASMCs. A: Effect of CCK-8 on LPS-induced HO-1 mRNA expression in PASMCs. n = 3. ^dP < 0.01 vs Control; ^bP < 0.01 vs LPS. B: CCK-8 increases JNK activation induced by LPS in PASMCs. n = 3. ^aP < 0.05 vs Control; ^bP < 0.01 vs LPS.