Palmitate up-regulates laminin expression via ROS/integrin αvβ3 pathway in HLSECs

Abstract

Aims/introduction: To investigate the roles of reactive oxygen species (ROS) and integrin αvβ3 in palmitate-induced laminin expression of human liver sinusoidal endothelial cells (HLSECs).

Results: The protein expression of integrin αv, integrin β3 and laminin are increased by palmitate in HLSECs in a time- and dose-dependent manner. NAC, the ROS inhibitor, significantly inhibited the up-regulation of protein expression of integrin αv, integrin β3 and laminin by palmitate (P < 0.05). Palmitate markedly enhanced ROS formation (P < 0.05), which was not inhibited by LM609, the antibody of integrin αvβ3. Palmitate significantly increased laminin synthesis (P < 0.05), which was attenuated by LM609 and NAC (P < 0.05).

Materials and methods: HLSECs were treated with palmitate in the presence or absence of LM609 (10 μg/ml) or N-acetylcysteine (NAC) (2 mM). Expression of integrin αv, integrin β3 and laminin were measured by RT-PCR and Western blot. Immunocytochemistry were used for examining laminin expression. The generation of ROS was measured using the fluorescent signal 2',7' dichloro-fluorescein diacetate (DCFH-DA).

Conclusions: The results suggested that palmitate increases laminin expression through ROS/integrin αv/β3 pathway.

Keywords: human liver sinusoidal endothelial cells; integrin αvβ3; laminin.

Figure 1. Palmitate induced ROS formation and the mRNA expression integrin αv, integrin β3 and laminin in HLSECs.

Cells were cultured with BSA and palmitate 50 umol/L, 100 umol/L 200 umol/L for 6, 24 and 48 h respectively. (A) Statistical data of ROS expression by the fluorescent signal DCFH-DA. (B) Statistical data of integrin αv mRNA expression by Quantitative RT-PCR. (C) Statistical data of integrin β3 mRNA expression by Quantitative RT-PCR. (D) Statistical data of laminin mRNA expression by Quantitative RT-PCR Data are expressed as mean ± S.D. from three independent experiments, ^*P < 0.05 vs control (BSA).

Figure 2. Palmitate increased the protein expression of integrin αv and integrin β3 in HLSECs.

(A) Representative photograph of Western blot. (B) Statistical data of integrin αv expression. (C) Statistical data of integrin β3 expression. Results are expressed as mean ± S.D. from three independent experiments, ^*P < 0.05 vs control (BSA).

Figure 3. Effect of NAC (2 mM, 24 h) on integrin αv and integrin β3 expression.

Cells were treated with 100 μmol/L palmitate for 48 h. (A) Representative photograph of Western blot. (B) Statistical data of integrin αv expression. (C) Statistical data of integrin β3 expression. Results are expressed as mean ± S.D. from three independent experiments, ^*P < 0.05 vs control (BSA), ^**P < 0.05 vs palmitate.

Figure 4. Palmitate induced ROS formation and effect of LM609 (10 μg/ml, 12 h) on ROS expression in HLSECs.

Cells were treated with 100 μmol/L palmitate for 48 h. Results are expressed as mean ± S.D. from three independent experiments, ^*P < 0.05 vs control (BSA).

Figure 5. Effect of palmitate, NAC and LM609 on protein expression of laminin in HLSCEs.

Cells were treated with 100 μmol/L palmitate for 48 h. (A) Representative photograph of Immunocytochemistry. (B) Statistical data of laminin expression. Results are expressed as mean ± S.D. from three independent experiments, ^*P < 0.05 vs control (BSA), ^**P < 0.05 vs palmitate.

Figure 6. Effect of palmitate, NAC and LM609 on protein expression of laminin in HLSCEs.

Cells were treated with 100 μmol/L palmitate for 48 h. (A) Representative photograph of Western-Blot. (B) Statistical data of laminin expression. Results are expressed as mean ± S.D. from three independent experiments, ^*P < 0.05 vs control (BSA), ^**P < 0.05 vs palmitate.