Effects of Na+ -H+ exchanger 1 knockdown on protein expression levels of ATP binding cassette transporter A1 and cholesterol efflux in hypoxic RAW264.7 cells

Abstract

Objective To explore the effects of Na+ -H+ exchanger 1(NHE1) knockdown on ATP binding cassette transporter A1 (ABCA1) protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells. Methods The RAW264.7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1(siNHE1) or scramble RNA (siNC). The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition.In the meantime, the methods of SNARF-1, Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity, intracellular Ca2+ ([Ca2+ ]i) and calpain activity, respectively.Furthermore, ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells.In parallel, the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HLPC and 3H-cholesterol. Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds, 1.28 folds and 61.96% (all P<0.05), and increased[Ca2+ ]i and calpain activity by 4.51 folds and 2.41 folds(all P<0.05). Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95%, 60.75% and 66.44%, respectively (all P<0.05) and[Ca2+ ]i and calpain activity were reduced by 59.23% and 54.66% (P<0.05). Furthermore, the ABCA1 protein level was 61.67% lower in the hypoxic cells than in the normoxic cells (P<0.05), and siNHE1 was increased by 56.52%after treatment of Hypoxia.Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57% and 101.81% (all P<0.05). Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24%及49.66%(all P<0.05). Hypoxia reduced the cholesterol efflux by 34.79%(P<0.05), which were partially reversed by siNHE1. Conclusions NHE1 might play an important role in hypoxia-induced ABCA1 protein attenuation and reverse cholesterol transport dysfunction through[Ca2+ ]i/calpain pathway. Key words: ATP binding cassette transporter A1; Na+ -H+ exchanger 1; Anoxia; Cholesterol