2-phenylethynesulfonamide Prevents Induction of Pro-inflammatory Factors and Attenuates LPS-induced Liver Injury by Targeting NHE1-Hsp70 Complex in Mice

The endotoxin-mediated production of pro-inflammatory cytokines plays an important role in the pathogenesis of liver disorders. Heat shock protein (Hsp70) overexpression has established functions in lipopolysaccharide (LPS)-mediated inflammatory response. However, little is known about the role of Hsp70 activity in LPS signaling. We hypothesized that inhibition of Hsp70 substrate binding activity can ameliorate LPS-induced liver injury by decreasing induction of pro-inflammatory factors. In this study, C57/BL6 mice were injected intraperitoneally with LPS and 2-phenylethynesulfonamide (PES), an inhibitor of Hsp70 substrate binding activity. We found that i. PES prevented LPS-induced increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, infiltration of inflammatory cells, and liver cell apoptosis; ii. PES reduced inducible nitric oxide synthase (iNOS) protein expression as well as serum nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) content in LPS-stimulated mice; iii. PES reduced the mRNA level of iNOS, TNF-alpha, and IL-6 in LPS-stimulated liver. iiii. PES attenuated the degradation of inhibitor of kappa beta-alpha (I kappa B-alpha) as well as the phosphorylation and nuclear translocation of nuclear factor-kappa B (NF-kappa B) in LPS-stimulated liver. Similar changes in the protein expression of inflammatory markers, IkB-alpha degradation, and NF-kappa B phosphorylation and nuclear translocation were observed in RAW 264.7 cells. Further mechanistic studies revealed that PES remarkably reduced the elevation of [Ca2+] i and intracellular pH value (pHi) in LPS-stimulated RAW 264.7 cells. Furthermore, PES significantly reduced the increase in Na+/H+ exchanger 1 (NHE1) association to Hsp70 in LPS-stimulated macrophages and liver, suggesting that NHE1-Hsp70 interaction is required for the involvement of NHE1 in the inflammation response. In conclusion, inhibition of Hsp70 substrate binding activity in vivo reduces the induction of pro-inflammatory factors and prevents LPS-induced liver injury likely by disrupting NHE1-Hsp70 interaction which consequently reduces the activation of I kappa B-alpha-NF-kappa B pathway in liver.