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

Abstract

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-α (TNF-α), and interleukin-6 (IL-6) content in LPS-stimulated mice; iii. PES reduced the mRNA level of iNOS, TNF-α, and IL-6 in LPS-stimulated liver. iiii. PES attenuated the degradation of inhibitor of κB-α (IκB-α) as well as the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB) in LPS-stimulated liver. Similar changes in the protein expression of inflammatory markers, IκB-α degradation, and NF-κB 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 IκB-α-NF-κB pathway in liver.

Pifithrin-μ Attenuates Acute Sickness Response to Lipopolysaccharide in C57BL/6J Mice

“…They also act on neurons with the assistance of endothelial cell transporters [6] . Our previous studies showed that pifithrin-μ attenuates the production of pro-inflammatory factors in both microglia and macrophages [14,15] , demonstrating that the anti-sickness effect of pifithrin-μ may be associated with its prevention on cytokine production. Reduction of food intake is one of the symptoms observed after the overproduction of pro-inflammatory cytokines in the innate immune system.…”

“…In this study, animals treated with pifithrin-μ prior to LPS administration displayed a decrease in serum corticosterone levels. Previous studies showed that pifithrin-μ exerts its pharmacological effects mainly through targeting the Hsp70 C terminal substrate binding activity [13] , and this targeting dissociates a battery of molecules such as apoptotic protease activating facter-1 (APAF1) and Na + /H + exchanger-1 from Hsp70 [13][14][15] . Hsp70 interacts physically with the intracellular glucocorticoid receptor (GR) and facilitates its activation by glucocorticoids [22,23] .…”

“…Pifithrin-μ exerts its pharmacological effects mainly through the suppression of the substrate-binding activity of heat shock protein 70 (Hsp70) [13] . Given the inhibitory role of pifithrin-μ in pro-inflammatory factor production [14,15] , we speculate that pifithrin-μ may attenuate sickness behavior. To test this possibility, the LPS-induced sickness model in C57BL/6J mice was employed in this study.…”

Defining the Na+/H+ exchanger NHE1 interactome in triple-negative breast cancer cells

“…NHE1 and HSP70 were previously shown to interact in PS127A Chinese hamster fibroblasts [17]. More recently [41], HSP70 was found to be dynamically associated with NHE1 in macrophages. Heat shock proteins are inducible molecular chaperones that facilitate the folding of proteins and their transport across membranes, and prevent protein aggregation in response to cellular stress.…”

Promoting Neuronal Tolerance of Diabetic Stress

“…Similarly, increases in macrophage phagocytosis and neutrophil microbicide activity have been reported after exogenous Hsp70 administration (Kovalchin et al, 2006; Ortega et al, 2006). eHsp70 also prompted pro-inflammatory cytokine release from epithelial, innate immune and adaptive immune cells, which may explain why eHsp70 is associated with inflammatory cytokine production in multiple mouse and human studies (Asea et al, 2000; Asea et al, 2002; Basu et al, 2001; Chase et al, 2007; Dvoriantchikova et al, 2014; Dybdahl et al, 2005; Huang et al, 2013; Qiao et al, 2008). Collectively, these data support a strong capacity of eHsp70 to initiate and escalate an immune-mediated inflammatory response.…”