Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

Morin,a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators,Intracellular ROS Levels and NF-κB Activation

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG₂), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.     

Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway

Excessive pulmonary inflammatory response is critical in the development of acute lung injury (ALI). Previously, microRNAs (miRNAs) have been recognized as an important regulator of inflammation in various diseases. However, the effects and mechanisms of miRNAs on inflammatory response in ALI remain unclear. Herein, we tried to screen miRNAs in the processes of ALI and elucidate the potential mechanism. Using a microarray assay, microRNA let-7e (let-7e) was chose as our target for its reported suppressive roles in several inflammatory diseases. Down-regulation of let-7e by antagomiR-let-7e injection attenuated LPS-induced acute lung injury. We also found that antagomiR-let-7e could obviously improve the survival rate in ALI mice. Moreover, antagomiR-let-7e treatment reduced the production of proinflammatory cytokines (i.e., TNF-α, IL-1β and IL-6) in bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice. Luciferase reporter assays confirmed that suppressor of cytokine signaling 1 (SOCS1), a powerful attenuator of nuclear factor kappa B (NF-κB) signaling pathway, was directly targeted and suppressed by let-7e in RAW264.7 cells. In addition, it was further observed that SOCS1 was down-regulated, and inversely correlated with let-7e expression levels in lung tissues of ALI mice. Finally, down-regulation of let-7e suppressed the activation of NF-κB pathway, as evidenced by the reduction of p-IκBα, and nuclear p-p65 expressions in ALI mice. Collectively, our findings indicate that let-7e antagomir protects mice against LPS-induced lung injury via repressing the pulmonary inflammation though regulation of SOCS1/NF-κB pathway, and let-7e may act as a potential therapeutic target for ALI.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1^TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1^TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1^TG bone marrow cells into non-transgenic (AEBP1^NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1^TG mammary macrophages and epithelium. Shh expression was induced in AEBP1^TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1^TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1^TG peritoneal macrophages. The conditioned AEBP1^TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1^TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages

C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway.     

Doxycycline Inhibits Inflammation-Induced Lymphangiogenesis in Mouse Cornea by Multiple Mechanisms

Lymphangiogenesis is significantly involved in the pathogenesis of diseases, including graft rejection, cancer metastasis and various inflammatory conditions. The inhibition of lymphangiogenesis has become a new therapeutic target for the treatment of these diseases. Here, we explored the anti-lymphangiogenic effects of doxycycline in inflammation-induced lymphangiogenesis (ILA) in the cornea and the underlying mechanisms. In the present study, mice with ILA of the cornea were treated with topical doxycycline (0.1%) or vehicle control. Lymphangiogenesis was quantified using corneal immunostaining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Human dermal lymphatic endothelial cells (HDLECs) and a murine macrophage cell line (RAW264.7) were used to further explore the underlying mechanisms of doxycycline-mediated anti-lymphangiogenesis in vitro. Our results showed that doxycycline treatment dramatically inhibited ILA in the mouse cornea (p<0.001), with a significant decrease in vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 signalling, macrophage infiltration and inflammatory cytokine expression. Doxycycline also significantly inhibited VEGF-C-induced HDLEC proliferation in vitro by modulating the PI3K/Akt/endothelial nitric oxide (NO) synthase (eNOS) pathway and significantly suppressed interleukin-1β (IL-1β), TNF-α and VEGF-C production in the RAW264.7 cell line by modulating the PI3K/Akt/nuclear factor-kappaB (NF-κB) pathway. Additionally, doxycycline treatment dramatically reduced the phosphorylation of NF-κBp65, Akt and eNOS in ILA and significantly inhibited matrix metalloproteinases (MMPs) activity in vitro and in ILA. In conclusion, doxycycline inhibited ILA, possibly through suppression of VEGF-C signalling, macrophage function and MMPs activity. This observation suggests that doxycycline is a potential therapeutic agent for lymphangiogenesis-related diseases.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

Nur77 Prevents Osteoporosis by Inhibiting the NF‐κB Signalling Pathway and Osteoclast Differentiation

Inflammation is a major risk factor for osteoporosis, and reducing inflammatory levels is important for the prevention of osteoporosis. Although nuclear receptor 77 (Nur77) protects against inflammation in a variety of diseases, its role in osteoporosis is unknown. Therefore, the main purpose of this study was to investigate the osteoprotective and anti‐inflammatory effects of Nur77. The microCT and haematoxylin and eosin staining results indicated that knockout of Nur77 accelerated femoral bone loss in mice. The enzyme‐linked immunosorbent assay (ELISA) results showed that knockout of Nur77 increased the serum levels of hsCRP and IL‐6. The expression levels of NF‐κB, IL‐6, TNF‐α and osteoclastogenesis factors (TRAP, NFATC1, Car2, Ctsk) in the femurs of Nur77 knockout mice were increased significantly. Furthermore, in vitro, shNur77 promoted the differentiation of RAW264.7 cells into osteoclasts by activating NF‐κB, which was confirmed by PDTC treatment. Mechanistically, Nur77 inhibited osteoclast differentiation by inducing IκB‐α and suppressing IKK‐β. In RAW264.7 cells, overexpression of Nur77 alleviated inflammation induced by siIκB‐α, while siIKK‐β alleviated inflammation induced by shNur77. Consistent with the in vivo studies, we found that compared with control group, older adults with high serum hsCRP levels were more likely to suffer from osteoporosis (OR = 1.76, p < 0.001). Our data suggest that Nur77 suppresses osteoclast differentiation by inhibiting the NF‐κB signalling pathway, strongly supporting the notion that Nur77 has the potential to prevent and treat osteoporosis.     

Genistein-3′-sodium sulfonate Attenuates Neuroinflammation in Stroke Rats by Down-Regulating Microglial M1 Polarization through α7nAChR-NF-κB Signaling Pathway

Microglial M1 depolarization mediated prolonged inflammation contributing to brain injury in ischemic stroke. Our previous study revealed that Genistein-3′-sodium sulfonate (GSS) exerted neuroprotective effects in ischemic stroke. This study aimed to explore whether GSS protected against brain injury in ischemic stroke by regulating microglial M1 depolarization and its underlying mechanisms. We established transient middle cerebral artery occlusion and reperfusion (tMCAO) model in rats and used lipopolysaccharide (LPS)-stimulated BV2 microglial cells as in vitro model. Our results showed that GSS treatment significantly reduced the brain infarcted volume and improved the neurological function in tMCAO rats. Meanwhile, GSS treatment also dramatically reduced microglia M1 depolarization and IL-1β level, reversed α7nAChR expression, and inhibited the activation of NF-κB signaling in the ischemic penumbra brain regions. These effects of GSS were further verified in LPS-induced M1 depolarization of BV2 cells. Furthermore, pretreatment of α7nAChR inhibitor (α-BTX) significantly restrained the neuroprotective effect of GSS treatment in tMCAO rats. α-BTX also blunted the regulating effects of GSS on neuroinflammation, M1 depolarization and NF-κB signaling activation. This study demonstrates that GSS protects against brain injury in ischemic stroke by reducing microglia M1 depolarization to suppress neuroinflammation in peri-infarcted brain regions through upregulating α7nAChR and thereby inhibition of NF-κB signaling. Our findings uncover a potential molecular mechanism for GSS treatment in ischemic stroke.     

Gprc5a-deficiency confers susceptibility to endotoxin-induced acute lung injury via NF-κB pathway

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1β and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.     

Hydrogen Sulfide Suppresses Oxidized Low-density Lipoprotein (Ox-LDL)-stimulated Monocyte Chemoattractant Protein 1 generation from Macrophages via the Nuclear Factor κB (NF-κB) Pathway

This study was designed to examine the role of hydrogen sulfide (H₂S) in the generation of oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein 1 (MCP-1) from macrophages and possible mechanisms. THP-1 cells and RAW macrophages were pretreated with sodium hydrosulfide (NaHS) and hexyl acrylate and then treated with ox-LDL. The results showed that ox-LDL treatment down-regulated the H₂S/cystathionine-β-synthase pathway, with increased MCP-1 protein and mRNA expression in both THP-1 cells and RAW macrophages. Hexyl acrylate promoted ox-LDL-induced inflammation, whereas the H₂S donor NaHS inhibited it. NaHS markedly suppressed NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter in ox-LDL-treated macrophages. Furthermore, NaHS decreased the ratio of free thiol groups in p65, whereas the thiol reductant DTT reversed the inhibiting effect of H₂S on the p65 DNA binding activity. Most importantly, site-specific mutation of cysteine 38 to serine in p65 abolished the effect of H₂S on the sulfhydration of NF-κB and ox-LDL-induced NF-κB activation. These results suggested that endogenous H₂S inhibited ox-LDL-induced macrophage inflammation by suppressing NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter. The sulfhydration of free thiol group on cysteine 38 in p65 served as a molecular mechanism by which H₂S inhibited NF-κB pathway activation in ox-LDL-induced macrophage inflammation.     

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-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 (pH_i) 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.