Pipoxolan suppresses the inflammatory factors of NF‐κB, AP‐1, and STATs, but activates the antioxidative factor Nrf2 in LPS‐stimulated RAW 264.7 murine macrophage cells

Abstract Although pipoxolan (PIPO) is a smooth muscle relaxant, its anti‐inflammatory capability has not been studied. Therefore, we investigated the anti‐inflammatory molecular mechanisms of PIPO in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages. In this study, we used the MTT assay to evaluate the cytotoxicity, applied the enzyme‐linked immunosorbent assay to determine the inflammatory cytokines, and performed Western blotting to assess protein expression. The results showed that PIPO significantly inhibited cytokine production, including nitric oxide, prostaglandin E2, tumor necrosis factor‐α, and interleukin‐6. PIPO also suppressed the pro‐inflammatory mediator expression with inducible nitric oxide synthase and cyclooxygenase‐2. Moreover, PIPO prohibited the multiple inflammatory transcription factor pathways, including inhibitor kappa B/nuclear factor of the κ light chain enhancer of B cells (NF‐κB), mitogen‐activated protein kinase/activator protein‐1 (AP‐1), Janus kinase/signal transducer and activator of transcription (STAT), and toll‐like receptor 4 (TLR4)/serine/threonine kinase (AKT). Besides, PIPO effectively activated the nuclear factor erythroid 2‐related factor 2 (Nrf2)/heme oxygenase‐1 antioxidative pathway. Collectively, PIPO may attenuate the inflammatory effects via influencing the LPS/TLR4 receptor binding; suppress the expression of anti‐inflammatory transcription factors NF‐κB, AP‐1, and STAT; and activating the antioxidative transcription factor Nrf2 in LPS‐stimulated mouse RAW 264.7 cells.


| INTRODUCTION
Inflammation is a defensive immune response of the animal body to stimuli. However, uncontrolled inflammation can lead to immunodeficiency and many chronic diseases. 1 In mammals, the most inflammatory process always initiated from lipopolysaccharide (LPS) binding to the TLR4 coreceptor. 2 During the inflammatory process, LPS binds to the transmembrane toll-like receptor 4 (TLR4) receptor and activates the myeloid differentiation primary response 88 (MyD88)-dependent or/and independent pathways. Through activating the Akt/MAPK pathway by (MyD88)-dependent activation, the transcription factors NF-κB and AP-1 translocate from the cytoplasm to the nucleus, leading to the production of inflammatory cytokines such as IL-1, IL-6, and TNF-α. 3 In contrast, the activated MyD88 independent pathway can prompt the interferon regulatory factor signal pathway, which enables the secondary wave of transcription factor STATs to transfer from the cytoplasm into the nucleus. 4 NF-κB, the multipotent transcriptional factor, plays a critical role in the pro-inflammatory processes. 5 Phosphorylated inhibition of kappa B (IκB) is the crucial step in starting the nuclear translocation of NF-κB from cytoplasm to the nucleus, which can regulate the transcription and expression of multiple genes for inflammation and immune responses. 6 Besides, the MAPK pathway regulates transcription and translation levels of inflammatory mediators by activating transcription factors AP-1 and NF-κB. 7 Abnormal activation of transcription factors AP-1 (c-Jun, c-Fos, Jun D, and ATF2) is associated with redox-mediated inflammation and related to the transcription of pro-inflammatory genes encoding enzymes (such as iNOS, COX-2). 8,9 In chronic inflammatory responses, the Janus kinase (JAK)/STAT signaling triggered the pro-inflammatory cytokines (TNF-α, IL-2, and IL-6). 8 JAKs are receptor tyrosine kinases that mediate phosphorylation of STATs. The activated STAT dimerizes and translocates to the nucleus and binds to the promoter of the target gene, which then regulates its transcription. 9 It has demonstrated that Nrf2 blocks the expression of pro-inflammatory cytokines and prevents inflammatory responses, Nrf2 cascade acts as a novel anti-inflammatory signal to heme oxygenase-1 (HO-1) expression in macrophages. 10 Moreover, MAPKs also assist TLR signaling in cross talking with the Nrf2 pathway. 11 Thus, Nrf2 can translocate into the nucleus and initiates the transcription of genes encoding cytoprotective proteins. Among these proteins, HO-1, superoxide dismutase (SOD), and catalase (CAT) play a vital role in anti-inflammation and antioxidation responses. 12 Pipoxolan (PIPO) ( Figure 1A) is a smooth muscle relaxant that inhibits phosphodiesterase and maintenance cyclic-AMP levels and prevents Ca 2+ entering the cell membrane in smooth muscle. 13,14 According to earlier studies, PIPO can inhibit the phosphorylation of JNK and p38 and inhibit the expression metalloproteinases-2 (MMP-2) and MMP-9. 15 PIPO also inhibited the phosphatidylinositol-4,-5-diphosphate 3-kinase (PI3K) protein expression, and the phosphorylation of protein kinase B (AKT), indicating that PIPO inhibits PI3K/ AKT intrinsic apoptotic signaling pathways. 16 Moreover, PIPO can also inhibit the Ras/MEK/ERK pathway preventing neuronal apoptosis, decreasing the migration and intimal thickening in vascular smooth muscle cells. 13 Therefore, this study evaluated the anti-inflammatory molecular mechanism of PIPO in inflammatory transcription factor and antioxidant transcription factor in LPS-stimulated mouse RAW264.7 cells.

| Nitrite assay
The production of nitrite was determined using Griess reagent. 18 RAW264.7 cells (3 × 10 4 /well) were seeded in 96-well cell culture plates overnight. Cells were treated with various concentrations of PIPO for 1 hour before incubating with LPS for 24 hours. Cell culture supernatants were collected and, subsequently, nitrite production was detected using the Griess reagent. The absorbance at 540 nm was measured with a Micro-Reader EPOCH2 plate reader.

| Enzyme-linked immunosorbent assay
The cytokines were produced according to the manufacturer's instructions. 19 RAW264.7 cells (3 × 10 4 /well) were seeded in 96-well cell culture plates overnight. Cells were treated with various concentrations of PIPO for 1 hour before incubating with LPS. Cell culture supernatants were collected and subsequently, the levels of cytokines (TNF-α, IL-1β, and IL-6) were detected using the enzyme-linked immunosorbent assay (ELISA) kits. The absorbance was measured with a Micro-Reader EPOCH2 plate reader.

| Western blotting assay
Total protein or nuclear protein extracts from cells were prepared as described previously. 21

| Immunofluorescence assay
Immunofluorescence assay was applied to detect the expression with confocal spectral microscopy, as described previously. 22 RAW 264.7 cells were seeded onto glass coverslips overnight and treated with PIPO for 1 hour before incubating with LPS. After exposure, cells were treated with cold 4% paraformaldehyde for 20 minutes and permeabilized using 0.5% Triton-X 100 for 30 minutes. Then, the cells were blocked with 5% bovine serum albumin for 1 hour and incubated with a primary antibody of p65, p50, c-Jun, c-Fos, p-Nrf2, p-STAT3 for overnight at 4 C. The cells were washed three times and incubated with a secondary antibody labeled with Alexa Fluor-594 for 1 hour. The cells were then washed another three times and DAPI (50 μg/mL) was added for 20 minutes at 37 C in the dark. The coverslips were sealed and the images were taken with a Leica TCS SP2 confocal spectral microscopy.

| Statistical analysis
The data were presented as mean ± SE and analyzed using one-way analysis of variance, and differences between the LPS-treated and LPS-untreated (control) groups were considered statistically significant at the level of #P < 0.05 vs negative LPS-stimulated group, *P < .05, **P < .01, and ***P < .001 the PIPO-treated vs LPSstimulated and PIPO-untreated group.

| PIPO inhibited the phosphorylation of IKKα/β and IκB and inhibited the nuclear translocation of NF-κB in LPS-stimulated RAW264.7 cells
In the natural state, NF-κB always stabilized in the cytoplasm by binding to the IκB junction. IKKβ is an IκB kinase and an essential factor in NF-βB activity. 26 The results demonstrated that treatment with PIPO inhibited LPS-stimulated IKK phosphorylation and IκBα phosphorylation, respectively ( Figure 3A).

| PIPO suppressed the LPS-stimulated inflammatory response through regulated Nrf2/HO-1 pathway
Nrf2/HO-1 is a critical antioxidant pathway, and the transcription factor Nrf2 suppresses oxidative stress and regulates antiinflammatory. 29 Moreover, Nrf2 is correlated with the induction of HO-1, GPx, and glutathione-S-transferase, allowing the scavenging of free radicals in cells caused by oxidative damage. 30 As shown in Figure 6A

| DISCUSSION
Inflammation is an essential immune response defense process of the body against the stimulus, infection, or injury. However, chronic inflammation also causes immune deficiency and promotes the development of many diseases or cancer. 31 LPS/TLR4 stimulates the inflammatory transcription factor signaling pathway (including NF-κB and AP-1 transcription factors) and causes the release of proinflammatory factors (iNOS, COX-2). 32 Therefore, overstimulation of the NF-κB transduction-signaling pathway thus produce a pathogenspecific essential immune response and release pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). 33 In the present study, we found that PIPO effectively inhibited LPS-stimulated production of cytokine nitrite and PGE 2 , which confirmed the protein and mRNA expression of the upstream pro-inflammatory mediator iNOS and COX-2.
Besides, PIPO also reduced the production of cytokine TNF-α and IL-6 in LPS-stimulated RAW264.7 cells.
In general, NF-κB always stabilized in the cytoplasm by binding to the IκB junction. 26 Two related kinases, IKKα and IKKβ, phosphorylate the IκB proteins, prime to their degradation and the subsequent stimulation of gene expression of NF-κB. 34 The translocation of the NF-κB p50-p65 dimer to the nucleus promotes the release of inflammatory mediators such as TNF-α, IL-6, IL-1β, NO, and iNOS. 35 Our results suggested that PIPO significantly inhibited LPS-induced phosphorylation of IκB and decreased the protein expression of transcription factor NF-κB (p65 and p50) and reversed the nuclear translocation of NF-κB in LPS-stimulated RAW264.7 cells. The results were confirmed by the immunofluorescence analysis in the cytoplasm and nucleus. As a result, PIPO inhibited the NF-κB pathway participated in the anti-inflammatory process.
However, p38 MAPK induced expressions of TNF-α, IL-1β, IL-6, and IL-8. 36 Therefore, our results showed that PIPO could inhibit MAPK signaling by decreasing the phosphorylation of JNK, ERK1/2, and p38 and reducing the nuclear translocations of c-Jun and c-Fos from the cytoplasm into the nucleus in LPS-stimulated RAW264.7 cells. It has been demonstrated that TNF-α as the critical trigger along with increased activation of JAK1/STAT1 plays a crucial role in tumor cell proliferation and invasiveness. 37 In particular, IL-6 is the primary mediator of inflammation, which is the key to trigger the JAK2/STAT3 pathway, and plays a crucial role in tumor cell proliferation and invasiveness. 38 This study shows that PIPO inhibited the cytokine TNF-α and IL-6 production, and PIPO is effective in reducing the phosphorylation and the nuclear translocations of JAK1, 2/STAT1, 3. This study assessed whether the antiinflammatory function of PIPO is associated with the inhibitions of the JAK/STAT pathway and participated in the anti-inflammatory process.
Interestingly, the oxidative stress pathway is the essential mechanism of the inflammatory response. Nrf2 plays a critical role in oxidative stress. 39 TLR4/MyD88 signaling can activate Nrf2 but independent from the reactive oxygen species. 40 Under the stimulation of oxidative stress, Nrf2 translocated from the cytoplasm into the F I G U R E 8 Proposed mechanism depicting the effect of pipoxolan (PIPO) suppressing the TLR4/AKT pathway, which inhibited the anti-inflammatory effects via the mitogen-activated protein kinase (MAPK)/ AP-1, inhibitor kappa B (IκB)/NF-κB, and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) transcription factor pathways, and activating the antioxidant effects via Nrf2/HO-1 signaling pathways on lipopolysaccharide (LPS)stimulated macrophages [Color figure can be viewed at wileyonlinelibrary.com] nucleus, stimulating the transcription of antioxidant response element-dependent genes, including HO-1, CAT, and SOD. 12 The data showed that PIPO attenuates the inflammatory effects via influencing LPS/TLR4 pathway, which significantly suppressed the antiinflammatory transcription factors NF-κB, AP-1, and STAT and activates the antioxidative transcription factor Nrf2 in LPS-stimulated RAW 264.7 cells (Figure 8).
In conclusion, our data demonstrate that PIPO attenuates the inflammatory effects via influencing the LPS/TLR4 pathway; suppresses the anti-inflammatory transcription factor NF-κB, AP-1, and STAT; and activates the antioxidative transcription factor Nrf2 in LPSstimulated RAW 264.7 cells.