Anna Ju

Anti-inflammatory effects of Crataeva nurvala Buch. Ham. are mediated via inactivation of ERK but not NF-κB

ETHNOPHARMACOLOGICAL RELEVANCE Crataeva nurvala Buch. Ham. is an important medicinal plant in India, and its extracts and components were used to treat various inflammatory diseases, such as urinary tract infection, rheumatoid arthritis, and colitis. However, no systemic studies about anti-inflammatory effects of Crataeva nurvala Buch. Ham. and its underlying mechanisms of action have been reported. This study aimed to explore the anti-inflammatory effects of ethanol extracts of Crataeva nurvala Buch. Ham. (ECN). MATERIALS AND METHODS The non-cytotoxic and maximal effective concentration of ECN was determined by measuring the formation of formazan from water-soluble tetrazolium salt in living cells. The inhibitory effect of ECN on nitric oxide (NO) synthesis was measured using Griess reagent, and Enzyme-linked immunosorbent assay (ELISA) was used to measure secreted tumor necrosis factor (TNF)-α and interleukin (IL)-6 protein levels. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting analysis were used to assess the mRNA and protein expression of each inflammatory mediator or relating signaling protein, respectively. RESULTS A non-cytotoxic concentration of ECN (≤200 μg/ml) significantly reduced the production of NO and IL-6, but not TNF-α, in lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Decreased production of NO by ECN was correlated with reduced expression of iNOS at the mRNA and protein levels. However, cyclooxygenase (COX)-2 expressions at mRNA and protein level were not regulated by ECN. The mRNA expression of IL-6 and IL-1β, but not TNF-α, was also inhibited by ECN treatment in LPS-stimulated RAW 264.7 macrophages. Reduced production of inflammatory mediators by ECN was followed by decreased activity of mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinase (ERK), but not nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). CONCLUSIONS These results indicate that ECN inhibits LPS-induced inflammatory responses via negative regulation of ERK in murine macrophages, suggesting that ECN is a candidate for alleviating severe inflammation.

Scaffold Role of DUSP22 in ASK1-MKK7-JNK Signaling Pathway

Mitogen-activated protein kinases (MAPKs) are involved in a variety of intracellular events such as gene expression, cell proliferation, and programmed cell death. MAPKs are activated by dual phosphorylation on threonine and tyrosine residues through sequential activation of protein kinases. Recent studies have shown that the protein kinases involved in MAPK signal transductions might be organized into signaling complexes by scaffold proteins. These scaffold proteins are essential regulators that function by assembling the relevant molecular components in mammalian cells. In this study, we report that dual-specificity phosphatase 22 (DUSP22), a member of the protein tyrosine phosphatase family, acts as a distinct scaffold protein in c-Jun N-terminal kinase (JNK) signaling. DUSP22 increased the phosphorylation in the activation loop of JNK regardless of its phosphatase activity but had no effect on phosphorylation levels of ERK and p38 in mammalian cells. Furthermore, DUSP22 selectively associated with apoptosis signal-regulating kinase 1 (ASK1), MAPK kinase 7 (MKK7), and JNK1/2. Both JNK phosphorylation and JNK-mediated apoptosis increased in a concentration-dependent manner regardless of DUSP22 phosphatase activity at low DUSP22 concentrations, but then decreased at higher DUSP22 concentrations, which is the prominent feature of a scaffold protein. Thus, our data suggest that DUSP22 regulates cell death by acting as a scaffold protein for the ASK1-MKK7-JNK signal transduction pathway independently of its phosphatase activity.

Methanol extracts of Xanthium sibiricum roots inhibit inflammatory responses via the inhibition of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) in murine macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Xanthium sibiricum has been used as a traditional Chinese medicine for the treatment of appendicitis, bronchitis, arthritis, and other inflammatory ailments. However, its pharmacological activity related to an anti-inflammatory effect remain unknown. This present study aims to investigate the anti-inflammatory effect of methanol extracts of X. sibiricum roots (MXS), and to further determine its underlying mechanism of action in order to assess the medicinal value of X. sibiricum roots. MATERIALS AND METHODS To assess the anti-inflammatory activity of MXS in lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages, the production of nitric oxide (NO) was measured using the Griess reagent system. The levels of pro-inflammatory cytokines and mediators were quantified using an Enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Subsequently, immunoblotting analyses were employed to detect inflammatory mediators as well as to elucidate the underlying regulatory mechanisms suppressed by MXS. RESULTS MXS inhibited LPS-stimulated NO production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages within the non-cytotoxic concentration range (50-400 μg/ml). In addition, mRNA and protein levels of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly suppressed by MXS at the concentration of 400 μg/ml. Furthermore, MXS (200 μg/ml) clearly reduced the phosphorylation levels of the inhibitor of kappa Bα (IκBα) and signal transducer and activator of transcription 3 (STAT3), without affecting changes in the phosphorylation levels of mitogen-activated protein kinases (MAPKs). When five major components (betulin, betulinic acid, β-sitosterol, stigmasterol, and scopoletin) of MXS were separately investigated, stigmasterol and β-sitosterol seemed to play major inhibitory roles in the LPS-induced production of inflammatory mediators such as NO, IL-6, and TNF-α. CONCLUSION Our results demonstrate that MXS has an anti-inflammatory property in LPS-stimulated RAW 264.7 macrophages, and its anti-inflammatory activity is exerted by the regulation of nuclear factor-κB (NF-κB) and STAT3 signaling pathways.

Methanol extracts of Euphorbia cooperi inhibit the production of inflammatory mediators by inhibiting the activation of c‑Jun N‑terminal kinase and p38 in murine macrophages

Numerous Euphorbiaceae plants have been used for the treatment of diseases, including liver diseases, asthma and rheumatism. The present study evaluated the effect of methanol extracts from Euphorbia cooperi (MEC), a member of the Euphorbiaceae plant family, on the production of inflammatory cytokines interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α, nitric oxide (NO) as well as the activation of mitogen‑activated protein kinase and nuclear factor (NF)‑κB signaling. Non‑cytotoxic concentrations of MEC significantly reduced the production of NO and IL‑6, but not TNF‑α, in lipopolysaccharide (LPS)‑stimulated RAW 264.7 macrophages. The decreased production of NO by MEC was due to alleviated expression of inducible NO synthase. Reporter assays with cells treated with MEC demonstrated reduced activator protein‑1 (AP-1) activity, while NF‑κB activity was not reduced. Furthermore, the phosphorylation levels of c‑Jun N‑terminal kinase (JNK) and p38 were suppressed by MEC while phosphorylation levels of inhibitor of κB were not reduced by MEC, suggesting that MEC‑mediated inactivation of JNK and p38 is the underlying regulatory mechanism for inflammatory mediators in LPS‑stimulated RAW 264.7 macrophages.

Dual-specificity phosphatase 5 acts as an anti-inflammatory regulator by inhibiting the ERK and NF-κB signaling pathways

Although dual-specificity phosphatase 5 (DUSP5), which inactivates extracellular signal-regulated kinase (ERK), suppresses tumors in several types of cancer, its functional roles remain largely unknown. Here, we show that DUSP5 is induced during lipopolysaccharide (LPS)-mediated inflammation and inhibits nuclear factor-κB (NF-κB) activity. DUSP5 mRNA and protein expression increased transiently in LPS-stimulated RAW 264.7 cells and then returned to basal levels. DUSP5 overexpression in RAW 264.7 cells suppressed the production of pro-inflammatory tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), whereas knockdown of DUSP5 increased their expression. Investigation of two major inflammatory signaling pathways, mitogen-activated protein kinase (MAPK) and NF-κB, using activator protein-1 (AP-1) and NF-κB reporter plasmids, respectively, showed that NF-κB transcription activity was downregulated by DUSP5 in a phosphatase activity-independent manner whereas AP-1 activity was inhibited by DUSP5 phosphatase activity towards ERK,. Further investigation showed that DUSP5 directly interacts with transforming growth factor beta-activated kinase 1 (TAK1) and inhibitor of κB (IκB) kinases (IKKs) but not with IκBα. DUSP5 binding to IKKs interfered with the association of TAK1 with IKKs, suggesting that DUSP5 might act as a competitive inhibitor of TAK1-IKKs association. Therefore, we propose that DUSP5 negatively regulates ERK and NF-κB in a phosphatase activity-dependent and -independent manner, respectively.