Amino acid sequences characterization and anti-inflammatory potency evaluation of Portulaca oleracea L. oligopeptides in macrophages

The Portulaca oleracea L. oligopeptides are seldom explored because they are often present in a complex matrix. In the current study, eleven novel Portulaca oleracea L. oligopeptides (POPs) were isolated and their mino acid sequence identified. Further, the anti-inflammatory potency was explored in lipopolysaccharide (LPS)-induced RAW264.7 cells. Results showed that POP-1∼[EHGEYE] possessed excellent anti-inflammatory potency by attenuating the pro-inflammatory cytokine expression (TNF-α, NO, IL-1β); inhibiting iNOS and COX-2 expressions and regulating the MAPK, PI3K/Akt and NF-κB signaling pathways. This may be an important molecular mechanism of POPs in anti-inflammatory damage.

POP-1 performed excellent anti-inflammatory potency by attenuating the pro-inflammatory cytokine expression (TNF-α, NO, IL-1β); inhibiting iNOS and COX-2 expressions and regulating the MAPK, PI3K/Akt and NF-κB signaling pathways.     

Xiao Qing Long Tang essential oil exhibits inhibitory effects on the release of pro-inflammatory mediators by suppressing NF-κB,AP-1, and IRF3 signalling in the lipopolysaccharide-stimulated RAW264.7 cells

Xiao Qing Long Tang (literally “Minor blue dragon decoction” in Chinese), a traditional Chinese formula, is prescribed to treat respiratory diseases. However, only few studies have been reported on its anti-inflammatory mechanisms. In this study, we investigated the inhibitory effects of Xiao Qing Long Tang essential oil on inflammatory mediators and explored the mechanisms of action of XQEO in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells. XQEO was prepared via steam distillation and characterized by GC-MS analysis. MTT and Griess assays were used to measure cell viability and NO production, respectively. The mRNA expression and the production of LPS-induced pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-10) and chemokines (MCP-1, Rantes, and MIP-1α) were determined by real-time PCR and enzyme-linked immunosorbent assay, respectively. Furthermore, we determined the protein levels of the components of NF-κB, AP-1 and IRF3 signalling by Western blotting. Immunofluorescence assay was used to estimate the nuclear translocation of NF-κB, AP-1 and IRF3. The results showed that XQEO inhibited the secretion of NO and PGE2 and down-regulated the mRNA and protein levels of iNOS and COX-2. We also found that XQEO suppressed the LPS-induced overproduction of pro-inflammatory mediators. Moreover, XQEO inhibited the phosphorylation of NF-κB/p65, AP-1/c-Jun, and IRF3 by suppressing their upstream kinases, such as MAPKs, TBK1, Akt, IKKα/β, and IκB, reducing the LPS-induced NF-κB, AP-1 and IRF3 translocation to the nucleus. These findings suggest that XQEO effectively suppresses the production of pro-inflammatory mediators possibly through the inhibition of NF-κB, AP-1, and IRF3 signalling in the LPS-stimulated RAW264.7 cells.

Xiao Qing Long Tang essential oil suppresses the production of inflammatory mediators via blocking NF-κB, AP-1, and IRF3 signalling in the lipopolysaccharide-stimulated RAW264.7 cells.     

Preventive effect of oligonol on nitric oxide and reactive oxygen species production through regulation of nuclear factor kappa B signaling pathway in RAW 264.7 macrophage cells against sodium nitroprusside

Oligonol, a low-molecular weight polyphenol isolated from lychee fruit, has been shown to possess beneficial properties, including anti-oxidative, anti-diabetic, and hepatoprotective properties in vitro and in vivo. This study was performed to investigate the anti-inflammatory effects of oligonol using sodium nitroprusside (SNP)-stimulated RAW 264.7 macrophage cells. Our results demonstrated that exposure of SNP to RAW 264.7 cells significantly decreased cell viability, and increased nitric oxide (NO) and reactive oxygen species (ROS) production. However, treatment with oligonol inhibited cell death and suppressed the over-production of NO and ROS induced by SNP in a dose-dependent manner. Consistent with these findings, oligonol significantly downregulated the mRNA levels of pro-inflammatory mediators, inducible nitric oxide synthase and cyclooxygenase-2, when compared with the SNP-treated control group. Furthermore, suppression of nuclear factor-κB (NF-κB) activation was also observed after treatment with oligonol in RAW 264.7 macrophage cells. These results suggest that oligonol attenuated SNP-induced oxidative stress and inflammatory responses via regulation of the NF-κB signalling pathway. On the basis of such potent anti-oxidant and anti-inflammatory properties, we propose that oligonol may contribute in the prevention and treatment of inflammation-related disorders.

Oligonol attenuated SNP-induced oxidative stress and inflammatory responses via regulation of the NF-κB signalling pathway in RAW 264.7 macrophage cells.     

Immunomodulatory mechanism of α-d-(1→6)-glucan isolated from banana

Banana is a delicious fruit with potent immunomodulatory function. In this study, α-d-(1→6)-glucan was purified from banana pulp. It could significantly promote pinocytic activity and production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The mRNA expression of nitric oxide synthase (iNOS), IL-6 and TNF-α was increased in RAW264.7 macrophages. α-d-(1→6)-glucan could not only increase the expression levels of p-p65 and p-IκBα, but also induce the translocation of nuclear factor-kappa B (NF-κB) p65 into the nucleus. Moreover, mitogen-activated protein kinases (MAPKs), including p-ERK, p-JNK and p-p38, were upregulated. These results suggested that NF-κB and MAPK signaling pathways were involved in the immunomodulatory mechanisms of α-d-(1→6)-glucan. The results revealed that α-d-(1→6)-glucan might be the critical component responsible for the health benefits of banana.

Banana is a delicious fruit with potent immunomodulatory function.     

Polyphenols extracted from huangjiu have anti-inflammatory activity in lipopolysaccharide stimulated RAW264.7 cells

In the present study, an extraction method, combining extraction by ethyl acetate + ethanol and purification by HPD400 resin, was established to obtain huangjiu polyphenol extract (HPE). After extraction and purification, the polyphenol yield was 22.57%, and 90.57% protein and 97.99% sugar were removed. HPLC analysis indicated that (+)-catechin (91.33 μg mL⁻¹) was the predominant phenolic compound among the 11 detected polyphenols. In LPS-stimulated RAW264.7 cells, HPE exhibits anti-inflammatory effects by inhibiting the production of NO and pro-inflammatory cytokines (TNF-α, interleukin IL-6 and IL-1β). The anti-inflammatory effect of HPE is associated with the inhibition of iNOS expression, the suppression of NF-κB translocation to the nucleus, and the inhibition of the phosphorylation of IκB and the MAPK family proteins, e.g. p-38, Erk 1/2, and JNK. Moreover, the activation of Nrf2 and HO-1 is also related to the anti-inflammatory effect of HPE.

HPE with the predominant polyphenol of (+)-catechin exhibits anti-inflammatory activity through the NF-κB pathway and MAPK signaling.     

Re-Du-Ning inhalation solution exerts suppressive effect on the secretion of inflammatory mediators via inhibiting IKKα/β/IκBα/NF-κB,MAPKs/AP-1, and TBK1/IRF3 signaling pathways in lipopolysaccharide stimulated RAW 264.7 macrophages

Background: Re-Du-Ning inhalation solution (RIS) is a novel preparation derived from the Re-Du-Ning injection, which has been clinically used to treat respiratory diseases such as pneumonia for more than twenty years in China. However, scant reports have been issued on its anti-inflammatory mechanisms. Aim: we investigated the suppressive effect of RIS on inflammatory mediators and explored the underlying mechanism of action. Methods: RIS freeze dried powder was characterized by HPLC analysis. Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage was selected as the cell model. The cell viability was determined by using the MTT assay. Moreover, the production of nitric oxide (NO) was measured by the Griess reaction. The protein secretions from inflammatory mediators were determined by the enzyme-linked immunosorbent assay (ELISA). The protein levels and enzyme activities were examined by Western blotting. The nuclear translocation of nuclear factor-kappa B (NF-κB), AP-1, and IRF3 was further explored by immunofluorescence assay. Results: the viability of the RAW 264.7 cells was not significantly changed after 24 h incubation with RIS concentration up to 400 μg mL⁻¹. The RIS remarkably reduced the production of NO and prostaglandin E₂ (PGE₂), and downregulated the expression of iNOS and COX-2. The concentrations of cytokines (IL-1β, IL-6, and TNF-α) and chemokines (MCP-1, CCL-5, and MIP-1α) in the culture medium were significantly decreased by the RIS treatment. Furthermore, the phosphorylation of IκB-α, IKKα/β, TBK1, ERK, p38, JNK, NF-κB, AP-1, and IRF3 was downregulated by the RIS treatment. The nuclear translocation of NF-κB, AP-1, and IRF3 was also inhibited after the RIS treatment. Conclusion: the suppressive effect of RIS is associated with the regulated NF-κB, AP-1, and IRF3 and their upstream proteins. This study provides a pharmacological basis for the application of RIS in the treatment of inflammatory disorders.

Re-Du-Ning inhalation solution suppresses the production of inflammatory mediators through TLR4 signaling pathway.     

Discovery and preliminary SAR of 14-aryloxy-andrographolide derivatives as antibacterial agents with immunosuppressant activity

Antibacterials (which restore gut flora balance) and immunosuppressants (which correct immune defects) are two important and effective therapeutic agents for the treatment of inflammatory bowel disease (IBD) in clinical use today. Since the structural skeleton of andrographolide, isolated from Andrographis paniculata, has become known as a natural antibiotic with anti-inflammation and heat-clearing and detoxifying properties, 14-aryloxy andrographolide derivatives have been designed, synthesized, and tested for their antibacterial effects on E. coli, S. aureus, and E. faecalis, which are related to IBD. It has been discovered in this study that the andrographolide skeleton is more selective against E. faecalis, the 14-aryloxy group with basicity is important for antibacterial functions, and the 14-(8′-quinolinyloxy) group is a good pharmacophore with antibacterial activity. In addition, we found that 7b1 and 8b1 are good and selective inhibitors of E. faecalis; two 14β-(8′-quinolinyloxy) andrographolide derivatives, 6b17 and 9b, exhibit good activity against E. coli, S. aureus, and E. faecalis. Likewise and importantly, further exploration of immunosuppressant activity for IBD shows that compound 7b1 is a selective inhibitor of the TNF-α/NF-κB signaling pathway, whereas 8b1 is selectively active against the TLR4/NF-κB signaling pathway; moreover, the compounds 6b17 and 9b are active in inhibiting the IL-6/STAT3, TLR4/NF-κB, and TNF-α/NF-κB signaling pathways. Based on these results, we have further focused on the development of dual function inhibitors of IBD as antibacterial and immunosuppressant agents by structural modification of andrographolide.

6b17 and 9b are active against 3 tested bacteria while they block IL-6/STAT3, TLR4/NF-κB and TNF-α/NF-κB pathways. 7b1 and 8b1 are selectively active against E. faecalis but 7b1 selectively blocks TNF-α/NF-κB pathway and 8b1 selectively blocks TLR4/NF-κB pathway.     

Retracted Article: Structural characterization and anti-inflammatory potency of Mesobuthus martensii Karsch oligopeptides in lipopolysaccharide (LPS)-induced RAW264.7 macrophages

Scorpion venom represents a significant source of bio-active peptides. However, the anti-inflammatory potency of scorpion venom oligopeptides (CMOs) has not been well explored. In the current study, thirty-five CMOs were isolated, the amino acid sequences were identified, and the anti-inflammatory potency was further explored in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. The results showed that CMO-1 (His-Tyr-Gly-His) demonstrated the best anti-inflammatory potency by attenuating inflammatory cytokine (NO, TNF-α, IL-6, and IL-1β) production. CMO-1 also inhibited IκBα degradation and p65 nuclear translocation and suppressed NF-κB activation. Moreover, CMO-1 inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. It is worth noting that CMO-1 exhibited anti-inflammatory potency; thus, it is a potential anti-inflammatory agent.

Scorpion venom represents a significant source of bio-active peptides.     

Inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal H1N1 influenza infection in mice

Introduction

Factors implicated in influenza-mediated morbidity and mortality include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction. Tumor necrosis factor-alpha (TNF-α) is an important pro-inflammatory cytokine present during influenza infection, but it is unclear whether direct inhibition of TNF-α can elicit protection against influenza infection.

Methods

In this study, the commercially available TNF-α inhibitor etanercept was used to inhibit TNF-α induced by lethal A/FM/1/47 (H1N1) influenza virus infection of mice. The effects of TNF-α inhibition on mouse survival, pathologic changes, immune cell infiltration, inflammatory cytokine secretion, Toll-like receptor expression, and activation of the NF-κB (nuclear factor kappa B) signaling pathway were evaluated.

Results

The intranasal delivery of etanercept provided significant protection against mortality (30% of mice survived up to 14 days after infection) in mice treated with etanercept. In contrast, no survivors were found beyond 6 days in mice treated with saline after lethal challenge with H1N1 influenza virus. It was observed that etanercept significantly reduced inflammatory cell infiltration (for example, macrophages and neutrophils), inflammatory cytokine secretion (for example, interleukin-6, TNF-α, and interferon gamma), and expression of Toll-like receptors (TLR-3, TLR-4, and TLR-7). Etanercept also downregulated and inhibited the cascade proteins of the NF-κB signaling pathway (for example, MyD88, TRIF, NF-κB, and p65), as well as enhanced host control of virus replication.

Conclusions

These findings indicate that etanercept, by blocking TNF-α, can significantly downregulate excessive inflammatory immune responses and provide protection against lethal influenza infection, making its use a novel strategy for controlling severe influenza-induced viral pneumonia.     

Structural characterization and anti-inflammatory effect in hepatocytes of a galactoglucan from Antrodia camphorata mycelium

The galactoglucan ACP2 was isolated from cultured Antrodia camphorata mycelium through anion-exchange column chromatography and Sephadex G-100 chromatography and shown to exhibit hepatoprotective function in L02 cells. Based on monosaccharide composition analysis, ACP2 was mainly composed of glucose, galactose, and 6-deoxyglucose in a molar ratio of 5 : 2 : 1. The average molecular weight of ACP2 was 1.93 × 10⁴ Da. The primary structure of ACP2 was elucidated with Fourier-transform infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. The results indicated the following composition: →6)-linked-β-d-Galp-(1→, →6)-linked-α-d-Glcp-(1→, →3)-linked-α-d-Glcp-(1→, and →2,4)-linked-β-d-Glcp-(1→, with terminal 6-deoxy-α-d-Glcp and α-d-Glcp. ACP2 alleviated lipopolysaccharide-induced hepatocyte inflammation by down-regulating the expressions of COX-2, IL-1β, TNF-α and IL-6. The decreased expressions of TLR4, MyD88, NF-κB, and phosphorylated p38 in ACP2-treated L02 cells indicated that ACP2 might ameliorate inflammation through the TLR4 and p38/NF-κB signaling pathways.

A previously undescribed polysaccharide ACP2 was isolated from Antrodia camphorata mycelium. ACP2 ameliorated hepatocyte inflammation through TLR4 and p38/NF-κB signal pathway.     

Retracted Article: Anti-inflammatory potency of Locusta migratoria manilensis cyclopeptides in mast cells and macrophages

Locusts are esteemed as a traditional Chinese medicine, as well as tonic foods in Asian countries. While searching for natural anti-inflammatory agents in natural products, we isolated four novel locust cyclopeptides (LCPs) and the results show that [cyclo-(Trp-Leu-His-Thr)]∼LCP-3 has potent anti-inflammatory potency in RAW264.7 and HMC-1 cells under LPS (lipopolysaccharide) stimuli. Furthermore, mechanistic studies show that LCP-3 attenuates pro-inflammatory cytokine (TNF-α, IL-6, IL-1β, NO and PGE2) expression. Moreover, LCP-3 attenuates inflammatory damage associated with the direct inhibition of iNOS and COX-2 expression. LCP-3 also regulates the MAPK, PI3K/AKT and NF-κB pathways to attenuate LPS-induced damage. Of note, our study first reports the anti-inflammatory potency of LCPs and elucidates their underlying molecular mechanisms.

Locusts are esteemed as a traditional Chinese medicine, as well as tonic foods in Asian countries.     

A simple and robust reporter gene assay for measuring the bioactivity of anti-RANKL therapeutic antibodies

RANKL (receptor activator of nuclear factor κB ligand) plays a key role in the differentiation, activation and survival of osteoclasts. Denosumab, which targets RANKL, is approved for osteoporosis or bone loss that has a high risk for fracture and bone metastases from solid tumors. Bioactivity determination is essential for the safety and efficacy of therapeutic antibodies. At present, the mechanism of action (MOA) based bioassay for anti-RANKL monoclonal antibodies (mAbs) is the measurement of tartrate resistant acid phosphatase (TRAP) activity, which takes about five days and has complex operation and relatively high variation. In this study, we developed a reporter gene assay (RGA) based on a RAW264.7 cell line stably expressing luciferase reporter under the control of nuclear factor-κB (NF-κB) response elements. After optimizing the key parameters, the validation results based on ICH-Q2 not only show superior specificity, precision, linearity, accuracy and passage stability, but also a short duration and simple operation. These results demonstrate the RGA based on the RANKL–RANK–NF-κB pathway can be an excellent alternative for measuring the bioactivity of anti-RANKL mAbs.

The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-RANKL mAbs.     

Retracted Article: PVT1 knockdown alleviates vancomycin-induced acute kidney injury by targeting miR-124 via inactivation of NF-κB signaling

Acute kidney injury (AKI) is a serious threat for human health and life. High dose of vancomycin (VAN) can give rise to AKI. The roles and molecular basis of long noncoding RNA plasmacytoma variant translocation 1 (PVT1) in VAN-induced AKI have been poorly defined till now. Protein levels of p65, phosphorylated p65 (p-p65), NF-κB inhibitor alpha (IκBα), phosphorylated IκBα (p-IκBα), Bcl-2 and Bax were measured by western blot assay. RNA levels of PVT1 and microRNA-124 (miR-124) were determined by RT-qPCR assay. HK-2 cell apoptosis was detected by an Annexin V-FITC apoptosis detection assay. Kidney functions were assessed by blood urea nitrogen (BUN) level, serum creatinine (Scr) level, histopathologic analysis, and TUNEL assay. Bioinformatical analysis, luciferase reporter assay, RIP and RNA pull down assays were performed to explore whether PVT1 could interact with miR-124. PVT1 was highly expressed in VAN-induced AKI models. Functional analysis revealed that PVT1 knockdown ameliorated VAN-induced AKI in vivo. Further exploration manifested that PVT1 directly interacted with miR-124. Moreover, the silencing of PVT1 abated VAN-induced HK-2 cell apoptosis in vitro, while this effect was reversed by the miR-124 inhibitor. Also, VAN treatment resulted in the reduction of miR-124 expression and the activation of NF-κB signaling in HK-2 cells. The inhibition of NF-κB alleviated VAN-induced HK-2 cell apoptosis. PVT1 activated NF-κB signaling by targeting miR-124 in VAN-induced HK-2 cells. PVT1 knockdown lessened VAN-induced AKI by targeting miR-124 via inactivating the NF-κB signaling, elucidating the critical roles and molecular basis of PVT1 in VAN-induced AKI and highlighting the diagnostic and therapeutic values of PVT1 in AKI.

PVT1 was highly expressed in the kidneys of VAN-induced AKI mice.