Translocation Of NF-κB Research Articles

USP10 regulates macrophage inflammation responses via stabilizing NEMO in LPS-induced sepsis.

Sepsis is a systemic inflammatory response syndrome characterized by persistent inflammation and immunosuppression, leading to septic shock and multiple organ dysfunctions. Ubiquitin-specific peptidase 10 (USP10), a deubiquitinase enzyme, plays a vital role in cancer and arterial restenosis, but its involvement in sepsis is unknown. In this study, we investigated the significance of USP10 in lipopolysaccharide (LPS)-stimulated macrophages and its biological roles in LPS-induced sepsis. Lipopolysaccharides (LPS) were used to establish sepsis models in vivo and in vitro. We use western blot to identify USP10 expression in macrophages. Spautin-1 and USP10-siRNA were utilized for USP10 inhibition. ELISA assays were used to assess for TNF-α and IL-6 in vitro and in vivo. Nuclear and cytoplasmic protein extraction and Confocal microscopy were applied to verify the translocation of NF-κB. Mechanically, co-immunoprecipitation and rescue experiments were used to validate the regulation of USP10 and NEMO. In macrophages, we found that LPS induced USP10 upregulation. The inhibition or knockdown of USP10 reduced the pro-inflammatory cytokines TNF-α and IL-6 and suppressed LPS-induced NF-κB activation by regulating the translocation of NF-κB. Furthermore, we found that NEMO, the regulatory subunit NF-κB essential modulator, was essential for the regulation of LPS-induced inflammation by USP10 in macrophages. NEMO protein evidently interacted with USP10, whereby USP10 inhibition accelerated the degradation of NEMO. Suppressing USP10 significantly attenuated inflammatory responses and improved the survival rate in LPS-induced sepsis mice. Overall, USP10 was shown to regulate inflammatory responses by stabilizing the NEMO protein, which may be a potential therapeutic target for sepsis-induced lung injury.

Standardized extract of Glehnia Littoralis abrogates memory impairment and neuroinflammation by regulation of CREB/BDNF and NF-κB/MAPK signaling in scopolamine-induced amnesic mice model

Mild cognitive impairment is a typical symptom of early Alzheimer’s disease (AD). Glehnia littoralis (G. littoralis), a medicinal halophyte plant commonly used to treat strokes, has been shown to possess some therapeutic qualities. In this study, we investigated the neuroprotective and anti-neuroinflammatory effects of a 50% ethanol extract of G. littoralis (GLE) on lipopolysccharide (LPS)-stimulated BV-2 cells and scopolamine-induced amnesic mice. In the in vitro study, GLE treatment (100, 200, and 400 µg/mL) markedly attenuated the translocation of NF-κB to the nucleus concomitantly with the significant mitigation of the LPS-induced production of inflammatory mediators, including NO, iNOS, COX-2, IL-6, and TNF-α. In addition, the GLE treatment suppressed the phosphorylation of MAPK signaling in the LPS-stimulated BV-2 cells. In the in vivo study, mice were orally administered with the GLE (50, 100, and 200 mg/kg) for 14 days, and cognitive loss was induced via the intraperitoneal injection of scopolamine (1 mg/kg) from 8 to 14 days. We found that GLE treatment ameliorated memory impairment and simultaneously improved memory function in the scopolamine-induced amnesic mice. Correspondingly, GLE treatment significantly decreased the AChE level and upregulated the protein expression of neuroprotective markers, such as BDNF and CREB, as well as Nrf2/HO-1 and decreased the levels of iNOS and COX-2 in the hippocampus and cortex. Furthermore, GLE treatment attenuated the increased phosphorylation of NF-κB/MAPK signaling in the hippocampus and cortex. These results suggest that GLE has a potential neuroprotective activity that may ameliorate learning and memory impairment by regulating AChE activity, promoting CREB/BDNF signaling, and inhibiting NF-κB/MAPK signaling and neuroinflammation.

Anti-inflammatory, anti-proliferative and anti-psoriatic potential of apigenin in RAW 264.7 cells, HaCaT cells and psoriasis like dermatitis in BALB/c mice

AimsPsoriasis is an immune-mediated skin disease characterized by keratinocytes hyperproliferation, abnormal differentiation and inflammation. Therefore, this study aimed to investigate in-vitro and in-vivo anti-inflammatory and anti-proliferative activity to evaluate anti-psoriatic potential of apigenin. Main methodsFor in-vivo study, 5 % imiquimod cream was used to induce psoriasis-like skin inflammation in BALB/c mice to mimic human psoriatic conditions. PASI score, CosCam score, histopathology, immunohistochemistry, qRT-PCR, and ELISA were done to evaluate the anti-psoriatic potential of topically applied apigenin. For in-vitro studies, LPS-induced inflammation in RAW 264.7 was done, and qRT-PCR, ELISA, and immunofluorescence were conducted to evaluate the anti-inflammatory activity of apigenin. Migration and cell doubling assay in HaCaT cells were performed to assess the anti-proliferative effect of apigenin. Acute dermal toxicity profile of apigenin has also been done as per OECD guidelines. Key findingsResults showed that apigenin significantly reduce the PASI and CosCam scores, ameliorate the deteriorating histopathology, and effectively downregulated the expression of CCR6, IL-17A, and NF-κB. Apigenin effectively downregulated the expression and secretion of pro-inflammatory cytokines through IL-23/IL-17/IL-22 axis. Apigenin suppressed nuclear translocation of NF-κB in LPS-induced RAW 264.7 cells. Cell migration and cell doubling assay in HaCaT cells showing the anti-proliferative potential of apigenin and it was found safe in acute dermal toxicity study. SignificanceApigenin was found effective against psoriasis in both in-vitro and in-vivo models suggesting apigenin as a potential candidate for the development of anti-psoriatic agent.

Review of Small Molecule Anticancer NF-κB Inhibitors

Cancer is one of the most common diseases in the world. Th e transcription factor NF-κB plays a key role in various physiological processes including immune response, cell proliferation, cell apoptosis and inflammation. Due to participation of NF-kappa B signaling pathways in carcinogenesis, angiogenesis, and tumor resistance to chemo- and radiotherapy, the factor is considered to be the ideal target for pharmacological treatment of cancer. Th e paper presents a literature review of the RSCI, PubMed, Scopus, Web of Science, Chemical Abstracts NCCN databases and other open access data. The carcinogenesis inhibitors were selected from all NF-κB inhibitors found. Inhibitors with antitumor activity of analytical interest include Bruton tyrosine kinase (BTK) inhibitors, cellular inhibitors of apoptosis proteins (c-IAP), proteasome inhibitors, and one inhibitor of NF-κB translocation to the nucleus. Th e authors analyzed products of diff erent development stages with recorded antitumor activity to varying degrees. NF-κB inhibitors are promising drug candidates, but since NF-κB is involved in most biological processes and the broad spectrum of action can cause side eff ects, the selective action of these compounds is to be explored.

Acyclic retinoid peretinoin reduces hemorrhage-associated brain injury in vitro and in vivo

Peretinoin is an acyclic retinoid that stimulates retinoic acid receptors (NR1Bs) and produces therapeutic effects on hepatocellular cancer. We have previously shown that NR1B agonists such as Am80 and all trans-retinoic acid suppress pathogenic events in intracerebral hemorrhage. The present study addressed the actions of peretinoin and Am80 against cytotoxicity of a blood protease thrombin on cortico-striatal slice cultures obtained from neonatal rat brains. Application of 100 U/ml thrombin to the slice cultures for 72 h caused cell death in the cortical region and tissue shrinkage in the striatal region. Peretinoin (50 μM) and Am80 (1 μM) counteracted these cytotoxic effects of thrombin, and the effect of peretinoin and Am80 was blocked by LE540, an NR1B antagonist. A broad-spectrum kinase inhibitor K252a (3 μM) attenuated the cytoprotective effect of peretinoin in the cortical region, whereas a specific protein kinase A inhibitor KT5720 (1 μM) attenuated the protective effect of peretinoin in the cortical and the striatal regions. On the other hand, nuclear factor-κB (NF-κB) inhibitors such as pyrrolidine dithiocarbamate (50 μM) and Bay11-7082 (10 μM) prevented thrombin-induced shrinkage of the striatal region. Peretinoin and Am80 as well as Bay11-7082 blocked thrombin-induced nuclear translocation of NF-κB in striatal microglia and loss of striatal neurons. We also found that daily administration of peretinoin reduced histopathological injury and alleviated motor deficits in a mouse model of intracerebral hemorrhage. These results indicate that NR1B agonists including peretinoin may serve as a therapeutic option for hemorrhagic brain injury.

Discovery of an effective anti-inflammatory agent for inhibiting the activation of NF-κB

In this study, based on the effect of compounds on the activation of NF-κB and NO release, compound 51 was discovered as the best one with NO release inhibition IC50 value was 3.1 ± 1.1 μM and NF-κB activity inhibition IC50 value was 172.2 ± 11.4 nM. Compound 51 could inhibit the activation of NF-κB through suppressing phosphorylation and nuclear translocation of NF-κB, and suppress LPS-induced inflammatory response in RAW264.7 cells, such as the over-expression of TNF-α and IL-6, which were target genes of NF-κB. This compound also showed preferable anti-inflammatory activity in vivo, including alleviating significantly gastric distention and splenomegaly caused by LPS stimulation, reducing the level of oxidative stress induced by LPS, and inhibiting the expression of IL-6 and TNF-α in serum. Thus, it’s reasonable to consider that this compound is a promising small molecule with anti-inflammatory effect for inhibiting the NF-κB signalling pathway.

Dietary flavonoid isoliquiritigenin inhibits microglia-mediated neuroinflammation in Parkinson's disease models via JNK/NFκB/AKT signaling pathway

Isoliquiritigenin (ISL) is a naturally-occurring flavonoid with numerous pharmacological properties such as anti-inflammation, yet its role in Parkinson’s disease (PD) with microglia-mediated neuroinflammation remains unknown. In this study, the effects of ISL on inhibiting microglia-mediated neuroinflammation in PD were evaluated in the 1-methyl-4-phenylpyridinium (MPTP)-induced mouse model of PD and the lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Our results showed that ISL had the capability to prevent behavioral deficits and the excessive microglial activation in MPTP-treated mice. Besides that, ISL was able to reduce MPTP-mediated neuronal cell death in the substantia nigra and striatum of mice. In addition, ISL was found to prevent the elevation of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and mitigate the phosphorylation of c-Jun N-terminal protein kinase (JNK), protein kinase B (AKT), nuclear factor kappa light-chain enhancer of activated B cells (NFκB) as well as inhibitor of NFκB protein ɑ in substantia nigra and striatum of MPTP-treated mice. In LPS-stimulated BV-2 cells, ISL inhibited production of inflammatory mediators such as interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), suppressed the phosphorylation and nuclear translocation of NFκB, and inhibited the activation of JNK and AKT signaling. Moreover, the agonists of JNK partly abolished the inhibitory effects of ISL in LPS-treated BV-2 cells. ISL inhibits microglia-mediated neuroinflammation in PD models probably through deactivating JNK/NFκB/AKT signaling pathway. The novel findings suggest the therapeutic potential of ISL for microglia-mediated neuroinflammation in PD.

Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

Cbf-14, a cationic peptide derived from cathelin-domain, exhibits anti-inflammation activity via inhibiting PI3K- Akt /ROS/ NF-κB signaling pathway

Cbf-14 with the sequence RLLRKFFRKLKKSV, is an effective antimicrobial peptide derived from a cathelin-like domain. Previous reports have demonstrated that Cbf-14 not only exerts antimicrobial activity against penicillin-resistant bacteria but also alleviates bacterial-induced inflammation in E. coli BL21 (DE3)-NDM-1-infected mice. In this article, we demonstrated that Cbf-14 can effectively reduce RAW 264.7 intracellular infection caused by clinical strain E. coli and alleviate the inflammatory response of cells and improve cell survival after infection. Therefore, we established the LPS-stimulated RAW 264.7 cell inflammation model to uncover the molecular mechanisms of the peptide Cbf-14 in anti-inflammatory activity. The results reveal that Cbf-14 can decrease LPS-induced ROS secretion by blocking the membrane translocation of p47-phox subunits and suppressing p47-phox protein phosphorylation. Meanwhile, this peptide can down-regulate the over-expression of iNOS, and finally inhibit the NO excessive secretion from RAW 264.7 macrophages stimulated by LPS. Moreover, Cbf-14 also down-regulates the expression levels of p-IκB and p-p65 and inhibits the nuclear translocation of NF-κB through blocking MAPK- and/or PI3K-Akt signaling pathways. Overall, Cbf-14 exhibits anti-inflammatory activity through inhibiting NF-κB activity and ROS production via PI3K- Akt signaling pathway.

Anti-inflammatory effects of 6S-5-methyltetrahydrofolate‐calcium on RAW264.7 cells and zebrafish

Aim6S-5-methyltetrahydrofolate is the predominant form of dietary folate in circulation and is used as a crystalline form of calcium salt (MTHF-Ca). Reports revealed that MTHF-Ca was more safe than folic acid, a synthetic and highly stable version of folate. Folic acid has been reported to have anti-inflammatory effects. The study's objective was to assess the anti-inflammatory effect of MTHF-Ca in vitro and in vivo. Main methodsIn vitro, the ROS production was assessed by H2DCFDA, and nuclear translocation of NF-κB were evaluated by the NF-κB nuclear translocation assay kit. Interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) were assessed using ELISA. In vivo, ROS production was assessed by H2DCFDA, neutrophils and macrophages recruitment were evaluated in tail transection-induced and CuSO4-induced zebrafish inflammation models. Expression of inflammation related genes were also investigated based on CuSO4-induced zebrafish inflammation model. Key findingsMTHF-Ca treatment decreased LPS-induced ROS production, inhibited nuclear translocation of NF-κB and decreased the levels of IL-6, IL-1β and TNF-α in RAW264.7 cells. In addition, MTHF-Ca treatment inhibited ROS production, suppressed the recruitment of neutrophils and macrophages, and reduced the expression of inflammation related genes, including jnk, erk, nf-κb, myd88, p65, tnf-α, and il-1b in zebrafish larvae. SignificanceMTHF-Ca may play an anti-inflammatory role by reducing the recruitment of neutrophils and macrophages and keeping the low levels of proinflammatory mediators and cytokines. MTHF-Ca may have a potential role in the treatment of inflammatory diseases.

Tocilizumab suppresses NF-kappa B activation via toll-like receptor 9 signaling by reducing cell-free DNA in rheumatoid arthritis.

Endogenous DNA is released into the bloodstream as cell-free DNA (cfDNA) following cell death and is associated with various pathological conditions. However, their association with therapeutic drugs against rheumatoid arthritis (RA) remains unknown. Therefore, we investigated the significance of cfDNA in RA treated with tocilizumab and tumour necrosis factor inhibitor (TNF-I). Biological DMARDs (bDMARDs), including tocilizumab and TNF-I, were administered to 77 and 59 RA patients, respectively. Plasma cfDNA levels were measured at weeks 0, 4, and 12 by quantitative polymerase chain reaction. Disease activity was evaluated at the same time point using DAS28ESR. cfDNA levels from RA synovial cells treated with tocilizumab or etanercept for 24 h were measured. Human toll-like receptor 9 (hTLR9)-expressing HEK293 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon NF-κB activation, were stimulated by cfDNA from RA patients, and subsequently, SEAP levels were determined. NF-κB translocation was evaluated by immunofluorescence staining with or without tocilizumab. The DAS28ESR significantly improved in both bDMARD groups at week 12. However, plasma cfDNA levels significantly decreased in the tocilizumab group at week 12 compared to that in week 0. cfDNA levels correlated with DAS28ESR in biological treatment-naïve patients administered tocilizumab. cfDNA levels in synovial cells were significantly suppressed by tocilizumab treatment and unaltered with etanercept. HEK293 cells released SEAP upon cfDNA stimulation, and the observed NF-κB nuclear translocation was suppressed by tocilizumab. Tocilizumab suppressed inflammation via the TLR9 pathway by decreasing cfDNA levels. Regulation of cfDNA may be a therapeutic target for RA.

Bioefficacy of Nga-Mon (Perilla frutescens) Fresh and Dry Leaf: Assessment of Antioxidant, Antimutagenicity, and Anti-Inflammatory Potential.

Perilla leaves are known to be a rich source of polyphenols, which have been shown to exhibit various biological effects. This study aimed to compare the bioefficacies and bioactivities of fresh (PLEf) and dry (PLEd) Thai perilla (Nga-mon) leaf extracts. Phytochemical analysis indicated that both PLEf and PLEd were abundant in rosmarinic acid and bioactive phenolic compounds. PLEd, which had higher levels of rosmarinic acid but lower levels of ferulic acid and luteolin than PLEf, exhibited greater effectiveness in a free radical scavenging assay. Furthermore, both extracts were found to suppress intracellular ROS generation and exhibit antimutagenic activity against food-borne carcinogens in S. typhimurium. They also attenuated lipopolysaccharide-induced inflammation in RAW 264.7 cells by inhibiting the expression of nitric oxide, iNOS, COX-2, TNF-α, IL-1β, and IL-6 through the suppression of NF-κB activation and translocation. However, PLEf exhibited a higher ability to suppress cellular ROS production and higher antimutagenic and anti-inflammatory activities than PLEd, which can be attributed to its combination of phytochemical components. Overall, PLEf and PLEd have the potential to serve as natural bioactive antioxidant, antimutagenic, and anti-inflammatory agents to achieve potential health benefits.

Fluoxetine inactivates STAT3/NF-κB signaling and promotes sensitivity to cisplatin in bladder cancer

Bladder cancer is known as one of the top ten most common cancer types worldwide and can be majorly divided into muscles invasive bladder cancer (MIBC) and non-muscles invasive type (NMIBC). However, the prognosis of BC remains poor under standard treatment including radical cystectomy or concurrent chemoradiotherapy. Numerous studies have reported that the prognosis of BC is associated with the activation of signal transducer and activator of transcription (STAT3) and nuclear factor kappa-B (NF-κB). Fluoxetine, a well-known anti-depressant, has been reported to against various type of cancers. However, it is unclear whether fluoxetine has the capacity to inhibit BC progression by targeting STAT3 and NF-κB-mediated signaling. Here, we used cell viability, apoptosis assay, wound healing assay, invasion/migration assay, Western blotting assay, immunofluorescence staining, as well as animal experiments, to elucidate the efficacy of fluoxetine on in vitro and in vivo BC models. We found that fluoxetine may induce cytotoxicity and intrinsic/extrinsic apoptosis in BC and enhance the potential of cisplatin. Fluoxetine promoted both caspase-dependent and caspase-independent apoptosis signaling by activating caspase-3, 8, 9, apoptosis-inducing factor (AIF), and EndG. Furthermore, fluoxetine suppressed invasion and migration ability and the expression of metastasis-associated genes. Fluoxetine was also found to inactivate the phosphorylation of STAT3 (Tyr705) and NF-κB (Ser536) and suppress the nuclear translocation of NF-κB. In MB49-bearing mice, fluoxetine effectively delayed the progression of BC without inducing general toxicity. In summary, the induction of apoptosis and the inhibition of invasion triggered by fluoxetine are associated with the inactivation of STAT3 and NF-κB.

Anti-inflammatory effects of Peucedanum japonicum Thunberg leaves extract in Lipopolysaccharide-stimulated RAW264.7cells.

Peucedanum japonicum Thunberg are perennial herbaceous plants known to be cultivated for food and traditional medicinal purposes. P. japonicum has been used in traditional medicine to soothe coughs and colds, and to treat many other inflammatory diseases. However, there are no studies on the anti-inflammatory effects of the leaves. Inflammation plays an important role in our body as a defense response of biological tissues to certain stimuli. However, the excessive inflammatory response can lead to various diseases. This study aimed to investigate the anti-inflammatory effects of P. japonicum leaves extract (PJLE) in LPS-stimulated RAW 264.7cells. Nitric Oxide (NO) production assay measured by NO assay. Inducible NO synthase (iNOS), COX-2, MAPKs, AKT, NF-κB, HO-1, Nrf-2 were examined by western blotting. PGE2, TNF-α, and IL-6 were analyzed by ELSIA. Nuclear translocation of NF-κB was detected by immunofluorescence staining. PJLE suppressed inducible nitric oxygen synthase (iNOS) and prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2, COX-2) expression, increased heme oxygenase 1 (HO-1) expression, and decreased nitric oxide production. And PJLE inhibited the phosphorylation of AKT, MAPK, and NF-κB. Taken together, PJLE down-regulated inflammatory factors such as iNOS and COX-2 by inhibiting the phosphorylation of AKT, MAPK, and NF-κB. These results suggest that PJLE can be used as a therapeutic material to modulate inflammatory diseases.

A p-Tyr42 RhoA Inhibitor Promotes the Regeneration of Human Corneal Endothelial Cells by Ameliorating Cellular Senescence.

The development of treatment strategies for human corneal endothelial cells (hCECs) disease is necessary because hCECs do not regenerate in vivo due to the properties that are similar to senescence. This study is performed to investigate the role of a p-Tyr42 RhoA inhibitor (MH4, ELMED Inc., Chuncheon) in transforming growth factor-beta (TGF-β)- or H2O2-induced cellular senescence of hCECs. Cultured hCECs were treated with MH4. The cell shape, proliferation rate, and cell cycle phases were analyzed. Moreover, cell adhesion assays and immunofluorescence staining for F-actin, Ki-67, and E-cadherin were performed. Additionally, the cells were treated with TGF-β or H2O2 to induce senescence, and mitochondrial oxidative reactive oxygen species (ROS) levels, mitochondrial membrane potential, and NF-κB translocation were evaluated. LC3II/LC3I levels were determined using Western blotting to analyze autophagy. MH4 promotes hCEC proliferation, shifts the cell cycle, attenuates actin distribution, and increases E-cadherin expression. TGF-β and H2O2 induce senescence by increasing mitochondrial ROS levels and NF-κB translocation into the nucleus; however, this effect is attenuated by MH4. Moreover, TGF-β and H2O2 decrease the mitochondrial membrane potential and induce autophagy, while MH4 reverses these effects. In conclusion, MH4, a p-Tyr42 RhoA inhibitor, promotes the regeneration of hCECs and protects hCECs against TGF-β- and H2O2-induced senescence via the ROS/NF-κB/mitochondrial pathway.

Estrogen-sensitive activation of SGK1 induces M2 macrophages with anti-inflammatory properties and a Th2 response at the maternal–fetal interface

BackgroundDecidual macrophages participate in immune regulation at the maternal–fetal interface. Abnormal M1/M2 polarization of decidual macrophages might predispose immune maladaptation in recurrent pregnancy loss (RPL). However, the mechanism of decidual macrophage polarization is unclear. We explored the role of Estradiol (E2)-sensitive serum-glucocorticoid regulated kinase (SGK) 1 in promoting macrophage polarization and suppressing inflammation at the maternal–fetal interface.MethodsWe assessed serum levels of E2 and progesterone during first trimester of pregnancy in women with or without threatened miscarriages (ended in live birth, n = 448; or early miscarriages, n = 68). For detection of SGK1 in decidual macrophages, we performed immunofluorescence labeling and western blot analysis applying decidual samples from RPL (n = 93) and early normal pregnancy (n = 66). Human monocytic THP-1 cells were differentiated into macrophages and treated with Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS), E2, inhibitors or siRNA for in vitro analysis. Flow cytometry analysis were conducted to detect macrophages polarization. We also applied ovariectomized (OVX) mice with hormones exploring the mechanisms underlying the regulation of SGK1 activation by E2 in the decidual macrophages in vivo.ResultsSGK1 expression down regulation in the decidual macrophages of RPL was consistent with the lower concentration and slower increment of serum E2 from 4 to 12 weeks of gestation seen in these compromised pregnancies. LPS reduced SGK1 activities, but induced the pro-inflammatory M1 phenotype of THP-1 monocyte-derived macrophages and T helper (Th) 1 cytokines that favored pregnancy loss. E2 pretreatment promoted SGK1 activation in the decidual macrophages of OVX mice in vivo. E2 pretreatment amplified SGK1 activation in TLR4-stimulated THP-1 macrophages in vitro through the estrogen receptor beta (ERβ) and PI3K pathway. E2-sensitive activation of SGK1 increased M2 macrophages and Th2 immune responses, which were beneficial to successful pregnancy, by inducing ARG1 and IRF4 transcription, which are implicated in normal pregnancy. The experiments on OVX mice have shown that pharmacological inhibition of E2 promoted nuclear translocation of NF-κB in the decidual macrophages. Further more, pharmacological inhibition or knockdown of SGK1 in TLR4-stimulated THP-1 macrophages activated NF-κB by promoting its nuclear translocation, leading to increased secretion of pro-inflammatory cytokines involved in pregnancy loss.ConclusionOur findings highlighted the immunomodulatory roles of E2-activated SGK1 in Th2 immune responses by priming anti-inflammatory M2 macrophages at the maternal–fetal interface, resulting in a balanced immune microenvironment during pregnancy. Our results suggest new perspectives on future preventative strategies for RPL.

JAC4 Alleviates Rotenone-Induced Parkinson's Disease through the Inactivation of the NLRP3 Signal Pathway.

Parkinson's disease (PD) is the fastest-growing neurodegeneration disease, characterized typically by a progressive loss of dopaminergic neurons in the substantia nigra, and there are no effective therapeutic agents to cure PD. Rotenone (Rot) is a common and widely used pesticide which can directly inhibit mitochondrial complex I, leading to a loss of dopaminergic neurons. Our previous studies proved that the JWA gene (arl6ip5) may play a prominent role in resisting aging, oxidative stress and inflammation, and JWA knockout in astrocytes increases the susceptibility of mice to 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. JWA-activating compound 4 (JAC4) is a small-molecule activator of the JWA gene, but its role in and mechanism against PD have not yet been clarified. In the present study, we showed that the JWA expression level is strongly related to tyrosine hydroxylase (TH) in different growth periods of mice. Additionally, we constructed models with Rot in vivo and in vitro to observe the neuroprotective effects of JAC4. Our results demonstrated that JAC4 prophylactic intervention improved motor dysfunction and dopaminergic neuron loss in mice. Mechanistically, JAC4 reduced oxidative stress damage by reversing mitochondrial complex I damage, reducing nuclear factor kappa-B (NF-κB) translocation and repressing nucleotide-binding domain, leucine-rich-containing family and pyrin domain-containing-3 (NLRP3) inflammasome activation. Overall, our results provide proof that JAC4 could serve as a novel effective agent for PD prevention.

Caffeic acid methyl ester inhibits mast cell activation through the suppresion of MAPKs and NF-κB signaling in RBL-2H3 cells

Anti-inflammatory effects of caffeic acid derivatives have been widely reported. However, the effect of caffeic acid methyl ester (CAME) on the anti-allergic effect in mast cells has not been elucidated. The present study was aimed to investigate the anti-allergic properties of CAME and its underlying mechanism. Rat basophilic leukemia (RBL-2H3) cells were incubated withphorbol-12-myristate-13-acetate (PMA) and a calcium ionophore, A23187 to induce mast cell activation. Anti-allergic effect of CAME was examined by measuring cytokine, histamine and β-hexosaminidase release. Western blotting was conducted to determine cyclooxygenase-2 (COX-2) expression, Mitogen-activated protein kinases (MAPKs) activation and nuclear factor-κB (NF-κB) translocation. CAME significantly suppressed PMA/A23187-induced TNF-α secretion, and β-hexosaminidase and histamine release in a concentration-dependent manner. Furthermore, CAME significantly attenuated PMA/A23187-induced COX-2 expression and nuclear translocation of NF-κB. CAME significantly suppressed PMA/A23187-induced increased phosphorylation of p38, ERK and JNK RBL-2H3 cells. The results demonstrate that CAME significantly attenuates anti-allergic action by suppressing degranulation of mast cells through the suppression of MAPKs/NF-κB signaling pathway in RBL-2H3 cells.

Polysaccharide from Abelmoschus esculentus L. flower ameliorates cyclophosphamide-induced immunosuppression via the TLR4/endocytosis and MAPK-NF-κB signaling pathways in mice

The present study was designed to investigate the immunomodulatory effects and mechanisms of a Abelmoschus esculentus L. flower polysaccharide (AEFP22) in the CTX-induced immunosuppressed mice. After intragastric gavage of AEFP22 for two weeks, nonspecific immunity, humoral immunity, cellular immunity and the status of immune organs were improved significantly in immunosuppressed mice. The immunoglobulins, cytokines and nitric oxide (NO) in serum also increased notably. Transcriptome analysis of the spleen showed that most immune-related differentially expressed genes (DEGs) were regulated inversely in the AEFP22-treated group and model group. Based on gene ontology (GO) analysis and Kyoto Encyclopedia of genes and genomes (KEGG) enrichment analysis of the transcriptome, the expression of key proteins in the toll-like receptor (TLR)4/endocytosis and mitogen-activated protein kinase (MAPK)-nuclear factor kappa B (NF-κB) signaling pathways were evaluated. The results showed that AEFP22 could ameliorate CTX-induced immunosuppression by increasing the expression of TLR4, myeloid differentiation primary response (MyD)88, TNF receptor associated factor (TRAF) 6 and major histocompatibility complex (MHC) class Ⅱ, enhance the phosphorylation of MAPKs and promote the nuclear translocation of NF-κB. This study indicated the immunoregulatory activity and potential mechanisms of AEFP22, which suggested the potential role of AEFP22 as an effective immunomodulator.

Size Effect of Silver Nanoparticles Derived from Olive Mill Wastewater in THP-1 Cell Lines

The constant demand of silver nanoparticles (AgNPs) for different applications requires a new selection of solvents and reagents for their synthesis, to make them less toxic to living organisms and the environment. Among the alternative technologies that can be used to exclude the use of toxic products, green chemistry is based on the employment of biomolecules derived from plants or microorganisms to achieve NPs. Therefore, with the aim of applying the principles of circular economy, the waste deriving from the production of olive oil represents a useful source of polyphenols to be used as reduction agents to obtain AgNPs. In our work, we employed the Olive Mill Wastewater (OMWW), the so-called vegetation water typical of the Mediterranean geographical area, to achieve two sizes of AgNPs, i.e., 50 nm and 30 nm. These NPs were tested on the human monocytic cell line (THP-1) using two concentrations (3 µM and 5 µM) to understand their ability to trigger or not the inflammatory response. This was undertaken following IL-6, IL-8, IL-5 and TNF-α secretion and the NF-kB translocation. We concluded that the AgNPs did not induce strong activation of these pathways, especially when the cells were treated with higher dimensional NPs. Consequently, the application of these NPs in vivo for therapeutic purpose could be significant.