The potential role of SNHG16/ miRNA-146a/ TRAF6 signaling pathway in the protective effect of zoledronate against colorectal cancer and associated osteoporosis in mouse model.

Bone fracture as a consequence of colorectal cancer (CRC) and associated osteoporosis (OP) is considered a risk factor for increasing the mortality rate among CRC patients. SNHG16/ miRNA-146a/ TRAF6 signaling pathway is a substantial contributor to neoplastic evolution, progression, and metastasis. Here, we investigated the effect of zoledronate (ZOL) on the growth of CRC and associated OP in a mouse model. Thirty Balb/c mice were divided into Naïve, azoxymethane (AOM)/dextran sodium sulfate (DSS), and ZOL groups. Body weight and small nucleolar RNA host gene 16 (SNHG16) expression, microRNA-146a, and TRAF6 in bone, colon, and stool were investigated. Samples of colon and bone were collected and processed for light microscopic, immunohistochemical staining for cytokeratin 20 (CK20), nuclear protein Ki67 (pKi-67), and caudal type homeobox transcription factor 2 (CDx2) in colon and receptor activator of nuclear factor kB (RANK) and osteoprotegerin (OPG) in bone. A computerized tomography (CT) scan of the femur and tibia was studied. ZOL produced a significant decrease in the expression of SNHG16 and TRAF6 and an increase in miRNA-146a in the colon and bone. ZOL administration improved the histopathological changes in the colon, produced a significant decrease in CK20 and Ki-67, and increased CDx2 expressions. In bone, ZOL prevented osteoporotic changes and tumour cell invasion produced a significant decrease in RANK and an increase in OPG expressions, alongside improved bone mineral density in CT scans. ZOL could be a promising preventive therapy against colitis-induced cancer and associated OP via modulation expression of SNHG16, miRNA-146a, and TRAF6.

Inhibition of the RLR signaling pathway by SARS-CoV-2 ORF7b is mediated by MAVS and abrogated by ORF7b-homologous interfering peptide.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and characterized by dysregulated immune response. Studies have shown that the SARS-CoV-2 accessory protein ORF7b induces host cell apoptosis through the tumor necrosis factor alpha (TNF-α) pathway and blocks the production of interferon beta (IFN-ß). The underlying mechanism remains to be investigated. In this study, we found that ORF7b facilitated viral infection and production, and inhibited the RIG-I-like receptor (RLR) signaling pathway through selectively interacting with mitochondrial antiviral-signaling protein (MAVS). MAVS439-466 region and MAVS Lys461 were essential for the physical association between MAVS and ORF7b, and the inhibition of the RLR signaling pathway by ORF7b. MAVSK461/K63 ubiquitination was essential for the RLR signaling regulated by the MAVS-ORF7b complex. ORF7b interfered with the recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) and the activation of the RLR signaling pathway by MAVS. Furthermore, interfering peptides targeting the ORF7b complex reversed the ORF7b-suppressed MAVS-RLR signaling pathway. The most potent interfering peptide V disrupts the formation of ORF7b tetramers, reverses the levels of the ORF7b-inhibited physical association between MAVS and TRAF6, leading to the suppression of viral growth and infection. Overall, this study provides a mechanism for the suppression of innate immunity by SARS-CoV-2 infection and the mechanism-based approach via interfering peptides to potentially prevent SARS-CoV-2 infection.IMPORTANCEThe pandemic coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and continues to be a threat to public health. It is imperative to understand the biology of SARS-CoV-2 infection and find approaches to prevent SARS-CoV-2 infection and ameliorate COVID-19. Multiple SARS-CoV-2 proteins are known to function on the innate immune response, but the underlying mechanism remains unknown. This study shows that ORF7b inhibits the RIG-I-like receptor (RLR) signaling pathway through the physical association between ORF7b and mitochondrial antiviral-signaling protein (MAVS), impairing the K63-linked MAVS polyubiquitination and its recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) to MAVS. The most potent interfering peptide V targeting the ORF7b-MAVS complex may reverse the suppression of the MAVS-mediated RLR signaling pathway by ORF7b and prevent viral infection and production. This study may provide new insights into the pathogenic mechanism of SARS-CoV-2 and a strategy to develop new drugs to prevent SARS-CoV-2 infection.

The crosstalk between macrophages and cancer cells potentiates pancreatic cancer cachexia.

With limited treatment options, cachexia remains a major challenge for patients with cancer. Characterizing the interplay between tumor cells and the immune microenvironment may help identify potential therapeutic targets for cancer cachexia. Herein, we investigate the critical role of macrophages in potentiating pancreatic cancer induced muscle wasting via promoting TWEAK (TNF-like weak inducer of apoptosis) secretion from the tumor. Specifically, depletion of macrophages reverses muscle degradation induced by tumor cells. Macrophages induce non-autonomous secretion of TWEAK through CCL5/TRAF6/NF-κB pathway. TWEAK promotes muscle atrophy by activating MuRF1 initiated muscle remodeling. Notably, tumor cells recruit and reprogram macrophages via the CCL2/CCR2 axis and disrupting the interplay between macrophages and tumor cells attenuates muscle wasting. Collectively, this study identifies a feedforward loop between pancreatic cancer cells and macrophages, underlying the non-autonomous activation of TWEAK secretion from tumor cells thereby providing promising therapeutic targets for pancreatic cancer cachexia.

TRAF6-mediated ubiquitination of AKT in the nucleus is a critical event underlying the desensitization of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of ß-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D2-like receptors (D2R, D3R, D4R), have suggested the existence of other desensitization mechanisms. The present study employed D2R/D3R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT. RESULTS: AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin ß1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of ß-Arr2. The deubiquitinated ß-Arr2 then forms a complex with Gßγ, which serves as a biomarker for GPCRs desensitization. Like in D3R, ubiquitination of AKT is also involved in the desensitization of ß2 adrenoceptors. CONCLUSION: Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression.

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

TRAF6 enhances PD-L1 expression through YAP1-TFCP2 signaling in melanoma.

Immunotherapy represented by programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. In this study, we performed a Stress and Proteostasis CRISPR interference screening to identify PD-L1 positive modulators. Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. As a non-conventional E3 ubiquitin ligase, TRAF6 is inclined to catalyze the synthesis and linkage of lysine-63 (K63) ubiquitin which is related to the stabilization of substrate proteins. Our results showed that suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in melanoma. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription. Inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent comparable to anti-PD-1 therapies with no obvious toxicity. Our findings reveal the potential of inhibiting TRAF6 to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

Investigating the Effects and Mechanisms of Cyclomorusin on Osteoclasts in a High Glucose Environment.

Diabetes mellitus is an endocrine disease characterized by prolonged hyperglycemia. Prolonged high blood sugar levels interfere with the differentiation and maturation process of OBs and OCs, leading to the onset of osteoporosis. However, OCs differentiation and maturation is a complex regulatory process. In this study, we used a co-culture system of RAW264.7 and MC3T3-E1 cells under HG concentration to explore the effect of CYM on OCs in a HG environment. The effects of CYM on the formation and function of OCs were observed using TRAP-positive cell counts and bone resorption pits. Then, mRNA and protein expression levels of OCs-related genes were detected by real-time qPCR and western blotting. The results showed that CYM had an inhibitory effect on OCs differentiation and bone resorption, reduced mRNAs expression of OCs-associated genes, and downregulated RANKL/RANK/TRAF6 pathway that mediates OCs differentiation. CYM could be a promising natural compound against diabetic osteoporosis.

Yigansan ameliorates maternal immune activation-induced autism-like behaviours by regulating the IL-17A/TRAF6/MMP9 pathway: Network analysis and experimental validation.

BACKGROUND: Maternal immune activation (MIA) is a significant factor inducing to autism spectrum disorder (ASD) in offspring. The fundamental principle underlying MIA is that inflammation during pregnancy impedes fetal brain development and triggers behavioural alterations in offspring. The intricate pathogenesis of ASD renders drug treatment effects unsatisfactory. Traditional Chinese medicine has strong potential due to its multiple therapeutic targets. Yigansan, composed of seven herbs, is one of the few that has been proven to be effective in treating neuro-psychiatric disorders among numerous traditional Chinese medicine compounds, but its therapeutic effect on ASD remains unknown. HYPOTHESIS: Yigansan improves MIA-induced ASD-like behaviours in offspring by regulating the IL-17 signalling pathway. METHODS: Pregnant C57BL/6J mice were intraperitoneally injected with poly(I:C) to construct MIA models and offspring ASD models. Network analysis identified that the IL-17A/TRAF6/MMP9 pathway is a crucial pathway, and molecular docking confirmed the binding affinity between the monomer of Yigansan and target proteins. qRT-PCR and Western blot were used to detect the expression levels of inflammatory factors and pathway proteins, immunofluorescence was used to detect the distribution of IL-17A, and behavioural tests were used to evaluate the ASD-like behaviours of offspring. RESULTS: We demonstrated that Yigansan can effectively alleviate MIA-induced neuroinflammation of adult offspring by regulating the IL-17A/TRAF6/MMP9 pathway, and the expression of IL-17A was reduced in the prefrontal cortex. Importantly, ASD-like behaviours have been significantly improved. Moreover, we identified that quercetin is the effective monomer for Yigansan to exert therapeutic effects. CONCLUSION: Overall, this study was firstly to corroborate the positive therapeutic effect of Yigansan in the treatment of ASD. We elucidated the relevant molecular mechanism and regulatory pathway involved, determined the optimal therapeutic dose and effective monomer, providing new solutions for the challenges of drug therapy for ASD.

Withaferin A alters the expression of microRNAs 146a-5p and 34a-5p and associated hub genes in MDA-MB-231 cells.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. Due to the absence of obvious therapeutic targets, microRNAs (miRNAs) provide possible hope to treat TNBC. Withaferin A (WA), a steroidal lactone, possesses potential anticancer activity with lesser side effects. The present study identifies hub genes (CDKN3, TRAF6, CCND1, JAK1, MET, AXIN2, JAG1, VEGFA, BRCA1, E2F3, WNT1, CDK6, KRAS, MYB, MYCN, TGFßR2, NOTCH1, SIRT1, MYCN, NOTCH2, WNT3A) from the list of predicted targets of the differentially expressed miRNAs (DEMs) in WA-treated MDA-MB-231 cells using in silico protein-protein interaction network analysis. CCND1, CDK6, and TRAF6 hub genes were predicted as targets of miR-34a-5p and miR-146a-5p, respectively. The study found the lower expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells, and further, it was observed that WA treatment effectively restored the lost expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells. An anti-correlation expression pattern was found among the miR-34a-5p and miR-146a-5p and the respective target hub genes in WA-treated TNBC cells. In conclusion, WA might exert anti-cancer effect in TNBC cells by inducing miR-34a-5p and miR-146a-5p expressions and decreasing CCND1, CDK6, and TARF6 target hub genes in TNBC cells.

Transcriptomic screening of novel targets of sericin in human hepatocellular carcinoma cells.

Sericin, a natural protein derived from Bombyx mori, is known to ameliorate liver tissue damage; however, its molecular mechanism remains unclear. Herein, we aimed to identify the possible novel targets of sericin in hepatocytes and related cellular pathways. RNA sequencing analysis indicated that a low dose of sericin resulted in 18 differentially expressed genes (DEGs) being upregulated and 68 DEGs being downregulated, while 61 DEGs were upregulated and 265 DEGs were downregulated in response to a high dose of sericin (FDR ≤ 0.05, fold change > 1.50). Functional analysis revealed that a low dose of sericin regulated pathways associated with the complement and coagulation cascade, metallothionine, and histone demethylate (HDMs), whereas a high dose of sericin was associated with pathways involved in lipid metabolism, mitogen-activated protein kinase (MAPK) signaling and autophagy. The gene network analysis highlighted twelve genes, A2M, SERPINA5, MT2A, MT1G, MT1E, ARID5B, POU2F1, APOB, TRAF6, HSPA8, FGFR1, and OGT, as novel targets of sericin. Network analysis of transcription factor activity revealed that sericin affects NFE2L2, TFAP2C, STAT1, GATA3, CREB1 and CEBPA. Additionally, the protective effects of sericin depended on the counterregulation of APOB, POU2F1, OGT, TRAF6, and HSPA5. These findings suggest that sericin exerts hepatoprotective effects through diverse pathways at different doses, providing novel potential targets for the treatment of liver diseases.

TIFAB regulates the TIFA-TRAF6 signaling pathway involved in innate immunity by forming a heterodimer complex with TIFA.

Nuclear factor κB (NF-κB) is activated by various inflammatory and infectious molecules and is involved in immune responses. It has been elucidated that ADP-ß-D-manno-heptose (ADP-Hep), a metabolite in gram-negative bacteria, activates NF-κB through alpha-kinase 1 (ALPK1)-TIFA-TRAF6 signaling. ADP-Hep stimulates the kinase activity of ALPK1 for TIFA phosphorylation. Complex formation between phosphorylation-dependent TIFA oligomer and TRAF6 promotes the polyubiquitination of TRAF6 for NF-κB activation. TIFAB, a TIFA homolog lacking a phosphorylation site and a TRAF6 binding motif, is a negative regulator of TIFA-TRAF6 signaling and is implicated in myeloid diseases. TIFAB is indicated to regulate TIFA-TRAF6 signaling through interactions with TIFA and TRAF6; however, little is known about its biological function. We demonstrated that TIFAB forms a complex not with the TIFA dimer, an intrinsic form of TIFA involved in NF-κB activation, but with monomeric TIFA. The structural analysis of the TIFA/TIFAB complex and the biochemical and cell-based analyses showed that TIFAB forms a stable heterodimer with TIFA, inhibits TIFA dimer formation, and suppresses TIFA-TRAF6 signaling. The resultant TIFA/TIFAB complex is a "pseudo-TIFA dimer" lacking the phosphorylation site and TRAF6 binding motif in TIFAB and cannot form the orderly structure as proposed for the phosphorylated TIFA oligomer involved in NF-κB activation. This study elucidated the molecular and structural basis for the regulation of TIFA-TRAF6 signaling by TIFAB.

Identification and Validation of the Pyroptosis-Related Hub Gene Signature and the Associated Regulation Axis in Diabetic Keratopathy.

Background: Diabetic keratopathy (DK) poses a significant challenge in diabetes mellitus, yet its molecular pathways and effective treatments remain elusive. The aim of our research was to explore the pyroptosis-related genes in the corneal epithelium of the streptozocin-induced diabetic rats. Methods: After sixteen weeks of streptozocin intraperitoneal injection, corneal epithelium from three diabetic rats and three normal groups underwent whole-transcriptome sequencing. An integrated bioinformatics pipeline, including differentially expressed gene (DEG) identification, enrichment analysis, protein-protein interaction (PPI) network, coexpression, drug prediction, and immune deconvolution analyses, identified hub genes and key drivers in DK pathogenesis. These hub genes were subsequently validated in vivo through RT-qPCR. Results: A total of 459 DEGs were screened out from the diabetic group and nondiabetic controls. Gene Set Enrichment Analysis highlighted significant enrichment of the NOD-like receptor, Toll-like receptor, and NF-kappa B signaling pathways. Intersection of DEGs and pyroptosis-related datasets showed 33 differentially expressed pyroptosis-related genes (DEPRGs) associated with pathways such as IL-17, NOD-like receptor, TNF, and Toll-like receptor signaling. A competing endogenous RNA network comprising 16 DEPRGs, 22 lncRNAs, 13 miRNAs, and 3 circRNAs was constructed. After PPI network, five hub genes (Nfkb1, Casp8, Traf6, Ptgs2, and Il18) were identified as upregulated in the diabetic group, and their expression was validated by RT-qPCR in streptozocin-induced rats. Immune infiltration characterization showed that diabetic corneas owned a higher proportion of resting mast cells, activated NK cells, and memory-resting CD4 T cells. Finally, several small compounds including all-trans-retinoic acid, Chaihu Shugan San, dexamethasone, and resveratrol were suggested as potential therapies targeting these hub genes for DK. Conclusions: The identified and validated hub genes, Nfkb1, Casp8, Traf6, Ptgs2, and Il18, may play crucial roles in DK pathogenesis and serve as therapeutic targets.

Effects of chronic Cr and Ni co-exposure on liver inflammation and autophagy in mice by regulating the TLR4/mTOR pathway.

Exposure to Cr and/or Ni can have widespread implications on the environment and health. However, the specific toxic effects of chronic Cr and Ni co-exposure on mice liver have not been reported. To ascertain the combined toxic effects of chronic Cr and Ni co-exposure on liver damage in mice, 80 6-week-old female C57BL/6 J mice were randomly divided into 4 groups: the Con group, Cr group (Cr+6 50 mg/L), Ni group (Ni+2 110 mg/L), and Cr + Ni group (Cr+6 50 mg/L + Ni+2 110 mg/L). The trial period lasted for 16 weeks. The results showed that Cr+6 and/or Ni+2 increased liver weight and liver index (P < 0.05) in mice, caused histological abnormality and ultrastructural damage, and micronutrients imbalance in mice liver. These findings serve as the basis for subsequent experiments. Compared with the individual exposure group, chronic Cr and Ni co-exposure resulted in decreased levels and activities of ALT, AST, MDA, T-AOC, and T-SOD (P < 0.05) in liver tissue, and decreased the mRNA expression levels of the TLR4/mTOR pathway related factors (TLR4, TRAM, TRIF, TBK-1, IRF-3, MyD88, IRAK-4, TRAF6, TAK-1, IKKß, NF-κB, IL-1ß, IL-6, TNFα, ULK1, Beclin 1, LC3) (P < 0.05) and decreased the protein expression levels of the factors (TLR4, MyD88, TRAF6, NF-κB p50, IL-6, TNFα, ULK1, LC3II/LC3I) (P < 0.05). Moreover, factorial analysis revealed the interaction between Cr and Ni, which was manifested as antagonistic effects on Cr concentration, Ni concentration, and TLR4, MyD88, NF-κB, mTOR, LC3, and p62 mRNA expression levels. In conclusion, the TLR4/mTOR pathway as a mechanism through which chronic Cr and Ni co-exposure induce liver inflammation and autophagy in mice, and there was an antagonistic effect between Cr and Ni. The above results provided a theoretical basis for understanding the underlying processes.

Gender-related variation expressions of neuroplastin TRAF6, GluA1, GABA(A) receptor, and PMCA in cortex, hippocampus, and brainstem in an experimental epilepsy model.

Epileptic seizures are seen as a result of changing excitability balance depending on the deterioration in synaptic plasticity in the brain. Neuroplastin, and its related molecules which are known to play a role in synaptic plasticity, neurotransmitter activities that provide balance of excitability and, different neurological diseases, have not been studied before in epilepsy. In this study, a total of 34 Sprague-Dawley male and female rats, 2 months old, weighing 250-300 g were used. The epilepsy model in rats was made via pentylenetetrazole (PTZ). After the completion of the experimental procedure, the brain tissue of the rats were taken and the histopathological changes in the hippocampus and cortex parts and the brain stem were investigated, as well as the immunoreactivity of the proteins related to the immunohistochemical methods. As a result of the histopathological evaluation, it was determined that neuron degeneration and the number of dilated blood vessels in the hippocampus, frontal cortex, and brain stem were higher in the PTZ status epilepticus (SE) groups than in the control groups. It was observed that neuroplastin and related proteins TNF receptor-associated factor 6 (TRAF6), Gamma amino butyric acid type A receptors [(GABA(A)], and plasma membrane Ca2+ ATPase (PMCA) protein immunoreactivity levels increased especially in the male hippocampus, and only AMPA receptor subunit type 1 (GluA1) immunoreactivity decreased, unlike other proteins. We believe this may be caused by a problem in the mechanisms regulating the interaction of neuroplastin and GluA1 and may cause problems in synaptic plasticity in the experimental epilepsy model. It may be useful to elucidate this mechanism and target GluA1 when determining treatment strategies.

ASFV pA151R negatively regulates type I IFN production via degrading E3 ligase TRAF6.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly mortal and hemorrhagic infectious disease in pigs. Previous studies have indicated that ASFV modulates interferon (IFN) production. In this study, we demonstrated that ASFV pA151R negatively regulated type I IFN production. Ectopic expression of pA151R dramatically inhibited K63-linked polyubiquitination and Ser172 phosphorylation of TANK-binding kinase 1 (TBK1). Mechanically, we demonstrated that E3 ligase TNF receptor-associated factor 6 (TRAF6) participated in the ubiquitination of TBK1 in cGAS-STING signaling pathway. We showed that pA151R interacted with TRAF6 and degraded it through apoptosis pathway, leading to the disruption of TBK1 and TRAF6 interaction. Moreover, we clarified that the amino acids H102, C109, C132, and C135 in pA151R were crucial for pA151R to inhibit type I interferon production. In addition, we verified that overexpression of pA151R facilitated DNA virus Herpes simplex virus 1 (HSV-1) replication by inhibiting IFN-ß production. Importantly, knockdown of pA151R inhibited ASFV replication and enhanced IFN-ß production in porcine alveolar macrophages (PAMs). Our findings will help understand how ASFV escapes host antiviral immune responses and develop effective ASFV vaccines.

LXA4 protected mice from renal ischemia/reperfusion injury by promoting IRG1/Nrf2 and IRAK-M-TRAF6 signal pathways.

Excessive inflammatory response and increased oxidative stress play an essential role in the pathophysiology of ischemia/reperfusion (I/R)-induced acute kidney injury (IRI-AKI). Emerging evidence suggests that lipoxin A4 (LXA4), as an endogenous negative regulator in inflammation, can ameliorate several I/R injuries. However, the mechanisms and effects of LXA4 on IRI-AKI remain unknown. In this study, A bilateral renal I/R mouse model was used to evaluate the role of LXA4 in wild-type, IRG1 knockout, and IRAK-M knockout mice. Our results showed that LXA4, as well as 5-LOX and ALXR, were quickly induced, and subsequently decreased by renal I/R. LXA4 pretreatment improved renal I/R-induced renal function impairment and renal damage and inhibited inflammatory responses and oxidative stresses in mice kidneys. Notably, LXA4 inhibited I/R-induced the activation of TLR4 signal pathway including decreased phosphorylation of TAK1, p36, and p65, but did not affect TLR4 and p-IRAK-1. The analysis of transcriptomic sequencing data and immunoblotting suggested that innate immune signal molecules interleukin-1 receptor-associated kinase-M (IRAK-M) and immunoresponsive gene 1 (IRG1) might be the key targets of LXA4. Further, the knockout of IRG1 or IRAK-M abolished the beneficial effects of LXA4 on IRI-AKI. In addition, IRG1 deficiency reversed the up-regulation of IRAK-M by LXA4, while IRAK-M knockout had no impact on the IRG1 expression, indicating that IRAK-M is a downstream molecule of IRG1. Mechanistically, we found that LXA4-promoted IRG1-itaconate not only enhanced Nrf2 activation and increased HO-1 and NQO1, but also upregulated IRAK-M, which interacted with TRAF6 by competing with IRAK-1, resulting in deactivation of TLR4 downstream signal in IRI-AKI. These data suggested that LXA4 protected against IRI-AKI via promoting IRG1/Itaconate-Nrf2 and IRAK-M-TRAF6 signaling pathways, providing the rationale for a novel strategy for preventing and treating IRI-AKI.

Nicorandil-Pretreated Mesenchymal Stem Cell-Derived Exosomes Facilitate Cardiac Repair After Myocardial Infarction via Promoting Macrophage M2 Polarization by Targeting miR-125a-5p/TRAF6/IRF5 Signaling Pathway.

Background: Exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) have been considered as a promising cell-free therapeutic strategy for ischemic heart disease. Cardioprotective drug pretreatment could be an effective approach to improve the efficacy of MSC-exo. Nicorandil has long been used in clinical practice for cardioprotection. This study aimed to investigate whether the effects of exosomes derived from nicorandil pretreated MSC (MSCNIC-exo) could be enhanced in facilitating cardiac repair after acute myocardial infarction (AMI). Methods: MSCNIC-exo and MSC-exo were collected and injected into the border zone of infarcted hearts 30 minutes after coronary ligation in rats. Macrophage polarization was detected 3 days post-infarction, cardiac function as well as histological pathology were measured on the 28th day after AMI. Macrophages were separated from the bone marrow of rats for in vitro model. Exosomal miRNA sequencing was conducted to identify differentially expressed miRNAs between MSCNIC-exo and MSC-exo. MiRNA mimics and inhibitors were transfected to MSCs or macrophages to explore the specific mechanism. Results: Compared to MSC-exo, MSCNIC-exo showed superior therapeutic effects on cardiac functional and structural recovery after AMI and markedly elevated the ratio of CD68+ CD206+/ CD68+cells in infarcted hearts 3 days post-infarction. The notable ability of MSCNIC-exo to promote macrophage M2 polarization was also confirmed in vitro. Exosomal miRNA sequencing and both in vivo and in vitro experiments identified and verified that miR-125a-5p was an effector of the roles of MSCNIC-exo in vivo and in vitro. Furthermore, we found miR-125a-5p promoted macrophage M2 polarization by inhibiting TRAF6/IRF5 signaling pathway. Conclusion: This study suggested that MSCNIC-exo could markedly facilitate cardiac repair post-infarction by promoting macrophage M2 polarization by upregulating miR-125a-5p targeting TRAF6/IRF5 signaling pathway, which has great potential for clinical translation.

Gastrodin regulates the TLR4/TRAF6/NF-κB pathway to reduce neuroinflammation and microglial activation in an AD model.

BACKGROUND: Gastrodia elata (Orchidaceae) is a medicinal plant used in traditional Chinese medicine. The rhizomes contain numerous active components, of which Gastrodin (p-hydroxymethylphenyl-B-D-glucopyranoside) forms the basis of the traditional medicine Gastrodiae Rhizoma. Gastrodin is also found in other medicinal plants and has neuroprotective, antioxidant, and anti-inflammatory effects. Neuroinflammation plays a crucial role in neurodegeneration. Research indicates that consuming meals and drinks containing Gastrodiaelata can enhance cognitive functioning and memory in elderly patients. The mechanisms relevant to the problem have not been completely understood. PURPOSE: The aim was to examine the in vivo and in vitro anti-neuroinflammatory effects of Gastrodin. STUDY DESIGN: The neuroprotective effects of Gastrodin on the TLR4/TRAF6/NF-κB pathway and Stat3 phosphorylation in LPS-treated C57BL/6 mice and BV-2 cells were investigated. METHODS: 1. C57BL/6 mice were assigned to model, gastrodin, donepezil, and control groups (n = 10 per group). The Gastrodin group received 100 mg/kg/d for five days, and the Dopenezil group 1.3 mg/kg/d. A neuroinflammation model was established by administering intraperitoneal injections of 2 mg/kg LPS to all groups, excluding the control. To induce microglial activation in Gastrodin-treated mouse microglial BV-2 cells, 1 µg/ml LPS was introduced for 24 h Morris water mazes were utilized to evaluate learning and spatial memory. Expression and subcellular localization of TLR4/TRAF6/NF-κB axis-related proteins and p-Stat3, Iba-1, GFAP, iNOS, and CD206 were assessed by immunofluorescence, western blots, and ELISA. qRT-PCR was performed to determine and measure IL-1ß, TNF-α, cell migration, and phagocytosis. Overexpression of TRAF6 was induced by transfection, and the effect of Gastrodin on IL-1ß and p-NF-κB p65 levels was assessed. RESULTS: 1. In mice, gastrodin treatment mitigated LPS-induced deficits in learning and spatial memory, as well as reducing neuroinflammation in the hippocampus, expression of TLR4/TRAF6/NF-κB pathway proteins, activation of microglia and astrocytes, and phosphorylation of Stat3. 2. Gastrodin pretreatment improved LPS-induced inflammation in vitro, reducing expression of TLR4/TRAF6/NF-κB-associated proteins and p-Stat3, inducing microglial transformation from M1 to M2, and inhibiting migration and phagocytosis. Overexpression of TRAF6 inhibited the Gastrodin-induced effects. CONCLUSION: Gastrodin suppresses neuroinflammation and microglial activation by modifying the TLR4/TRAF6/NF-κB pathway and Stat3 phosphorylation.

TGFßR1 inhibition drives hepatocellular carcinoma proliferation through induction of toll-like-receptor signalling.

Transforming growth factor (TGF)-ß and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFßR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFß1 cytokine or TGFßR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFßR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFßR1 kinase inhibition abolished the cytostatic effects of TGFß1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFß1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFßR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFß signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.

Triclosan induces liver injury in long-life exposed mice via activation of TLR4/NF-κB/NLRP3 pathway.

Triclosan (TCS) is a widely used synthetic, with broad-spectrum antibacterial properties found in both pharmaceuticals and personal care products. More specifically, it is hepatotoxic in rodents and exhibits differential effects in mice and humans. However, the mechanisms underlying TCS-induced liver toxicity have not been elucidated. This study examined the role of the toll-like receptor 4 (TLR4)/ nuclear factor kappa B (NF-κB)/ nod-like receptor protein 3 (NLRP3) pathway in TCS-exposed liver toxicity by established a long-life TCS-exposed mice liver injury model. The 24 C57BL/6 pregnant mice exposed to TCS (0, 50 and 100 mg/kg) every day during the gestation and nursing period. After weaning, the male mice were left to continue administrate with TCS until 8 weeks of age. Then, mice in each group were sacrificed for investigation. Long-life exposure to TCS resulted in a reduction of body weight in growth mice. TCS exposure caused the increase of serum ALT, AST and ALP. The situation of inflammatory cell infiltration, macrophage recruitment and collagen fiber deposition in TCS-exposed mice liver tissues were performed by histological analysis including hematoxylin-eosin, Masson, Sirius red, and immunohistochemistry staining. Protein expression levels in TLR4/NF-κB/NLRP3 pathway was measured through Western blot, and the NLRP3 inflammasome activation was measured using real-time quantitative PCR (RT-qPCR). The results showed that exposure to TCS elevated TLR4, myeloid differentiation factor 88 (Myd88), TNF receptor associated factor 6 (TRAF6), enhanced NF-κB activation, and affected NLRP3 inflammasome activation in mice liver. Collectively, these findings indicate that long-life exposure to TCS-induced mice by upregulating the TLR4-Myd88-TRAF6 pathway, activating the NF-κB signaling cascade, initiating the NLRP3 inflammasome pathway, and ultimately leading to liver injury, including inflammation, hepatocyte pyroptosis and hepatofibrosis. Henceforth, the TLR4/NF-κB/NLRP3 pathway may now provide a theoretical basis and valuable therapeutic targets for overcoming TCS-induced liver toxicity.