NF-κB Phosphorylation Research Articles

Morin Mitigates 5-Fluorouracil-Induced Nephrotoxicity by Activating Nrf2/HO-1 and FXR, and Suppressing ERK/VCAM-1 and NF-κB Pathways.

5-Fluorouracil (5-FU) is a DNA analog used in chemotherapy to treat various tumors. However, the clinical use of 5-FU is limited due to its severe adverse effects, particularly its nephrotoxicity. Morin (MRN) is a flavanol found in many different plants, including those in the Moraceae family, and has anti-inflammatory and antioxidant bioactivities. The protective effects of MRN against experimental 5-FU-induced kidney injury were investigated in this work. The rats were assigned to four groups in our study: control, MRN (50mg/kg), 5-FU (30mg/kg), and 5-FU+MRN. The administration of MRN caused a significant (P<0.05) decrease in the serum urea and creatinine levels and a reduction in the histopathological changes induced by 5-FU, as shown by H&E, PAS, and Sirius red staining. IHC shows that MRN attenuates renal oxidative stress induced by 5-FU via co-activation of Nrf2, HO-1, and FXR. MRN protects against renal inflammation induced by 5-FU, as evidenced by decreased TNF-α and IL-6 levels in the rat kidney mediated by the downregulation of the ERK1/2 and VCAM-1 proteins and decreased NF-κB phosphorylation as shown by Western blotting. These findings support using MRN as a novel and promising treatment for 5-FU-induced nephrotoxicity.

Icariside II Alleviates Chondrocyte Inflammatory Injury by Inhibiting the TNIP2/NF-κB Pathway.

Icariside II exerts protective effects against various diseases; however, its specific effects on osteoarthritis (OA) remain unclear. Therefore, in this study, we aimed to investigate the effects of icariside II in an in vitro model of OA and analyze its action mechanisms. We established an in vitro OA model by treating a human chondrocyte cell line (CHON-001) with interleukin (IL)-1β, followed by treatment with different concentrations of icariside II. Cell viability was measured using the methyl thiazolyl tetrazolium assay, and the level of lactate dehydrogenase (LDH) released from cells was determined using the appropriate kit. Tumor necrosis factor (TNF)-α, IL-6, and IL-8 levels were determined via enzyme-linked immunosorbent assay. Flow cytometry was used to assess apoptosis. Apoptosisrelated protein expression levels and TNFAIP3-interacting protein 2 (TNIP2)/nuclear factor (NF)-κB signaling pathway were analyzed via reverse transcription-quantitative polymerase chain reaction and western blotting. Furthermore, TNIP2-small interfering RNA (siRNA) was used to determine whether the TNIP2/NF-κB pathway influences the effects of icariside II on OA. Results indicated that Icariside II did not exert any significant toxic effects on CHON-001 cells. It inhibited IL-1β-induced apoptosis and increase in LDH levels and enhanced the inflammatory response. Additionally, icariside II reversed the IL-1β-induced decrease in TNIP2 levels and increase in NF-κB phosphorylation. TNIP2-siRNA revealed that the TNIP2/NF-κB signaling pathway influenced the alleviating effects of icariside II on OA. In conclusion, our results revealed that icariside II attenuated IL-1β-induced inflammatory injury in chondrocytes by increasing TNIP2 expression and inhibiting NF-κB pathway activation, highlighting its therapeutic potential for OA.

1,25(OH)2D3 induces chondrocyte autophagy and reduces the loss of proteoglycans in osteoarthritis through inhibiting the NF-κB pathway.

Nuclear transcription factor-κB (NF-κB) activation is a pivotal event in the pathogenesis of osteoarthritis (OA). OA patients frequently exhibit vitamin D (VD) deficiency, which is commonly associated with NF-κB activation. Our study aimed to investigate whether VD could protect against OA by modulating NF-κB pathway and to explore the underlying mechanisms. Proteins levels were assessed by western blot analysis, gene expression was quantified by quantitative real-time polymerase chain reaction (qRT‒PCR) in vivo and in vitro. The expression of phosphorylated-p65 (p-p65) in knee OA rats was detected by immunohistochemistry, and an NF-κB nuclear translocation assay was validated in chondrocytes. Immunoprecipitation was employed to detect the interaction between NF-κB and vitamin D receptor (VDR) in vivo and in vitro. Small interfering RNA (Si-NF-κB and Si-VDR) transfection was used to investigate the role of NF-κB and VDR signaling pathway in knee OA rats under VD influence. Cartilage changes were visualized of knee OA rats using hematoxylin and eosin as well as safranin-O/fast green of staining. Our findings indicated that VD alleviates OA by inhibiting NF-κBpathway, which in turn reduces chondrocyte apoptosis and extracellular matrix (ECM) degradation. Further analysis revealed that VD primarily stabilizes NF-κB through the interaction of VDR and NF-κB, modulating the AMPK/mTOR signaling pathway to enhance autophagy and delay the progression of OA. This study highlights the protective role of VD in OA by stabilization of NF-κB, mainly through the interaction between VDR and NF-κB. This interaction regulates the AMPK/mTOR signaling pathway, promoting autophagy and suggesting a potential therapeutic strategy for OA management. Key Points • VD confers a protective effect on OA by primarily stabilizing NF-κB through the interaction between VDR and NF-κB, which in turn inhibits NF-κB phosphorylation and nuclear translocation. • In chondrocytes, VD helps shield against OA by blocking NF-κB's entry into the nucleus, subsequently regulating autophagy via the AMPK/mTOR signaling pathway.

CYBC1 Drives Glioblastoma Progression via ROS and NF-κB Pathways.

This study aims to investigate the role of Cytochrome b-245 chaperone 1 (CYBC1) in glioblastoma (GBM) progression, focusing on its involvement in reactive oxygen species (ROS) production and associated signaling pathways. Understanding the molecular mechanisms driven by CYBC1 could provide new therapeutic targets and prognostic markers for GBM. Publicly available datasets were analyzed to assess CYBC1 expression in GBM and its correlation with patient survival. GBM cell lines were genetically manipulated using the CRISPR/Cas9 system to deplete CYBC1. The effects of CYBC1 deficiency on cell proliferation, migration, invasion, and cell cycle dynamics were experimentally evaluated. Additionally, the impact of CYBC1 on the expression of NOXA1, a subunit of NADPH oxidase, and downstream signaling pathways such as NF-κB was explored. CYBC1 expression was significantly elevated in GBM tissues and correlated with poor patient survival. CYBC1 deficiency in GBM cells resulted in reduced cell viability, migration, and invasion. Mechanistically, CYBC1 positively regulated NOXA1 expression, which in turn enhanced ROS production and activated the ERK·AKT/NF-κB pathways. The suppression of CYBC1 led to decreased ROS levels, reduced phosphorylation of NF-κB, and downregulation of genes involved in epithelial-mesenchymal transition. CYBC1 is implicated in GBM progression by regulating NOXA1-mediated ROS production and activating the ERK·AKT/NF-κB pathways. This study suggests that CYBC1 could serve as a potential therapeutic target and prognostic marker in GBM, warranting further investigation into its molecular mechanisms and therapeutic potential.

PS-MPs Induced Inflammation and Phosphorylation of Inflammatory Signalling Pathways in Liver.

As new pollutants, microplastics (MPs) have attracted much attention worldwide because they cause serious environmental pollution and pose potential health risks to humans. However, the toxic effects of MPs are still unclear. In this study, we analysed the inflammatory effects of 0.1 μm polystyrene microplastics (PS-MPs) on mouse and human liver cell lines. After 28 days of exposure to PS-MPs, the mice presented decreased liver index values and increased AST/ALT values. HL7702 and HepG2 were treated with PS-MPs for 24 h, and the cytotoxicity, the expression levels of inflammatory factors, and the phosphorylation of proteins in inflammation related pathways were confirmed. Compared with the control, the cell viability of these two cells significantly decreased after exposure to the PS-MPs at 1000 μm/cm2, and the BMD model also exhibited a similar dose. LDH leakage and AST also increased in a dose-dependent increase after PS-MPs exposure. The relative levels of chemokines such as GM-CSF, IL-6, IL-8, and IL-12p70 were significantly greater than those in the control. Furthermore, the PS-MPs can increase the expression levels of TLR4, MyD88, and NF-κB and activate the phosphorylation of NF-κB and STATs. Based on these results, exposure to PS-MPs can stimulate liver inflammation and activate the TLR4/MyD88/NF-κB and JAK-STAT pathways.

Roux-en-Y gastric bypass alleviates kidney inflammation and improves kidney function in db/db mice by activating TLCA/TGR5 pathway.

Diabetic kidney disease (DKD) is a severe diabetic microvascular complication featured by chronic low-grade inflammation. Roux-en-Y gastric bypass (RYGB) surgery has gained importance as a safe and effective surgery to treat DKD. Bile acids are significantly changed after RYGB, which brings a series of metabolic benefits, but the relationship with the improvement of DKD is unclear. Therefore, this study performed RYGB surgery on db/db mice to observe the beneficial effects of the surgery on the kidneys, and performed bile acid targeted metabolomics analysis to explore bile acid changes. We found that RYGB significantly reduced albuminuria in db/db mice, improved renal function, reversed renal structural lesions, and attenuated podocyte injury, inflammation. Notably, bile acid metabolomic analysis revealed taurolithocholic acid (TLCA) as the most significantly altered bile acid after RYGB. Further in vitro and in vivo validation experiments revealed that TLCA supplementation improved renal function and reduced renal inflammatory damage in db/db mice. In addition, TLCA inhibited high glucose-induced inflammatory damage in MPC-5 cells, and its mechanism of action may be related to activating Takeda G protein-coupled receptor 5 (TGR5), inhibiting NF-κB phosphorylation, and thus inhibiting inflammatory response. In conclusion, RYGB may play a protective role in the kidneys of diabetic mice by activating the TLCA/TGR5 pathway.

Analgesic effect of Dahuang Fuzi Decoction in neuropathic pain through inhibiting TNF-α and PI3K-AKT signaling.

Neuropathic pain (NeP) presents considerable challenges in terms of effective management and significantly impacts the quality of life for affected patients. The current treatment options for NeP are limited, highlighting the need for alternative therapeutic approaches. Dahuang Fuzi Decoction (DF), a formula from traditional Chinese medicine, has shown potential in relieving pain symptoms associated with various types of NeP. However, the mechanisms through which DF exerts its effects remain largely unknown. In this study, we employed ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) to analyze the chemical composition of DF. A chronic sciatic nerve compression injury (CCI) rat mode was used to assess the analgesic efficacy of DF for NeP. Network pharmacology analysis was performed to identify the potential signaling pathways affected by DF. DF treatment significantly increased the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in CCI rats, indicating its analgesic effect. Network pharmacology analysis suggested that DF potentially modulated TNF-α and PI3K-AKT signaling pathways. Furthermore, DF treatment decreased the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in spinal cord tissues of CCI rats, suggesting an anti-inflammatory effect. Western blot analysis revealed that DF treatment reduced the expression of TNF-α, TNFR1, and phosphorylated forms of PI3K, AKT, IKKα/β, IKBα, and NF-κB in the spinal cord of CCI rats. Immunofluorescence analysis confirmed significant reductions in TNF-α and TNFR1 expression, as well as in AKT and NF-κB phosphorylation within astrocytes following DF administration. Our findings characterize the chemical constituents of DF and elucidate its underlying mechanism for relieving NeP. The analgesic effect of DF involves the inhibition of TNF-α and PI3K-AKT signaling pathways, providing a potential therapeutic approach for NeP management.

An Anti-RAGE Chimeric antibody alleviates CCl4-induced liver fibrosis via RAGE/NF-kB pathway in mice

Liver fibrosis is a progressive condition characterized by excessive deposition of extracellular matrix components, leading to organ dysfunction. Chronic inflammation and activation of hepatic stellate cells (HSCs) are two dominant events in all stages of fibrosis development. The receptor for advanced glycation end products (RAGE) pathway is involved in modulating liver injury and fibrosis, and preventing it, or deletion of Ager gene can protect the liver against fibrosis progression. To investigate functions and mechanism of chimeric anti-RAGE monoclonal antibody against liver fibrosis, murine-derived monoclonal anti-RAGE antibodies were used to construct murine-human chimeric antibodies. The properties of the chimeric antibody were characterized, and the biological functions of antibody A5 or its evolved humanized molecule, huA5, were investigated in cell or animal model. The data showed that blocking the RAGE pathway with huA5 robustly reduced liver injury and fibrosis. Furthermore, huA5 significantly suppressed the activation of HSCs and inhibited expression of fibrosis-associated genes, including COL1A1,TIMP1, and ACTA2. huA5 also interfered with RAGE downstream signal transduction and down-regulate both ERK and NF-κB phosphorylation, inhibited the RAGE/NF-kB pathway, leading to reduced expression of pro-inflammatory cytokines and profibrotic markers. Finally, RAGE silencing significantly decreased the expression of activation-related genes in HSCs, inhibiting HSCs proliferation and migration. These results clearly revealed that the anti-RAGE chimeric antibody exerted antifibrotic efficacy in vitro and attenuated liver fibrosis in vivo. HuA5 can be further developed as a lead molecule of drug to treat patients with liver fibrosis.

Therapeutic potential of monomethyl fumarate and aluminum ion combination in alleviating inflammation and oxidative stress in psoriasis.

Psoriasis is a chronic inflammatory skin condition characterized by erythematous plaques with white scales. Its pathogenesis is closely linked to oxidative stress and an imbalance in Th1/Th2 immune responses. Current treatments for psoriasis, such as topical agents, systemic therapies and phototherapy, frequently fail to achieve complete remission in clinical settings. Monomethyl fumarate (MMF), which has been approved by the US Food and Drug Administration in 2020 for multiple sclerosis, has demonstrated efficacy in psoriasis management. Additionally, our previous studies have identified aluminum ions as beneficial in psoriasis treatment. This present study investigates the combined therapeutic effects of MMF and aluminum ions and observed that the combination treatment achieves superior efficacy compared to either treatment alone in a psoriasis mouse model through the modulation of the Nrf2/NF-κB signaling pathway, as demonstrated in cellular models. The combination first activates Nrf2 nuclear translocation and induces antioxidant gene expression, followed by the inhibition of NF-κB nuclear translocation and phosphorylation, which reduces Th1 cytokine production and cellular chemotaxis. Concurrently, the treatment elevates Th2 cytokine secretion, thereby increasing the anti-inflammatory response in HaCaT cells. Overall, these findings support the MMF and aluminum ions combination (MMFAL) as a potential therapeutic strategy for psoriasis, effectively diminishing inflammation and oxidative stress.

Calycosin Ameliorates Neuroinflammation via TLR4-Mediated Signal Following Cerebral Ischemia/Reperfusion Injury in vivo and in vitro.

Cerebral ischemia-reperfusion injury (CIRI) is a key pathophysiological process that leads to stroke mortality, with TLR4-mediated inflammation playing a crucial role. Our previous research highlighted the neuroprotective effects of the phytoestrogen calycosin on CIRI, although the precise mechanism remains unclear. This study aimed to explore the effects of calycosin on the HMGB1/TLR4/NF-κB signaling pathway in rat models of CIRI, both in vivo and in vitro. In vivo, a rat CIRI model was established using middle cerebral artery occlusion (MCAO), inducing ischemia for 1.5h followed by 24h of reperfusion. Calycosin was administered intraperitoneally 1h after ischemia. Neurological deficits and brain infarct volumes were evaluated. Histological changes and key protein expressions around the ischemic penumbra were assessed by H&E staining and immunofluorescence. In vitro, primary neurons and PC12 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic CIRI. Cell viability was measured using a CCK8 assay, and alterations in HMGB1/TLR4/NF-κB pathway components were analyzed using qRT-PCR, Western blotting, and ELISA. In the MCAO rat model, calycosin significantly reduced neurological deficits and infarct sizes, and improved brain tissue damage following reperfusion. Similarly, in the OGD/R model, calycosin attenuated neuronal injury in PC12 cells and in primary neurons. Additionally, calycosin inhibited LPS-induced activation of the HMGB1/TLR4/NF-κB signaling pathway in PC12 cells. Both in vitro and in vivo studies have shown that calycosin effectively downregulates HMGB1 and TLR4 expression, decreases NF-κB and IκB phosphorylation, and reduces the secretion of inflammatory cytokines such as IL-6 and IL-18. These findings suggest that calycosin mitigates cerebral ischemia-reperfusion injury and neuroinflammation by inhibiting the HMGB1/TLR4/NF-κB signaling pathway, thereby providing neuroprotection.

Novel Glycyrrhetin Ureas Possessing 2-Hydroxy-3-enone A Ring: Modification, Anti-inflammatory Activity, and Targeted STING for the Remedy of Acute Kidney Injury.

Glycyrrhetin urea has emerged as a privileged scaffold with anti-inflammatory activity for the treatment and prevention of acute kidney injury (AKI). In this study, structural modifications of the A ring of glycyrrhetinic acid yielded a series of urea derivatives, among which compound 7o exhibited the most promising anti-inflammatory activity. 7o was confirmed to interact with STING through a cellular heat shift assay and to inhibit the STING/NF-κB pathway in RAW264.7 cells. It acted on the STING pathway, inhibited NF-κB phosphorylation, and subsequently reduced the level of release of inflammatory factors. Additionally, 7o significantly increased the survival rate of renal tubular epithelial cells, demonstrating a protective effect against cisplatin-induced cell death and mitigating inflammation activation. The in vivo AKI mouse model showed that 7o significantly downregulated serum creatinine (Scr), blood urea nitrogen (BUN), and levels of inflammatory factors (IL-1β, IL-6, and TNF-α), thereby improving renal function. Morphological analysis revealed that 7o attenuated the cisplatin-induced renal tubular injury. Therefore, 7o represents a promising lead for the prevention and treatment of AKI.

Gut microbiota analysis and LC-MS-based metabolomics to investigate AMPK/NF-κB regulated by Clostridium butyricum in the treatment of acute pancreatitis

BackgroundAcute pancreatitis (AP) is an inflammatory condition with potentially life-threatening complications. This study investigates the therapeutic potential of Clostridium butyricum for modulating the inflammatory cascade through the AMPK/NF-κB signaling pathway, focusing on inflammation induced by AP. LC-MS analysis of serum samples from AP patients highlighted the regulation of lipid metabolism and inflammation, and found that metabolites involved in the inhibition of NF-κB phosphorylation and the AMPK activation pathway were downregulated. We hypothesized that pre-administration of Clostridium butyricum and its culture supernatant could mitigate AP-induced damage by modulating the AMPK/NF-κB pathway.MethodsLipopolysaccharide (LPS)-induced cell inflammation models. LPS combined with CAE induced acute pancreatitis in mice. We divided mice into four groups: Con, AP, AP + C.Buty (AP with Clostridium butyricum treatment), and AP + CFS (AP with culture supernatant treatment). Analyses were performed using WB, RT-qPCR, Elisa, flow cytometry, IHC, and HE, respectively.ResultsOur study shows that CFS can reduce the apoptosis of LPS-induced cellular inflammation and reduce the release of LPS-induced cytoinflammatory factors through the AMPK/NF-κB pathway in vitro. In vivo, Clostridium butyricum and its supernatant significantly reduced inflammatory markers, and corrected histopathological alterations in AP mice. Gut microbiota analysis further supported these results, showing that Clostridium butyricum and its supernatant could restore the balance of intestinal flora disrupted by AP.ConclusionsMechanistically, our results indicated that the therapeutic effects of Clostridium butyricum are mediated through the activation of AMPK, leading to the inhibition of the NF-κB pathway, thereby reducing the production of pro-inflammatory cytokines. Clostridium butyricum and its culture supernatant exert a protective effect against AP-induced damage by modulating the AMPK/NF-κB signaling pathway. Future studies will further elucidate the molecular mechanisms underlying the beneficial effects of Clostridium butyricum in AP and explore its clinical applicability in human subjects.

Study on the mechanism of Fuzheng Huayu formula against biliary fibrosis in mice

Objective: To investigate the effect and mechanisms of Fuzheng huayu formula (FZHY) on biliary fibrosis in mice. Methods: Mdr2 gene knowout (Mdr2-/-) mice were randomly divided into model group, FZHY-treated group and obeticholic acid (OCA)-treated group. Wide type C57BL/6J (WT) mice of the same age were used as the control group. Mdr2-/- mice were treated with the corresponding drugs by gavage from the first day of the 9th week old. The WT and model groups were given 0.3% sodium carboxymethyl cellulose by gavage, and the mice were sacrificed at the end of 12th week old. The activities of serum alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were detected by high-speed biochemical analyzer. H&E staining and sirius red staining were used to observe the pathological changes of liver tissues. The hepatic hydroxyproline content was determined. The hepatic expressions of Col-Ⅰ and α-SMA, ductular reaction markers Epcam, CK7, CK19, and the expression of Pcna, Mki67 and Ccnd1; as well as the expression of inflammatory cytokines Ccl2, Ccl5, Tnf-α, Il10 and Cxcl4 were detected by immunohistochemical staining, western blot and qRT-PCR, respectively. Furthermore, the expression of PPARα and the phosphorylation NF-κB were detected by western blot. Results: Compared with WT mice, the serum ALT and ALP activities of Mdr2-/- mice were significantly increased (P<0.001). The percentage of SR positive stained area and hepatic Hyp content were significantly increased (P<0.01). The hepatic expression of Col-Ⅰ and α-SMA; Epcam, CK7, CK19, Pcna, Mki67 and Ccnd1; Ccl2, Ccl5, Tnf-α, Il10 and Cxcl4 were significantly increased (P<0.01). However, both FZHY and OCA significantly reversed the elevation of these aforementioned indicators (P<0.05; P<0.01). Further study showed that the hepatic expression of PPARα in Mdr2-/- mice was significantly lower than that of WT mice, however the phosphorylation of NF-κB was significantly enhanced (P<0.01). The expression of PPARα in liver tissues of FZHY mice was significantly increased (P<0.05), and the NF-κB phosphorylation was significantly inhibited compared with Mdr2-/- mice (P<0.05). Conclusion: FZHY significantly ameliorated liver fibrosis, ductular reaction and inflammation in Mdr2-/- spontaneous biliary fibrosis mice, and its mechanism was related to the regulation of PPARα/NF-κB pathway.

HM-chromanone attenuates obesity and adipose tissue inflammation by downregulating SREBP-1c and NF-κb pathway in high-fat diet-fed mice

Background: Obese adipose tissue produces various pro-inflammatory cytokines that are major contributors to adipose tissue inflammation. Objective: The present study aimed to determine the effects of HM-chromanone (HMC) against obesity and adipose tissue inflammation in high-fat diet-fed mice. Materials and methods: Twenty-four C57BL/6J male mice were divided into three groups: ND (normal diet), HFD (high-fat diet), and HFD + HMC. The ND group was fed a normal diet, whereas the HFD and HFD + HMC groups were fed a high-fat diet. After 10 weeks of feeding, the animals were orally administered the treatments daily for 9 weeks. The ND and HFD group received distilled water as treatment. The HFD+HMC group was treated with HM-chromaone (50 mg/kg). Results: HM-chromanone administration decreased body weight, fat mass, and adipocyte diameter. HM-chromanone also improved plasma lipid profiles, decreased leptin levels, and increased adiponectin levels. The inhibiting effect of HM-chromanone on SREBP-1c, PPARγ, C/EBPα, and FAS decreased adipogenesis, thereby alleviating lipid accumulation. Furthermore, HM-chromanone administration exhibited a reduction in macrophage infiltration and the expression of pro-inflammatory cytokines. HM-chromanone suppressed the phosphorylation of IκBα and NF-κB, leading to the inhibition of iNOS and COX2 expressions, resulting in decreased inflammation in adipose tissue. Discussion and conclusion: These results highlight the anti-obesity and anti-inflammatory properties of HM-chromanone, achieved through the downregulation of the SREBP-1c and NF-κB pathway in high-fat diet-fed mice.

Cardiomyocyte-derived small extracellular vesicle-transported let-7b-5p modulates cardiac remodeling viaTLR7 signaling pathway.

Cardiac remodeling is the major pathological change of heart failure. And let-7 family has been implicated in the development and pathogenesis of cardiovascular diseases. However, the mechanisms underlying let-7b-5p-mediated cellular pathogenesis of cardiac remodeling are not well understood. The present study aimed to explore the effects of let-7b-5p on cardiac remodeling and the corresponding regulatory mechanism. Invivo results indicated that cardiac let-7b-5p was upregulated in the mouse model of Angiotensin II (Ang II)-induced cardiac remodeling. Additionally, let-7b-5p knockdown ameliorated the effects of Ang II-induced cardiac remodeling, whereas let-7b-5p overexpression facilitated cardiac remodeling. Invitro, let-7b-5p mimics induced cardiomyocyte hypertrophy, fibroblast transdifferentiation, and the expression of inflammatory factors in neonatal mouse cardiomyocytes (NMCMs), neonatal mouse cardiac fibroblasts (NMCFs), and bone marrow-derived macrophages (BMDMs), respectively. Furthermore, let-7b-5p exerted its cardiac pro-remodeling effects at least partially through a small extracellular vesicle (SEV)-based delivery strategy. We found that let-7b-5p was enriched in SEVs derived from Ang II-treated NMCMs (NMCM-SEV) but not from Ang II-treated NMCFs (NMCF-SEV). Mechanistic analyses revealed that NMCM-SEV promoted TLR7 and MyD88 expression, which increased NF-κB phosphorylation levels. Knockdown of let-7b-5p, TLR7 or MyD88 in NMCMs, NMCFs, and BMDMs abolished the cardiac pro-remodeling effects of NMCM-SEV. These results uncover that let-7b-5p-containing NMCM-SEVs promote cardiac remodeling via the TLR7/MyD88/NF-κB pathway, implicating let-7b-5p as a potential therapeutic target for heart failure.

The role of NF-κB transcription factor in the regulation of cytokine induced thermal hyperalgesia in a Leishmania major model in BALB/c mice

Cutaneous leishmaniasis caused mainly by Leishmania major (L. major) is one of the trending models used to investigate induced hyperalgesia and the involved cytokines. Previous studies approached the role of several cytokines in the observed hyperalgesia, but the molecular mechanisms orchestrating such a response still needed to be addressed. In this study, we inspect the role of the NF-κB in the modulation of L. major-prompted hyperalgesia and cytokine expression in BALB/c mice by administering celastrol, a potent blocker of this transcription factor. Intraperitoneal injection of 0.5 mg/kg and 1 mg/kg of celastrol attenuated the L. major-induced thermal hyperalgesia in BALB/c mice for 15 days and 21 days, respectively, as detected by hot plate and tail flick behavioral assessments. Cytokine levels were quantified in the infected paws of BALB/c mice using Sandwich ELISA. The administration of 1 mg/kg celastrol decreased TNF-α levels in L. major infected mice for 23 days, and IL-1β expression declined significantly for 23 days using both celastrol dosages. However, no significant change was observed in the levels of IL-10 in our experimental groups. The activation of NF-κB was detected by observing the phosphorylation levels of the p65 subunit using PathScan phospho-ELISA. The level of NF-κB phosphorylation was elevated in L. major infected BALB/c mice. Only administering 1 mg/kg celastrol suppressed the phosphorylation of p65, thus inactivating NF-kB. In conclusion, our results provide new insights into the correlation between the activation of NF-kB, the induction of thermal hyperalgesia, and the expression of TNF-α and IL-1β in the L. major-induced hyperalgesia model.

MiRNA-431-5p enriched in EVs derived from IFN-β stimulated MSCs potently inhibited ZIKV through CD95 downregulation

BackgroundZika virus (ZIKV) primarily spreads through mosquito bites and can lead to microcephaly in infants and Guillain-Barre syndrome in adults. It is noteworthy that ZIKV can persist in the semen of infected males for extended periods and can be sexually transmitted. Infection with ZIKV has severe pathological manifestations on the testicular tissues of male mice, resulting in reduced sperm motility and fertility. However, there are no approved prophylactic vaccines or therapeutics available to treat Zika virus infection.MethodsUsing a male type I and II interferon receptor-deficient (ifnar1(-/-) ifngr1(-/-)) C57BL/6 (AG6) mouse model infected with ZIKV as a representative model, we evaluated the degree of testicular damage and viral replication in various organs in mice treated with EVs derived from MSC-stimulated with IFN-β (IFNβ-EVs) and treated with controls. We measured testicle size, detected viral load in various organs, and analyzed gene expression to assess treatment efficacy.ResultsOur findings demonstrated that intravenous administration of IFNβ-EVs effectively suppressed ZIKV replication in the testes. Investigation with in-depth RNA sequencing analysis found that IFN-β treatment changed the cargo miRNA of EVs. Notably, miR-431-5p was identified to be significantly enriched in IFNβ-EVs and exhibited potent antiviral activity in vitro. We showed that CD95 was a direct downstream target for miR-431-5p and played a role in facilitating ZIKV replication. miR-431-5p effectively downregulated the expression of CD95 protein, consequently promoted the phosphorylation and nuclear localization of NF-kB, which resulted in the activation of anti-viral status, leading to the suppression of viral replication.ConclusionsOur study demonstrated that the EVs produced by IFNβ-treated MSCs could effectively convey antiviral activity.

VCPIP1 negatively regulates NF-κB signaling pathways by deubiquitinating and stabilizing Erbin in MDP-stimulated macrophages

Macrophages are present in all tissues and body compartments under homeostatic physiological conditions. Importantly, they play a key role in pathological inflammatory processes when disturbed. They can quickly produce large amounts of inflammatory cytokines in response to danger signals. Macrophages can recognize muramyl dipeptide (MDP) through nucleotide-binding oligomerization domain (NOD)-like receptors, subsequently activating the NF-κB signaling pathway and producing proinflammatory cytokines. Erbin can bind to NOD2 and inhibit MDP-induced NF-κB activation, thus participating in the regulation of inflammatory response. Stabilizing or enhancing Erbin expression is essential for suppressing inflammatory responses. In this study, we used a deubiquitination enzyme plasmid library to screen for a key deubiquitinase, VCPIP1, which interacts with Erbin and influences its stability through deubiquitination modification. We investigated whether VCPIP1 affects inflammation using MDP-stimulated RAW 264.7 and BMDMs cells. The results showed that VCPIP1 deficiency reduced Erbin expression and increased NF-κB phosphorylation. Additionally, VCPIP1 deficiency promoted the release of inflammatory factors (IL-1β, IL-6, and TNF-α) in RAW 264.7 cells and BMDMs. This study further expands the role of deubiquitinases (DUBs) in inflammation, providing new insights for the prevention and treatment of sepsis, tumors, immune diseases, and other inflammatory reactions.

Antidepressant and anxiolytic effects of Wuling Capsule in CSDS mice: Alleviating HPA axis hyperactivity via the Nesfatin-1/NF-κB signaling pathway

Ethnopharmacological relevanceWuling capsule, composed of the traditional Chinese medicine Wuling mycelia powder, is made by fermenting and processing Xylaria nigripes (Kl.) Sacc. Clinically, it is commonly used to alleviate depression and anxiety. However, the mechanisms through which Wuling capsule exerts its therapeutic benefits have not been clearly defined. Aim of the studyThis study aims to elucidate the mechanisms by which Wuling capsule alleviates depression and anxiety like behaviors induced by chronic social defeat stress (CSDS) in mice. Materials and methodsHigh-performance liquid chromatography-tandem mass spectrometry system was used to identify the components of the Wuling capsule. Mice were subjected to CSDS to induce depression and anxiety-like behaviors. The expression of hypothalamic corticotropin-releasing hormone (CRH) and Nesfatin-1, as well as serum levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were quantified. Behavioral assessments included the open field test, elevated plus maze, sucrose preference test, and forced swim test to evaluate depression and anxiety levels. Specific manipulation of Nesfatin-1 in the paraventricular nucleus of the hypothalamus (PVN) or cells was achieved through stereotaxic virus injection or plasmid transfection. Intracerebral cannula implantation was used for PVN-specific drug administration. ResultsA total of 123 different components were identified in Wuling capsule. CSDS induced depression and anxiety-like behaviors in mice, along with hyperactivation of the HPA axis, increased hypothalamic Nesfatin-1 levels, and elevated nuclear factor kappa-B (NF-κB) phosphorylation. Overexpression of Nesfatin-1 in the hypothalamus mimicked the effects of CSDS. Conversely, knockdown of hypothalamic Nesfatin-1 or PVN-specific administration of Nesfatin-1 antibody or NF-κB antagonist mitigated CSDS-induced depression and anxiety-like behaviors and hypothalamic-pituitary-adrenal (HPA) axis hyperactivity. In neuroblastoma-2a (N2a) cells, overexpression of Nesfatin-1 led to increased NF-κB phosphorylation and CRH levels, whereas knockdown of Nesfatin-1 produced the opposite effects. Treatment with Wuling capsule alleviated CSDS-induced depression and anxiety-like behaviors, HPA axis hyperactivity, and activation of the hypothalamic Nesfatin-1/NF-κB pathway. ConclusionsThe hypothalamic Nesfatin-1/NF-κB pathway plays a significant role in depression by modulating the HPA axis and is involved in the antidepressant and anxiolytic effects of Wuling capsule.

Sodium Danshensu Inhibits Macrophage Inflammation in Atherosclerosis via the miR-200a-3p/MEKK3/NF-κB Signaling Pathway.

Macrophages are fundamental cellular components of atherosclerotic plaques, and inhibition of macrophage inflammation can delay the development of atherosclerotic plaques. Sodium danshensu (SDSS) can inhibit inflammatory responses and thus delay atherosclerosis, but the specific mechanism remains unclear.The effect of SDSS in inhibiting atherosclerosis was confirmed by observing and detecting atherosclerotic plaque area, morphology and lipid levels in the aorta. The mechanism by which SDSS attenuated atherosclerotic plaques was elucidated by in vivo and in vitro detection of inflammation-related mRNA and protein expression. In addition, bioinformatics analysis, RT-qPCR and dual-luciferase assays were used to predict and validate the potential miRNAs of SDSS to attenuate atherosclerosis. miR-200a-2p mimic and inhibitor were then compared for their effects on the efficacy of SDSS.SDSS inhibited atherosclerotic plaque formation and suppressed the expression of MEKK3, TNF-α, and IL-1β as well as nuclear factor-κB (NF-κB) phosphorylation and nuclear translocation to attenuate inflammatory responses. Bioinformatic predictions combined with RT-qPCR results and dual-luciferase assays indicated that miR-200a-3p negatively regulated MEKK3 expression by directly targeting the 3'UTR region of MEKK3, thereby blocking MEKK3. Further studies showed that miR-200a-3p inhibitor, but not miR-200a-3p mimic, reversed the beneficial effects of SDSS on inflammation.SDSS inhibited macrophage inflammation by modulating the miR-200a-3p/MEKK3/NF-κB signaling pathway.