Bacteroidetes promotes esophageal squamous carcinoma invasion and metastasis through LPS-mediated TLR4/Myd88/NF-κB pathway and inflammatory changes.

Gut microbiota plays a crucial role in gastrointestinal tumors. Additionally, gut microbes influence the progression of esophageal cancer. However, the major bacterial genera that affect the invasion and metastasis of esophageal cancer remain unknown, and the underlying mechanisms remain unclear. Here, we investigated the gut flora and metabolites of patients with esophageal squamous cell carcinoma and found abundant Bacteroides and increased secretion and entry of the surface antigen lipopolysaccharide (LPS) into the blood, causing inflammatory changes in the body. We confirmed these results in a mouse model of 4NQO-induced esophageal carcinoma in situ and further identified epithelial-mesenchymal transition (EMT) occurrence and TLR4/Myd88/NF-κB pathway activation in mouse esophageal tumors. Additionally, in vitro experiments revealed that LPS from Bacteroides fragile promoted esophageal cancer cell proliferation, migration, and invasion, and induced EMT by activating the TLR4/Myd88/NF-κB pathway. These results reveal that Bacteroides are closely associated with esophageal cancer progression through a higher inflammatory response level and signaling pathway activation that are both common to inflammation and tumors induced by LPS, providing a new biological target for esophageal cancer prevention or treatment.

ROS-responsive celastrol-nanomedicine alleviates inflammation for dry eye disease.

Dry eye disease (DED) is a major global eye disease leading to severe eye discomfort and even vision impairment. The incidence of DED has been gradually increasing with the high frequency of use of electronic devices. It has been demonstrated that celastrol (Cel) has excellent therapeutic efficacy in ocular disorders. However, the poor water solubility and short half-life of Cel limit its further therapeutic applications. In this work, a reactive oxygen species (ROS) sensitive polymeric micelle was fabricated for Cel delivery. The micelles improve the solubility of Cel, and the resulting Cel loaded micelles exhibit an enhanced intervention effect for DED. Thein vitroresults demonstrated that Cel-nanomedicine had a marked ROS responsive release behavior. The results ofin vitroandin vivoexperiments demonstrated that Cel has excellent biological activities to alleviate inflammation in DED by inhibiting TLR4 signaling activation and reducing pro-inflammatory cytokine expression. Therefore, the Cel nanomedicine can effectively eliminate ocular inflammation, promote corneal epithelial repair, and restore the number of goblet cells and tear secretion, providing a new option for the treatment of DED.

Solanum nigrum L. berries extract ameliorated the alcoholic liver injury by regulating gut microbiota, lipid metabolism, inflammation, and oxidative stress.

Solanum nigrum L. (SN) berry is an edible berry containing abundant polyphenols and bioactive compounds, which possess antioxidant and antiinflammatory properties. However, the effects of SN on alcohol-induced biochemical changes in the enterohepatic axis remain unclear. In the current study, a chronic ethanol-fed mice ALD model was used to test the protective mechanisms of SN berries. Microbiota composition was determined via 16S rRNA sequencing, we found that SN berries extract (SNE) improved intestinal imbalance by reducing the Firmicutes to Bacteroides ratio, restoring the abundance of Akkermansia microbiota, and reducing the abundance of Allobaculum and Shigella. SNE restored the intestinal short-chain fatty acids content. In addition, liver transcriptome data analysis revealed that SNE primarily affected the genes involved in lipid metabolism and inflammatory responses. Furthermore, SNE ameliorated hepatic steatosis in alcohol-fed mice by activating AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), peroxisome proliferator-activated receptor α (PPAR-α). SNE reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor-88 (MyD88) nuclear factor kappa-B (NF-κB), which can indicate that SNE mainly adjusted LPS/TLR4/MyD88/NF-κB pathway to reduce liver inflammation. SNE enhanced hepatic antioxidant capacity by regulating NRF2-related protein expression. SNE alleviates alcoholic liver injury by regulating of gut microbiota, lipid metabolism, inflammation, and oxidative stress. This study may provide a reference for the development and utilization of SN resources.

Anti-inflammatory effects of steroidal alkaloids of Solanum lyratum Thunb.

Solanum lyratum Thunb., a traditional Chinese herbal medicine, has a promising background. However, the anti-inflammatory effects of its component steroid alkaloid have not been explored. In this study, animal and cell experiments were performed to investigate the anti-inflammatory effects and mechanism of action of Solanum lyratum Thunb steroid alkaloid (SLTSA), in order to provide evidence for its potential utilization. SLTSA effectively inhibited ear swelling and acute abdominal inflammation of mice. We observed concentration-dependent inhibition of pro-inflammatory cytokines by SLTSA, as confirmed by the ELISA and RT-qPCR results. Flow cytometry, immunofluorescence and RT-qPCR analyses revealed that SLTSA suppressed TLR4 expression. Western blot results indicated that SLTSA inhibited the activation of the TLR4/MyD88/NF-κB signaling pathway. Our study demonstrated that SLTSA possesses anti-inflammatory properties.

Growth inhibition of subcutaneous tumor with glioma cells in nude mice by silencing TLR4.

This study investigated the regulatory impact of Toll-like receptor 4 (TLR4) gene on glioma cell proliferation and apoptosis, elucidating the molecular mechanisms underlying TLR4-induced growth inhibition in vivo. U-87MG-Sh and U-87MG-NC cells, with silenced TLR4 and negative control plasmid respectively, were established. Eighteen nude mice, divided into transfection, negative control, and blank control groups, were inoculated with corresponding cells. Over four weeks, the transfection group exhibited significantly reduced tumor growth rates, smaller mass and volume, and lower growth activity compared to controls. Histological analysis revealed sparse tumor cells, increased fibrous connective tissue, and slower angiogenesis in the transfection group. Flow cytometry demonstrated a lower proliferation index and increased G0/1 cell count in the transfection group. mRNA levels of TLR4, NF-κB, and CyclinD1 were significantly lower in the transfection group. TLR4 silencing correlated with U-87MG cell proliferation regulation, growth inhibition, NF-κB and CyclinD1 modulation, and induction of cell cycle arrest and apoptosis. These findings suggest TLR4 as a potential gene therapy target for glioma.

Exosomes derived from miR-146a-overexpressing fibroblast-like synoviocytes in cartilage degradation and macrophage M1 polarization: a novel protective agent for osteoarthritis?

Introduction: Pathological changes in the articular cartilage (AC) and synovium are major manifestations of osteoarthritis (OA) and are strongly associated with pain and functional limitations. Exosome-derived microRNAs (miRNAs) are crucial regulatory factors in intercellular communication and can influence the progression of OA by participating in the degradation of chondrocytes and the phenotypic transformation in the polarization of synovial macrophages. However, the specific relationships and pathways of action of exosomal miRNAs in the pathological progression of OA in both cartilage and synovium remain unclear. Methods: This study evaluates the effects of fibroblast-like synoviocyte (FLS)-derived exosomes (FLS-Exos), influenced by miR-146a, on AC degradation and synovial macrophage polarization. We investigated the targeted relationship between miR-146a and TRAF6, both in vivo and in vitro, along with the involvement of the NF-κB signaling pathway. Results: The expression of miR-146a in the synovial exosomes of OA rats was significantly higher than in healthy rats. In vitro, the upregulation of miR-146a reduced chondrocyte apoptosis, whereas its downregulation had the opposite effect. In vivo, exosomes derived from miR-146a-overexpressing FLSs (miR-146a-FLS-Exos) reduced AC injury and chondrocyte apoptosis in OA. Furthermore, synovial proliferation was reduced, and the polarization of synovial macrophages shifted from M1 to M2. Mechanistically, the expression of TRAF6 was inhibited by targeting miR-146a, thereby modulating the Toll-like receptor 4/TRAF6/NF-κB pathway in the innate immune response. Discussion: These findings suggest that miR-146a, mediated through FLS-Exos, may alleviate OA progression by modulating cartilage degradation and macrophage polarization, implicating the NF-κB pathway in the innate immune response. These insights highlight the therapeutic potential of miR-146a as a protective agent in OA, underscoring the importance of exosomal miRNAs in the pathogenesis and potential treatment of the disease.

Wogonin upregulates SOCS3 to alleviate the injury in Diabetic Nephropathy by inhibiting TLR4-mediated JAK/STAT/AIM2 signaling pathway.

BACKGROUND: Diabetic nephropathy (DN) is a life-threatening renal disease and needs urgent therapies. Wogonin is renoprotective in DN. This study aimed to explore the mechanism of how wogonin regulated high glucose (HG)-induced renal cell injury. METHODS: Diabetic mice (db/db), control db/m mice, and normal glucose (NG)- or HG-treated human tubule epithelial cells (HK-2) were used to evaluate the levels of suppressor of cytokine signaling 3 (SOCS3), Toll-like receptor 4 (TLR4), inflammation and fibrosis. Lentivirus was used to regulate SOCS3 and TLR4 expressions. After oral gavage of wogonin (10 mg/kg) or vehicle in db/db mice, histological morphologies, blood glucose, urinary protein, serum creatinine values (Scr), blood urea nitrogen (BUN), superoxide dismutase (SOD), glutathione (GSH), and reactive oxygen species (ROS) were assessed. RT-qPCR and Western blot evaluated inflammation and fibrosis-related molecules. RESULTS: HG exposure induced high blood glucose, severe renal injuries, high serumal Src and BUN, low SOD and GSH, and increased ROS. HG downregulated SOCS3 but upregulated TLR4 and JAK/STAT, fibrosis, and inflammasome-related proteins. Wogonin alleviated HG-induced renal injuries by decreasing cytokines, ROS, Src, and MDA and increasing SOD and GSH. Meanwhile, wogonin upregulated SOCS3 and downregulated TLR4 under HG conditions. Wogonin-induced SOCS3 overexpression directly decreased TLR4 levels and attenuated JAK/STAT signaling pathway-related inflammation and fibrosis, but SOCS3 knockdown significantly antagonized the protective effects of wogonin. However, TLR4 knockdown diminished SOCS3 knockdown-induced renal injuries. CONCLUSION: Wogonin attenuates renal inflammation and fibrosis by upregulating SOCS3 to inhibit TLR4 and JAK/STAT pathway.

Fosfenopril Attenuates Inflammatory Response in Diabetic Dry Eye Models by Inhibiting the TLR4/NF-κB/NLRP3 Signaling Pathway.

Purpose: The purpose of this study was to investigate the involvement of the TLR4/NF-κB/NLRP3 signaling pathway and its underlying mechanism in diabetic dry eye. Methods: Two models of diabetic dry eye were established in high glucose-induced human corneal epithelial (HCE-T) cells and streptozotocin (STZ)-induced C57BL/6 mice, and the TLR4 inhibitor fosfenopril (FOS) was utilized to suppress the TLR4/NF-κB/NLRP3 signaling pathway. The expression changes in TLR4, NF-κB, NLRP3, and IL-1ß, and other factors were detected by Western blot and RT‒qPCR, the wound healing rate was evaluated by cell scratch assay, and the symptoms of diabetic mice were evaluated by corneal sodium fluorescein staining and tear secretion assay. Results: In the diabetic dry eye model, the transcript levels of TLR4, NF-κB, NLRP3, and IL-1ß were raised, and further application of FOS, a TLR4 inhibitor, downregulated the levels of these pathway factors. In addition, FOS was found to be effective in increasing the wound healing rate of high glucose-induced HCE-T cells, increasing tear production, and decreasing corneal fluorescence staining scores in diabetic mice, as measured by cell scratch assay, corneal sodium fluorescein staining assay, and tear production. Conclusions: The current study found that the TLR4/NF-κB/NLRP3 signaling pathway regulates diabetic dry eye in an in vitro and in vivo model, and that FOS reduces the signs of dry eye in diabetic mice, providing a new treatment option for diabetic dry eye.

Butyrate inhibits the malignant biological behaviors of breast cancer cells by facilitating cuproptosis-associated gene expression.

BACKGROUND: Butyrate is a common short-chain fatty acids (SCFA), and it has been demonstrated to regulate the development of breast cancer (BC), while the underlying mechanism is still unreported. METHODS: Gas chromatography was used to measure the amounts of SCFA (acetate, propionate, and butyrate) in the feces. Cell viability was measured by the CCK-8 assay. The wound healing assay demonstrated cell migration, and the transwell assay demonstrated cell invasion. The levels of protein and gene were determined by western blot assay and RT-qPCR assay, respectively. RESULTS: The levels of SCFA were lower in the faecal samples from BC patients compared to control samples. In cellular experiments, butyrate significantly suppressed the cell viability, migration and invasion of T47D in a dose-dependent manner. In animal experiments, butyrate effectively impeded the growth of BC tumors. Toll like receptor 4 (TLR4) was highly expressed in the tumors from BC patients. Butyrate inhibited the expression of TLR4. In addition, butyrate promoted the expression of cuproptosis-related genes including PDXK (pyridoxal kinase) and SLC25A28 (solute carrier family 25 member 28), which was lowly expressed in BC tumors. Importantly, overexpression of TLR4 can reverses the promotion of butyrate to PDXK and SLC25A28 expression and the prevention of butyrate to the malignant biological behaviors of T47D cells. CONCLUSION: In summary, butyrate inhibits the development of BC by facilitating the expression of PDXK and SLC25A28 through inhibition of TLR4. Our investigation first identified a connection among butyrate, TLR4 and cuproptosis-related genes in BC progression. These findings may provide novel target for the treatment of BC.

E3 ubiquitin ligase BCA2 promotes breast cancer stemness by up-regulation of SOX9 by LPS.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. Breast cancer stem cells (BCSCs) are believed to play a crucial role in the carcinogenesis, therapy resistance, and metastasis of TNBC. It is well known that inflammation promotes stemness. Several studies have identified breast cancer-associated gene 2 (BCA2) as a potential risk factor for breast cancer incidence and prognosis. However, whether and how BCA2 promotes BCSCs has not been elucidated. Here, we demonstrated that BCA2 specifically promotes lipopolysaccharide (LPS)-induced BCSCs through LPS induced SOX9 expression. BCA2 enhances the interaction between myeloid differentiation primary response protein 88 (MyD88) and Toll-like receptor 4 (TLR4) and inhibits the interaction of MyD88 with deubiquitinase OTUD4 in the LPS-mediated NF-κB signaling pathway. And SOX9, an NF-κB target gene, mediates BCA2's pro-stemness function in TNBC. Our findings provide new insights into the molecular mechanisms by which BCA2 promotes breast cancer and potential therapeutic targets for the treatment of breast cancer.

[Experimental study on the inhibition of the NLRP3 signaling pathway with Shengsan Jiedu Huayu decoction to alleviate inflammatory injury in rats with acute-on-chronic liver failure].

Objective: To observe the therapeutic effect of Shengsan Jiedu Huayu decoction in alleviating inflammatory liver injury in rats with acute-on-chronic liver failure (ACLF) and its effect on the activation intensity for the NLRP3 signaling pathway. Methods: 63 SD rats were randomly divided into a blank group, a model group, and low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction (7.29 g/kg/d, 14.58 g/kg/d, and 29.16 g/kg/d). The ACLF rat model was replicated using carbon tetrachloride combined with d-galactosamine and lipopolysaccharide. Different dose gradients of the Shengsan Jiedu Huayu decoction were used for a five-day intervention treatment, and then rat serum and tissue samples were collected. A biochemical analyzer was used to detect the serum levels of ALT, AST, and TBIL in rats. ELISA was used to detect serum IL-18 and IL-1ß content. HE staining was used to observe histomorphological changes in liver tissue. Immunohistochemistry was used to detect GSDMD expression in liver tissue. Western blot and PCR were used to detect NLRP3, Caspase1, ASC, TLR4, IL-1ß, IL-18 protein, and mRNA expression levels.The groups were compared using analysis of variance and the rank-sum test. Results: Compared with the blank group, the model group's rat liver tissue was severely injured. Serum levels of ALT, AST, and TBIL, inflammatory factors IL-1ß and IL-18, and the GSDMD protein expression level, NLRP3 expression level, TLR4, caspase 1, ASC, IL-1ß, IL-18 protein, and mRNA (P<0.01) were all significantly increased in the model than the blank group (P<0.01). Additionally, compared with the model group, the low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction had improved liver tissue injury in ACLF rats, while the serum levels of ALT, AST, TBIL, IL-1ß, IL-18, liver tissue GSDMD protein, NLRP3, TLR4, caspase 1, and ASC expressions were all lower in the different dose gradients of the Shengsan Jiedu Huayu decoction than the model group, with the most evident reduction in the high-dose group (P<0.01). Conclusion: Shengsan Jiedu Huayu decoction can weaken the activation intensity of the NLRP3 signaling pathway, alleviate liver tissue pathological injury, reduce inflammatory factor release, and alleviate inflammatory liver injury in ACLF rats.

Designing of a multi-epitopes based vaccine against Haemophilius parainfluenzae and its validation through integrated computational approaches.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

The role of ZC3H12D-regulated TLR4-NF-κB pathway in LPS-induced pro-inflammatory microglial activation.

Lipopolysaccharide (LPS) is an important neurotoxin that can cause inflammatory activation of microglia. ZC3H12D is a novel immunomodulator, which plays a remarkable role in neurological pathologies. It has not been characterized whether ZC3H12D is involved in the regulation of microglial activation. The aim of this study was to investigate the role of ZC3H12D in LPS-induced pro-inflammatory microglial activation and its potential mechanism. To elucidate this, we established animal models of inflammatory injury by intraperitoneal injection of LPS (10 mg/kg). The results of the open-field test showed that LPS caused impaired motor function in mice. Meanwhile, LPS caused pro-inflammatory activation of microglia in the mice cerebral cortex and inhibited the expression of ZC3H12D. We also constructed in vitro inflammatory injury models by treating BV-2 microglia with LPS (0.5 µg/mL). The results showed that down-regulated ZC3H12D expression was associated with LPS-induced pro-inflammatory microglial activation, and further intervention of ZC3H12D expression could inhibited LPS-induced pro-inflammatory activation of microglia. In addition, LPS activated the TLR4-NF-κB signaling pathway, and this process can also be reversed by promoting ZC3H12D expression. At the same time, the addition of resveratrol, a nutrient previously proven to inhibit pro-inflammatory microglial activation, can also reverse this process by increasing the expression of ZC3H12D. Summarized, our data elucidated that ZC3H12D in LPS-induced pro-inflammatory activation of brain microglia via restraining the TLR4-NF-κB pathway. This study may provide a valuable clue for potential therapeutic targets for neuroinflammation-related injuries.

RG-I pectin-like polysaccharide from Rosa chinensis inhibits inflammation and fibrosis associated to HMGB1/TLR4/NF-κB signaling pathway to improve non-alcoholic steatohepatitis.

A novel RG-I pectin-like polysaccharide, YJ3A1, was purified from the flowers of Rosa chinensis and its structure and hepatoprotective effect in vivo and in vitro were investigated. The backbone of this polysaccharide is mainly composed of 1, 4-galactan, 1, 4-linked α-GalpA and 1, 2-linked α-Rhap disaccharide repeating unit attached by 1, 6-linked ß-Galp or 1, 5-linked α-Araf on C-4 of the Rhap. Interestingly, oral administration of YJ3A1 significantly ameliorates NASH-associated inflammation, oxidative stress and fibrosis and does not affect the liver morphology of normal mice at a dose of 50 mg/kg. The mechanism study suggests that the biological activity may associate to inactivating of high-mobility group box 1 protein (HMGB1)/TLR4/NF-κB and Akt signaling pathways by restraining the expression and release of HMGB1, thereby impeding the effect of NASH. The current findings outline a novel leading polysaccharide for new drug candidate development against NASH.

Hydrogen gas inhalation ameliorates LPS-induced BPD by inhibiting inflammation via regulating the TLR4-NFκB-IL6/NLRP3 signaling pathway in the placenta.

INTRODUCTION: Hydrogen (H2) is regarded as a novel therapeutic agent against several diseases owing to its inherent biosafety. Bronchopulmonary dysplasia (BPD) has been widely considered among adverse pregnancy outcomes, without effective treatment. Placenta plays a role in defense, synthesis, and immunity, which provides a new perspective for the treatment of BPD. This study aimed to investigate if H2 reduced the placental inflammation to protect the neonatal rat against BPD damage and potential mechanisms. METHODS: We induced neonatal BPD model by injecting lipopolysaccharide (LPS, 1 µg) into the amniotic fluid at embryonic day 16.5 as LPS group. LPS + H2 group inhaled 42% H2 gas (4 h/day) until the samples were collected. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from the control group (CON), LPS group and LPS + H2 group. HE staining was performed to evaluate inflammatory levels. RNA sequencing revealed dominant differentially expressed genes. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to mine the signaling pathways involved in protective effect of H2 on the development of LPS-induced BPD. We further used qRT-PCR, Western blot and ELISA methods to verify differential expression of mRNA and proteins. Moreover, we verified the correlation between the upstream signaling pathways and the downstream targets in LPS-induced BPD model. RESULTS: Upon administration of H2, the inflammatory infiltration degree of the LPS-induced placenta was reduced, and infiltration significantly narrowed. Hydrogen normalized LPS-induced perturbed lung development and reduced the death ratio of the fetus and neonate. RNA-seq results revealed the importance of inflammatory response biological processes and Toll-like receptor signaling pathway in protective effect of hydrogen on BPD. The over-activated upstream signals [Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), Caspase1 (Casp1) and NLR family pyrin domain containing 3 (NLRP3) inflammasome] in LPS placenta were attenuated by H2 inhalation. The downstream targets, inflammatory cytokines/chemokines [interleukin (IL)-6, IL-18, IL-1ß, C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1)], were decreased both in mRNA and protein levels by H2 inhalation in LPS-induced placentas to rescue them from BPD. Correlation analysis displayed a positive association of TLR4-mediated signaling pathway both proinflammatory cytokines and chemokines in placenta. CONCLUSION: H2 inhalation ameliorates LPS-induced BPD by inhibiting excessive inflammatory cytokines and chemokines via the TLR4-NFκB-IL6/NLRP3 signaling pathway in placenta and may be a potential therapeutic strategy for BPD.

Acteoside and isoacteoside alleviate renal dysfunction and inflammation in lipopolysaccharide-induced acute kidney injuries through inhibition of NF-κB signaling pathway.

Acute kidney injury (AKI) is a sudden loss of renal function with a high mortality rate and inflammation is thought to be the underlying cause. The phenylpropanoid components acteoside (ACT) and isoacteoside (ISO), which were isolated from Cistanche deserticola Y.C.Ma, have been reported to have preventive effects against kidney disorders. This study aimed to investigate the anti-inflammatory properties and protective mechanisms of ACT and ISO. In this investigation, kidney function was assessed using a semi-automatic biochemical analyzer, histopathology was examined using Hematoxylin-Eosin staining and immunohistochemistry, and the concentration of inflammatory cytokines was assessed using an enzyme-linked immunosorbent assay (ELISA) test. In addition, using Western blot and q-PCR, the expression of proteins and genes connected to the NF-κB signaling pathway in mice with lipopolysaccharide (LPS)-induced AKI was found. The findings showed that under AKI intervention in LPS group, ACT group and ISO group, the expression of Rela (Rela gene is responsible for the expression of NFκB p65 protein) and Tlr4 mRNA was considerably elevated (P<0.01), which led to a significant improvement in the expression of MyD88, TLR4, Iκ-Bɑ and NF-κB p65 protein (P<0.001). The levels of Alb, Crea and BUN (P<0.001) increased along with the release of downstream inflammatory factors such as IL-1ß, IL-6, Cys-C, SOD1 and TNF-α (P<0.001). More importantly, the study showed that ISO had a more favorable impact on LPS-induced AKI mice than ACT. In conclusion, by inhibiting NF-κB signaling pathway, ACT and ISO could relieve renal failure and inflammation in AKI, offering a fresh possibility for the therapeutic management of the condition.

Cholesterol accumulation impairs HIF-1α-dependent immunometabolic reprogramming of LPS-stimulated macrophages by upregulating the NRF2 pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis. Yet, how lipid loading modulates Mφ inflammatory responses remains unclear. We endeavored to gain mechanistic insights into how pre-loading with free cholesterol modulates Mφ metabolism upon LPS-induced TLR4 signaling. We found that activities of prolyl hydroxylases (PHDs) and factor inhibiting HIF (FIH) are higher in cholesterol loaded Mφs post-LPS stimulation, resulting in impaired HIF-1α stability, transactivation capacity and glycolysis. In RAW264.7 cells expressing mutated HIF-1α proteins resistant to PHDs and FIH activities, cholesterol loading failed to suppress HIF-1α function. Cholesterol accumulation induced oxidative stress that enhanced NRF2 protein stability and triggered a NRF2-mediated antioxidative response prior to and in conjunction with LPS stimulation. LPS stimulation increased NRF2 mRNA and protein expression, but it did not enhance NRF2 protein stability further. NRF2 deficiency in Mφs alleviated the inhibitory effects of cholesterol loading on HIF-1α function. Mutated KEAP1 proteins defective in redox sensing expressed in RAW264.7 cells partially reversed the effects of cholesterol loading on NRF2 activation. Collectively, we showed that cholesterol accumulation in Mφs induces oxidative stress and NRF2 stabilization, which when combined with LPS-induced NRF2 expression leads to enhanced NRF2-mediated transcription that ultimately impairs HIF-1α-dependent glycolytic and inflammatory responses.

Probiotic-derived amphiphilic exopolysaccharide self-assembling adjuvant delivery platform for enhancing immune responses.

Enhancing immune response activation through the synergy of effective antigen delivery and immune enhancement using natural, biodegradable materials with immune-adjuvant capabilities is challenging. Here, we present NAPSL.p that can activate the Toll-like receptor 4 (TLR4) pathway, an amphiphilic exopolysaccharide, as a potential self-assembly adjuvant delivery platform. Its molecular structure and unique properties exhibited remarkable self-assembly, forming a homogeneous nanovaccine with ovalbumin (OVA) as the model antigen. When used as an adjuvant, NAPSL.p significantly increased OVA uptake by dendritic cells. In vivo imaging revealed prolonged pharmacokinetics of NAPSL. p-delivered OVA compared to OVA alone. Notably, NAPSL.p induced elevated levels of specific serum IgG and isotype titers, enhancing rejection of B16-OVA melanoma xenografts in vaccinated mice. Additionally, NAPSL.p formulation improved therapeutic effects, inhibiting tumor growth, and increasing animal survival rates. The nanovaccine elicited CD4+ and CD8+ T cell-based immune responses, demonstrating the potential for melanoma prevention. Furthermore, NAPSL.p-based vaccination showed stronger protective effects against influenza compared to Al (OH)3 adjuvant. Our findings suggest NAPSL.p as a promising, natural self-adjuvanting delivery platform to enhance vaccine design across applications.

Improving stroke prognosis by TLR4 KO to enhance N2 neutrophil infiltration and reduce M1 macrophage polarization.

Cerebral ischemic stroke remains a leading cause of mortality and morbidity worldwide. Toll-like receptor 4 (TLR4) has been implicated in neuroinflammatory responses poststroke, particularly in the infiltration of immune cells and polarization of macrophages. This study aimed to elucidate the impact of TLR4 deficiency on neutrophil infiltration and subsequent macrophage polarization after middle cerebral artery occlusion (MCAO), exploring its role in stroke prognosis. The objective was to investigate how TLR4 deficiency influences neutrophil behavior poststroke, its role in macrophage polarization, and its impact on stroke prognosis using murine models. Wild-type and TLR4-deficient adult male mice underwent MCAO induction, followed by various analyses, including flow cytometry to assess immune cell populations, bone marrow transplantation experiments to evaluate TLR4-deficient neutrophil behaviors, and enzyme-linked immunosorbent assay and Western blot analysis for cytokine and protein expression profiling. Neurobehavioral tests and infarct volume analysis were performed to assess the functional and anatomical prognosis poststroke. TLR4-deficient mice exhibited reduced infarct volumes, increased neutrophil infiltration, and reduced M1-type macrophage polarization post-MCAO compared to wild-type mice. Moreover, the depletion of neutrophils reversed the neuroprotective effects observed in TLR4-deficient mice, suggesting the involvement of neutrophils in mediating TLR4's protective role. Additionally, N1-type neutrophils were found to promote M1 macrophage polarization via neutrophil gelatinase-associated lipocalin (NGAL) secretion, a process blocked by TLR4 deficiency. The study underscores the protective role of TLR4 deficiency in ischemic stroke, delineating its association with increased N2-type neutrophil infiltration, diminished M1 macrophage polarization, and reduced neuroinflammatory responses. Understanding the interplay between TLR4, neutrophils, and macrophages sheds light on potential therapeutic targets for stroke management, highlighting TLR4 as a promising avenue for intervention in stroke-associated neuroinflammation and tissue damage.

[Aloperine suppresses TLR4/NF-κB/NLRP3 signaling to ameliorate cigarette smoke-induced injury to human bronchial epithelial cells].

Objective To explore the effects of aloperine (Alo) on cigarette smoke-induced injury in human bronchial epithelial cells and its potential mechanism. Methods After human bronchial epithelial 16HBE cells were co-treated by 100 mL/L cigarette smoke extract (CSE) and various concentrations (50,100 and 200 µmol/L) of Alo, cell viability was assessed using CCK-8 assay. Lactate dehydrogenase (LDH) activity was measured with a related kit. Cell apoptosis was evaluated using the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and Western blot analysis. The levels of inflammatory factors were detected by ELISA. Oxidative stress levels were assessed using 2'7'-dichlorofluorescin diacetate (DCFH-DA) staining. The expression of Toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling-associated proteins was measured by Western blot analysis. After cells were co-treated with 100 mL/L CSE and 200 µmol/L Alo, the aforementioned assays were applied to evaluate the effects of TLR4 overexpression on the TLR4/NF-κB/NLRP3 signaling, LDH activity, apoptosis, inflammatory response and oxidative stress in cells. Results CSE exposure might inhibit 16HBE cell viability, increase LDH activity, apoptosis, inflammatory response and oxidative stress levels and activate TLR4/NF-κB/NLRP3 signaling. Treatment with Alo promoted cell viability, decreased LDH activity, cell apoptosis, inflammation and oxidative stress levels, and inactivated TLR4/NF-κB/NLRP3 signaling. Furthermore, TLR4 overexpression might reverse the protective role of Alo treatment in CSE-induced injury in 16HBE cells. Conclusion Alo may ameliorate CSE-induced injury in human bronchial epithelial cells via inhibiting TLR4/NF-κB/NLRP3 signaling.