Lipopolysaccharide Stimulation Research Articles

Biochanin A mitigates ulcerative colitis and intestinal inflammation in mice by inhibiting MAPK/NF-kB (p65) axis.

Ulcerative colitis (UC) is a chronic problem of the intestine and relapsing in nature. Biochanin A is a nature-derived isoflavonoid and has numerous bioactivities. However, its role against UC and intestinal inflammation remains obscure. We aimed to comprehensively explore the pharmacological effect of biochanin A in alleviating colitis and to evaluate the potential mechanisms. Initially, we explored the anti-inflammatory action of biochanin A (15, 30, and 60 μM) by employing lipopolysaccharide (LPS)-activated RAW 264.7 cells. In RAW 264.7 cells under LPS stimulation, biochanin A inhibited the elevation of reactive oxygen species (ROS) (p < 0.0001), interleukin (IL)-1β(p < 0.0001), IL-18 (p < 0.01), and tumor necrosis factor (TNF)-α (p < 0.01) release, nitrite production (p < 0.0001), and the expression of inducible nitric oxide synthase (iNOS)and cyclooxygenase-2 (COX-2)proteins. Next, we studied the effectiveness of biochanin A (20 and 40 mg/kg) in mouse colitis induced with dextran sulfate sodium (DSS)by assessing colon length, disease activity index (DAI)scoring, and performing colonoscopy and histological analysis. The pro-inflammatory cytokines were estimated using ELISA. Western blot studies were performed to assess underlying mechanisms. In mice, biochanin A treatment alleviated DAI score (p < 0.0001), restored colon length (p < 0.05) and morphology, and re-established colon histopathology. Biochanin A affects the phosphorylation of proteins associated with NF-κB (p65) and mitogen-activated protein kinase (MAPK)axis and regulates colonic inflammation by reducing the expression of inflammatory cytokines and myeloperoxidase (MPO)activity. Altogether, our findings support the idea that the anticolitis potential of biochanin A is allied with anti-inflammatory activity by inhibiting the MAPK/NF-κB(p65) axis. Hence, biochanin A may be an alternative option to alleviate the risk of colitis.

Copper Chelation Therapy Attenuates Periodontitis Inflammation through the Cuproptosis/Autophagy/Lysosome Axis

Periodontitis development arises from the intricate interplay between bacterial biofilms and the host’s immune response, where macrophages serve pivotal roles in defense and tissue homeostasis. Here, we uncover the mitigative effect of copper chelator Tetrathiomolybdate (TTM) on periodontitis through inhibiting cuproptosis, a newly identified form of cell death which is dependent on copper. Our study reveals concurrent cuproptosis and a macrophage marker within murine models. In response to lipopolysaccharide (LPS) stimulation, macrophages exhibit elevated cuproptosis-associated markers, which are mitigated by the administration of TTM. TTM treatment enhances autophagosome expression and mitophagy-related gene expression, countering the LPS-induced inhibition of autophagy flux. TTM also attenuates the LPS-induced fusion of autophagosomes and lysosomes, the degradation of lysosomal acidic environments, lysosomal membrane permeability increase, and cathepsin B secretion. In mice with periodontitis, TTM reduces cuproptosis, enhances autophagy flux, and decreases Ctsb levels. Our findings underscore the crucial role of copper-chelating agent TTM in regulating the cuproptosis/mitophagy/lysosome pathway during periodontitis inflammation, suggesting TTM as a promising approach to alleviate macrophage dysfunction. Modulating cuproptosis through TTM treatment holds potential for periodontitis intervention.

CD62-L down-regulation after L18-MDP stimulation as a complementary flow cytometry functional assay for the diagnosis of XIAP deficiency.

X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.

Oroxylin A inhibits inflammatory cytokines in periodontitis via HO‑1.

Periodontal disease is a common infectious disease that can lead to the loss of teeth. Hower how to effectively suppress the inflammation with medication is unclear. The aim of the present study was to investigate the anti‑inflammatory effect of Oroxylin A in periodontitis and its potential role through heme oxygenase‑1 (HO‑1). Primary rat gingival fibroblasts (RGFs) were cultured using the tissue block method and identified by immunofluorescence. Following lipopolysaccharide (LPS) stimulation of RGFs, Oroxylin A was administered at 50, 100, 200 or 400 µg/ml. Reverse transcription‑quantitative PCR was used to assess mRNA expression of cyclooxygenase (COX)‑2, TNF‑α, RANKL and osteoprotegerin (OPG). Western blotting was used to detect protein expression levels of COX ‑2, TNF‑α, RANKL and OPG. Following HO‑1 knockdown, the same treatment was performed. The expression of COX‑2 in rat gingival tissue was observed by immunohistochemistry. One‑way analysis of variance and Student's t test were used for statistical analysis. Oroxylin A downregulated mRNA expression of COX‑2, TNF‑α, RANKL and OPG in LPS‑induced RGFs. With increase of Oroxylin A dose, the expression of HO‑1 was gradually upregulated. When HO‑1 was knocked down, Oroxylin A did not downregulate the expression of COX‑2, TNF‑α, RANKL and OPG in LPS‑induced RGFs. Immunohistochemical results showed that expression of COX‑2 was downregulated by Oroxylin A, and the expression of TNF‑α, RANKL and OPG were also downregulated. Oroxylin A decreased expression of inflammatory cytokines in LPS‑induced RGFs and had a good inhibitory effect on periodontitis in rats.

Liquiritin Alleviates Inflammation in Lipopolysaccharide-Induced Human Corneal Epithelial Cells

Purpose This research was designed to elucidate the anti-inflammatory impacts of liquiritin on lipopolysaccharide (LPS)-activated human corneal epithelial cells (HCECs). Methods The Cell Counting kit-8 (CCK-8) assay was adopted to assess cell viability. The enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α. Transcriptome analysis was conducted to identify the genes that exhibited differential expression between different treatment. The model group included cells treated with LPS (10 µg/mL), the treatment group comprised cells treated with liquiritin (80 µM) and LPS (10 µg/mL), and the control group consisted of untreated cells. To further validate the expression levels of the selected genes, including CSF2, CXCL1, CXCL2, CXCL8, IL1A, IL1B, IL24, IL6, and LTB, quantitative real-time PCR was performed. The expression of proteins related to the Akt/NF-κB signaling pathway was assessed through western blot analysis. NF-κB nuclear translocation was evaluated through immunofluorescence staining. Results The secretion of IL-6, IL-8, and TNF-α in LPS-induced HCECs was significantly downregulated by liquiritin. Based on the transcriptome analysis, the mRNA expression of pro-inflammatory cytokines, namely IL-6, IL-8, IL-1β, IL-24, TNF-α, and IL-1α was overproduced by LPS stimulation, and suppressed after liquiritin treatment. Furthermore, the Western blot results revealed a remarkable reduction in the phosphorylation degrees of NF-κB p65, IκB, and Akt upon treatment with liquiritin. Additionally, immunofluorescence analysis confirmed liquiritin’s inhibition of LPS-induced p65 nuclear translocation. Conclusions Collectively, these findings imply that liquiritin suppresses the expression of proinflammatory cytokines, and the anti-inflammatory impacts of liquiritin may be caused by its repression of the Akt/NF-κB signaling pathway in LPS-induced HCECs. These data indicate that liquiritin could provide a potential therapeutic application for inflammation-associated corneal diseases.

Okra extract alleviates lipopolysaccharide-induced inflammation response through the regulation of bile acids, the receptor-mediated pathway, and gut microbiota.

Okra contains flavonoids and vitamin C as antioxidants and it contains polysaccharides as immunomodulators. Flavonoids regulate the inflammatory response in mice and may be related to gut microbiota. This study therefore aimed to investigate the impact of okra extract (OE) on inflammation in mice and to elucidate its underlying mechanism. Forty male Kunming (KM) mice were categorized into four groups: the control (CON) group, the lipopolysaccharide stimulation (LPS) group, the 5 mg mL-1 OE intervention (LPS + OE) group, and the 5 mg mL-1 OE supplementation plus mixed antibiotics (LPS + OE + ABX) group. The results showed that, compared with the OE group, the expression of inflammatory signaling pathway genes was upregulated and gut barrier genes were inhibited in the OE + ABX group. The Fxr receptor was activated and the abundance of Akkermansia was increased after OE supplementation, whereas the effect was reversed in the OE + ABX group. Meanwhile, Fxr was correlated positively with Akkermansia. The OE supplementation alleviated the inflammatory response in mice under LPS stimulation, accompanied by changes in gut microbiota and bile acid receptors, whereas the addition of antibiotics caused a disturbance to the gut microbiota in the OE group, thus reducing the effect of OE in alleviating the inflammatory response. © 2024 Society of Chemical Industry.

A Novel Bifunctional Fusion Protein (Anti-IL-17A-sST2) Protects against Acute Liver Failure, Modulating the TLR4/MyD88 Pathway and NLRP3 Inflammasome Activation

Acute liver failure (ALF) is a serious inflammatory disorder with high mortality rates, which poses a significant threat to human health. The IL-33/ST2 signal is a crucial regulator in inflammation responses associated with lipopolysaccharide (LPS)-induced macrophages. The IL-17A signaling pathway promotes the release of chemokines and inflammatory cytokines, recruiting neutrophils and T cells under LPS stimulation, thus facilitating inflammatory responses. Here, the potential therapeutic benefits of neutralizing the IL-17A signal and modulating the IL-33/ST2 signal in ALF were investigated. A novel dual-functional fusion protein, anti-IL-17A-sST2, was constructed, which displayed high purity and biological activities. The administration of anti-IL-17A-sST2 resulted in significant anti-inflammatory benefits in ALF mice, amelioration of hepatocyte necrosis and interstitial congestion, and reduction in TNF-α and IL-6. Furthermore, anti-IL-17A-sST2 injection downregulated the expression of TLR4 and NLRP3 as well as important molecules such as MyD88, caspase-1, and IL-1β. The results suggest that anti-IL-17A-sST2 reduced the secretion of inflammatory factors, attenuated the inflammatory response, and protected hepatic function by regulating the TLR4/MyD88 pathway and inhibiting the NLRP3 inflammasome, providing a new therapeutic approach for ALF.

Deregulated miR-146a-3p alleviates disease progression in atherosclerosis through inactivating NF-κB: An experimental study.

Atherosclerosis (AS), as a complex chronic inflammatory disease, is 1 of the main causes of cardiovascular and cerebrovascular diseases. This study aimed to confirm the direct interaction between miR-146a-3p and NF-κB, and explore the role of miR-146a-3p/NF-κB in the regulation of inflammation in AS. Bioinformatic prediction and dual-luciferase reporter assay were used to confirm the interaction between miR-146a-3p and NF-κB. Lipopolysaccharides stimulation was performed to establish AS inflammatory cell model, and the levels of pro-inflammatory cytokines were estimated using an enzyme-linked immunosorbent assay. miR-146a-3p and NF-κB expression were evaluated using reverse transcription quantitative PCR, and their clinical value was examined using a receiver operating characteristic curve. Inflammatory cell model showed increased IL-1β, IL-6, and TNF-α. NF-κB was a target gene of miR-146a-3p, and mediated the inhibitory effects of miR-146a-3p on inflammatory responses in the cell model. In patients with AS, miR-146a-3p/NF-κB was associated with patients' clinical data and inflammatory cytokine levels, and aberrant miR-146a-3p and NF-κB showed diagnostic accuracy to distinguish AS patients from healthy populations. miR-146a-3p might inhibit inflammation by targeting NF-κB in AS progression, and miR-146a-3p/ NF-κB might provide novel biomarkers and therapeutic targets for the prevention of AS and related vascular events.

Low-molecular weight branched polyethylenimine reduces cytokine secretion from human immune system monocytes stimulated with bacterial and fungal PAMPs.

Adaptive immunity recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs.

Polarized M2 macrophages induced by glycosylated nano-hydroxyapatites activate bone regeneration in periodontitis therapy.

To investigate the association between periodontal macrophage polarization states and the alveolar bone levels, and to assess whether glycosylated nano-hydroxyapatites (GHANPs) could improve bone regeneration in periodontitis by inducing macrophage M2 polarization. The change of macrophage polarization state in inflammatory periodontal tissues (with bone loss) was examined using clinical gingival samples. The relationship between macrophage phenotype and bone level in periodontal bone loss and repair was evaluated using a mouse periodontitis model. The effect of GHANPs on macrophage polarization was assessed by the in vitro model of lipopolysaccharide (LPS)-stimulated inflammation. The polarization-related markers were detected by immunofluorescence staining, real-time polymerase chain reaction and enzyme-linked immunosorbent assay analysis. The therapeutic effect of GHANPs on alveolar bone loss was explored in experimental periodontitis by histological staining and micro-CT analysis. A lower macrophage M2/M1 ratio was observed in periodontitis-affected human gingival tissues. The results of animal experiments demonstrated a positive correlation between a lower Arg-1/iNOS ratio and accelerated alveolar bone loss; also, the proportion of Arg-1-positive macrophages increased during bone repair and regeneration. The administration of GHANPs partially restored M2 macrophage polarization after LPS stimulation. GHANPs increased alveolar bone repair and regeneration in experimental periodontitis induced by ligation, potentially related to their macrophage M2 transition regulation. The findings of this study indicate that the induction of macrophage M2 polarization can be considered a viable approach for enhancing inflammatory bone repair. Additionally, GHANPs show potential in the clinical treatment of periodontitis.

Lipopolysaccharide Stimulation of Peripheral Blood Mononuclear Cells of Indigenous Pigs Causes Enhanced Expression of Pro-inflammatory Cytokines Over Exotic Pigs

Background: Selective breeding in pigs based on a higher potential of disease resistance can be achieved by studying the association between immune responsiveness and susceptibility to infectious diseases. The early immune responsiveness of pigs assessed in a quantifiable scale for different determinant cytokine genes’ expression involved in regulation of immune response can be a major determinant for evaluating the immune responsiveness. Methods: In this study, we carried out the quantitative relative expression of candidate cytokine genes [Interleukin-1 beta-(IL1b), IL6, IL10 and tumor necrosis factor-alpha (TNFa) and toll-like receptor-4 (TLR4) gene expression in the lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) of Zovawk (indigenous) and Large White Yorkshire (LWY-exotic) pig breeds using real-time PCR to understand the differences in swift immune responsiveness against gram-negative bacteria. Results: Zovawk PBMCs’ pro-inflammatory cytokines IL1b and IL6 expression increased rapidly with LPS stimulation up to 12 h, being significantly higher as early as 4 h compared to that of the LWY PBMCs. In Zovawk pigs, the pro-inflammatory cytokine genes’ expression was found to be quick and high; whereas, in LWY pigs the response was slow and low. Interestingly, the expression of IL10 was not detected in LPS-stimulated PBMCs of Zovawk pigs indicating that the remarkably higher expressions of IL6 and IL1b in response to LPS are within the threshold of the Zovawk pigs which have evolved through generations. Our findings clearly indicate the sturdy and early immune responsiveness of indigenous pigs against gram-negative bacteria over the exotic pigs.

Inhibition of LPS-Induced Skin Inflammatory Response and Barrier Damage via MAPK/NF-κB Signaling Pathway by Houttuynia cordata Thunb Fermentation Broth.

Houttuynia cordata Thunb is rich in active substances and has excellent antioxidant and anti-inflammatory activity. Scanning electron microscopy and gel permeation chromatography were used to analyze the molecular characteristics of the fermentation broth of Houttuynia cordata Thunb obtained through fermentation with Clavispora lusitaniae (HCT-f). The molecular weight of HCT-f was 2.64265 × 105 Da, and the polydispersity coefficient was 183.10, which were higher than that of unfermented broth of Houttuynia cordata Thunb (HCT). By investigating the active substance content and in vitro antioxidant activity of HCT-f and HCT, the results indicated that HCT-f had a higher active substance content and exhibited a superior scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radicals and hydroxyl radicals, with IC50 values of 11.85% and 9.01%, respectively. Our results showed that HCT-f could effectively alleviate the increase in the secretion of inflammatory factors and apoptotic factors caused by lipopolysaccharide (LPS) stimulation, and had a certain effect on repairing skin barrier damage. HCT-f could exert an anti-inflammatory effect by down-regulating signaling in the MAPK/NF-κB pathway. The results of erythrocyte hemolysis and chicken embryo experiments showed that HCT-f had a high safety profile. Therefore, this study provides a theoretical basis for the application of HCT-f as an effective ingredient in food and cosmetics.

Lobetyolin protects mice against LPS-induced sepsis by downregulating the production of inflammatory cytokines in macrophage.

Introduction: Sepsis is a clinical syndrome characterized by dysregulation of the host immune response due to infection, resulting in life-threatening organ damage. Despite active promotion and implementation of early preventative measures and bundle treatments, sepsis continues to exhibit high morbidity and mortality rates with no optimal pharmacological intervention available. Lobetyolin (LBT), the crucial component of polyacetylenes found in Codonopsis pilosula, has been scientifically proven to possess potent antioxidant and antitumor properties. However, its therapeutic potential for sepsis remains unknown. Methods: The mice received pretreatment with intraperitoneal injections of LBT, followed by injection with lipopolysaccharide (LPS) to induce sepsis. Peripheral blood samples were collected to detect TNF-α, IL-1β, and IL-6 levels. The survival status of different groups was recorded at various time intervals. RNA-Seq was utilized for the analysis of gene expression in peritoneal macrophages treated with LBT or LPS. Results: In this study, we observed a significant increase in the survival rate of mice pretreated with LBT in LPS induced sepsis mouse model. LBT demonstrated a remarkable reduction in the production of IL-6, TNF-α, and IL-1β in the serum, along with mitigated lung and liver tissue damage characterized by reduced inflammatory cell infiltration. Additionally, through RNA-seq analysis coupled with GO and KEGG analysis, it was revealed that LBT effectively suppressed genes associated with bacterium presence, cellular response to lipopolysaccharide stimulation, as well as cytokine-cytokine receptor interaction involving Cxcl10, Tgtp1, Gbp5, Tnf, Il1b and IRF7 specifically within macrophages. We also confirmed that LBT significantly downregulates the expression of IL-6, TNF-α, and IL-1β in macrophage activation induced by LPS. Discussion: Therefore, our findings demonstrated that LBT effectively inhibits the production of inflammatory cytokines (IL-6, TNF-α, and IL-1β) and mitigates sepsis induced by LPS through modulating macrophages' ability to generate these cytokines. These results suggest that LBT holds promise as a potential therapeutic agent for sepsis treatment.

Forsythiaside A attenuates lipopolysaccharide-induced mouse mastitis by activating autophagy and regulating gut microbiota and metabolism

Mastitis is an inflammatory disease of the mammary gland with a high incidence in lactating animals, significantly impacting their health and breastfeeding. Moreover, mastitis adversely affects milk quality and yield, resulting in substantial economic losses for the dairy farming industry. Forsythiaside A (FTA), a phenylethanol glycoside analog extracted from Forsythia, exhibits notable anti-inflammatory and antioxidant properties. However, its protective effects and specific mechanisms against mastitis remain unclear. In this study, a lipopolysaccharide (LPS)-induced mouse mastitis model was used to investigate the protective effect of FTA on LPS-induced mastitis and its potential mechanism using histological assays, Western blot, qRT-PCR, FITC-albumin permeability test, 16s rRNA gene sequencing analysis and non-targeted metabolomics assays to investigate the protective effect of FTA on LPS-induced mastitis model and its potential mechanism. The results demonstrated that FTA significantly mitigated LPS-induced mouse mastitis by reducing inflammation and apoptosis levels, modulating the PI3K/AKT/mTOR signaling pathways, inducing autophagy, and enhancing antioxidant capacity and the expression of tight junction proteins. Furthermore, FTA increased the abundance of beneficial microbiota while decreasing the levels of harmful microbiota in mice, thus counteracting the gut microbiota disruption induced by LPS stimulation. Intestinal metabolomics analysis revealed that FTA primarily regulated LPS-induced metabolite alterations through key metabolic pathways, such as tryptophan metabolism. This study confirms the anti-inflammatory and antioxidant effects of FTA on mouse mastitis, which are associated with key metabolic pathways, including the restoration of gut microbiota balance and the regulation of tryptophan metabolism. These findings provide a novel foundation for the treatment and prevention of mammalian mastitis using FTA.

LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor α degradation and LITAF activation.

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results showthat substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

ProBDNF-p75NTR signaling in sterile inflammation

Trauma, injury, and surgical procedures cause tissue damage, sterile inflammatory responses, and significant immune system stress leading to immune dysregulation and increased susceptibility to secondary infections. The spleen is a reservoir of myeloid immune cells that are involved in inflammation following various tissue injuries. One potential signaling pathway that may be involved in this process is proBDNF-p75NTR signaling in myeloid cells. Brain derived neurotrophic factor (BDNF) is a previously reported myokine, however there is little known about its role in skeletal muscle injury. We recently found that p75NTR, the receptor for proBDNF, was significantly upregulated (p=0.004) in myeloid cells isolated from the spleen of animals with hindlimb IR injury compared to sham controls, potentially in a sexually dimorphic manner. Our previous studies have shown that proBDNF, the precursor protein to BDNF, is the predominant form of BDNF expressed in skeletal muscle tissue and is significantly upregulated following tourniquet induced hind limb IR injury in mice (p=0.013). We hypothesize that proBDNF released from injured skeletal muscle could act through p75NTR in myeloid cells to regulate inflammatory responses following IR injury. The hind limb IR injury was induced using a tourniquet model in which an orthodontic rubber band placed on the hind limb at the level of the hip and left in place for 1.5 hours followed by tourniquet removal and reperfusion. A series of in vitro experiments were conducted on myeloid cells isolated from the spleen of sham mice, or mice with skeletal muscle IR injury. As a model of secondary infection, myeloid cells were stimulated with lipopolysaccharide (LPS) and cytokine release into culture media was measured using ELISA assays. We found that in myeloid cells isolated from IR injured animals, LPS stimulated cytokine release was increased. Further, treating isolated myeloid cells from IR injured animals with recombinant cleavage resistant proBDNF increased cytokine release compared to controls. To confirm that p75NTR on myeloid cells was at least partially responsible for the induction of cytokine expression following LPS or proBDNF stimulation, myeloid cells were treated with LM11A-31, a modulator of p75NTR in combination with LPS or proBDNF. Treatment with C31 (100ng) blunted cytokine release following LPS and proBDNF stimulation. These results suggest that the p75NTR pathway is involved in the innate immune function of splenic myeloid cells in injury induced inflammation and therefore is potentially a novel therapeutic target. This work was funded by NIH grant R01HL147105, NIH Training Grant T32-GM136503, and NIH F31 Fellowship F31HL167480. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of tumor necrosis factor receptor 1 and its association with LPS-induced apoptosis in Onchidium reevesii

The sea slug (Onchidium reevesii) inhabits intertidal environments and face environmental pressures and disease threats. The immune system of mollusks plays a crucial role in their defense against pathogens and maintaining homeostasis. The molecular mechanisms underlying its immune defense and response to pathogens remain largely unexplored. This study aimed to isolate and identify the cDNA sequence of tumor necrosis factor receptor 1 (TNFR1) in the sea slug. The obtained cDNA sequence was named OrTNFR1. Like other members of the TNF receptor superfamily, OrTNFR1 contains a signal peptide, TNF homologous domain, and death-related domain. qPCR results showed that OrTNFR1 was highly expressed in the hepatopancreas, blood, and intestines of sea slug. This indicates that OrTNFR1 plays a role in the immune-related tissues of sea slug and is consistent with the broad expression of TNFR1 in bivalves. To investigate the inducible expression pattern and downstream effects of OrTNFR1 in response to lipopolysaccharide (LPS) stimulation, we studied the functional role of the OrTNFR1 gene by stimulating with LPS in vivo and suppressing OrTNFR1 expression using siRNA technology. Our findings demonstrate that LPS stimulation rapidly up-regulates the expression of OrTNFR1 in the hepatopancreas, blood, and intestine of the sea slug. Moreover, si-TNFR1 effectively inhibits LPS-induced TNFR1 expression and its downstream genes. Additionally, we discovered that inhibiting OrTNFR1 reduces LPS-induced cell apoptosis and the expression of apoptosis-associated protein Caspase.TNFR1 is involved in the immune defense of the sea slug, and blocking TNFR1 effectively attenuates LPS-induced cell apoptosis through the TNFR1 pathway. This finding suggests that inhibiting TNFR1 in the sea slug may serve as a potential approach to reduce cell apoptosis. As a shell-less living in harsh intertidal environments, TNFR1 likely participates in its innate immunity. Therefore, studying the TNFR1 pathway contributes to understanding the innate immune signaling pathway in the sea slug, further elucidating host defense against pathogens and apoptotic functions.

Extraction of lipopolysaccharide from fish-derived Aeromonas hydrophila and their immunostimulatory effect on Qihe crucian carp

Purified lipopolysaccharide (LPS) was first obtained from fish-derived Aeromonas hydrophila (A. hydrophila) by hot phenol-water combined with enzymatic hydrolysis. Subsequently, different concentrations of LPS (0, 0.2, 0.4, 0.8, and 1.2 mg/mL) were injected intraperitoneally into Qihe crucian carp (Carassius auratus var. Qihe) with a dose of 3 μL/g body weight to evaluated their immunostimulatory activity by detecting the expression of immune-related genes in the spleen, head kidney, and hepatopancreas. The results showed that the average extraction rate of LPS was about 1.063% based on the precipitation weight of wet bacteria, of which the polysaccharide content was 9.39%. The silver staining band of LPS after SDS-PAGE electrophoresis showed a ladder distribution typically characterized by smooth-type LPS. Protein and nucleic acid electrophoresis results showed very low amounts of residual protein and nucleic acid, and high-performance liquid chromatography (HPLC) results showed that the purity of the extracted A. hydrophila LPS was similar to that of high-purity commercial LPS. Pro-inflammatory cytokine genes such as IL-1, IL-8, TNF-α, and IL-6 were more sensitive to LPS stimulation than anti-inflammatory factors such as IL-10 and TGF-β. After 2 h of injection, their expression levels in the spleen, head kidney, and hepatopancreas were significantly increased (P < 0.05), with the 0.8 and 1.2 mg/mL injection groups showing the most significant increase. The gene expression of IL-10 and TGF-β showed slight fluctuations in response to A. hydrophila LPS stimulation (P < 0.05). In conclusion, high-purity LPS was extracted from A. hydrophila derived from fish by modified hot phenol-water method, it demonstrating that LPS product could significantly regulate the innate immunity of Qihe crucian carp, with the most effective vaccination dose being 2 mg/kg/BW. These findings provide useful information for future research on endotoxin vaccines.

Gibberellic acid targeting ZBTB16 reduces NF-κB dependent inflammatory stress in sepsis-induced neuroinflammation

ObjectiveSepsis is frequently complicated by neuroinflammation. Gibberellic acid (GA3) is recognized for its anti-inflammatory properties. In this study, our objective was to investigate whether GA3 could alleviate Nuclear factor-kappa B (NF-κB) -dependent inflammatory stress in sepsis-induced neuroinflammation. MethodsC57BL/6J mice were administered 10 mg/kg lipopolysaccharide (LPS) to induce sepsis. BV2 cells were pre-incubated with GA3 and subjected lipopolysaccharide stimulation to replicate the inflammatory microglia during sepsis. Subsequently, we assessed the release of IL-6, TNF-α, and IL-1β, along with the expression of Zbtb16, NF-κB, and IκB. To investigate whether any observed anti-inflammatory effects of GA3 were mediated through a Zbtb16-dependent mechanism, Zbtb16 was silenced using siRNA. ResultsGA3 improved the survival of sepsis mice and alleviated post-sepsis cognitive impairment. Additionally, GA3 attenuated microglial M1 activation (pro-inflammatory phenotype), inflammation, and neuronal damage in the brain. Moreover, GA3 inhibited the release of TNF-α, IL-6, and IL-1β in microglia stimulated with LPS. The NF-κB signaling pathway emerged as one of the key molecular pathways associated with the impact of GA3 on LPS-stimulated microglia. Lastly, GA3 upregulated Zbtb16 expression in microglia that had been downregulated by LPS. The inhibitory effects of GA3 on microglial M1 activation were partially reversed through siRNA knockdown of Zbtb16. ConclusionsPre-incubation of microglia with GA3 led to the upregulation of the NF-κB regulator, Zbtb16. This process counteracted LPS-induced microglial M1 activation, resulting in an anti-inflammatory effect upon subsequent LPS stimulation.