Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis.

Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

Resveratrol prevents the release of neutrophil extracellular traps (NETs) by controlling hydrogen peroxide levels and nuclear elastase migration.

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.

Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1ß, IL-6, IL-12p40, IFN-ß) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Lipopolysaccharide regulation of antiinflammatory tristetraprolin family and proinflammatory gene expression in mouse macrophages.

OBJECTIVE: Tristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin lipopolysaccharides (LPS) stimulated TTP and tumor necrosis factor (TNF) gene expression, but less was known about LPS effects on TTP homologues and other proinflammatory gene expression in macrophages. The objective was to investigate LPS regulation of TTP family gene and TTP-targeted gene expression in mouse RAW264.7 macrophages using much higher concentrations of LPS and much longer treatment time than previous studies. RESULTS: MTT assay showed that LPS was not toxic to the cells under LPS treatment up to 1000 ng/mL for 2-24 h. LPS mildly affected the soluble protein content in the cells. qPCR assay showed that LPS stimulated TTP mRNA rapidly but not sustainably with 40, 10, and 3 fold of the DMSO control after 2, 8 and 24 h treatment, respectively. Immunoblotting confirmed qPCR results on LPS stimulation of TTP gene expression in the mouse macrophages. LPS exhibited minimal effects on ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels. LPS increased mRNA levels of TNF, COX2, GM-CSF, INFγ and IL12b up to 311, 418, 11, 9 and 4 fold, respectively. This study demonstrated that LPS did not affect macrophage viability, dramatically increased antiinflammatory TTP gene expression as well as proinflammatory TNF and COX2 gene expression but had only mild effects on TTP homologues and other proinflammatory cytokine gene expression in the mouse macrophages.

Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced endothelial cell injury via activating PI3K/AKT signaling pathway.

BACKGROUND: Lung endothelial barrier injury plays an important role in the pathophysiology of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) therapy has shown promise in ARDS treatment and restoration of the impaired barrier function. It has been reported that Wnt5a shows protective effects on endothelial cells. Therefore, the study aimed to investigate whether overexpression of Wnt5a could promote the protective effects of MSCs on Lipopolysaccharide (LPS)-induced endothelial cell injury. METHODS: To evaluate the protective effects of MSCs overexpressing Wnt5a, we assessed the migration, proliferation, apoptosis, and angiogenic ability of endothelial cells. We assessed the transcription of protective cellular factors using qPCR and determined the molecular mechanism using Western blot analysis. RESULTS: Overexpression of Wnt5a upregulated the transcription of protective cellular factors in MSCs. Co-culture of MSCWnt5a promoted endothelial migration, proliferation and angiogenesis, and inhibited endothelial cell apoptosis through the PI3K/AKT pathway. CONCLUSIONS: Overexpression of Wnt5a promoted the therapeutic effect of MSCs on endothelial cell injury through the PI3K/AKT signaling. Our study provides a novel approach for utilizing genetically modified MSCs in the transplantation therapy for ARDS.

Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Upregulated ATF1 Promotes Lipopolysaccharide Induced Inflammatory Response and Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Regulating NF-κB Pathway.

BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from bacterial plaque infection. While the involvement of activating transcription factor 1 (ATF1) has been extensively explored in various human diseases, its specific role in periodontitis remains unclear. This study aims to elucidate the expression and biological function of ATF1 in the context of periodontitis. METHODS: Primary human periodontal ligament cells (hPDLCs) were procured from clinical samples and subsequently characterized. Following treatment with P. gingivalis lipopolysaccharide (LPS, 10 µg/mL), hPDLCs underwent transfection with either ATF1 vector or siRNA. The expression levels of ATF1 in LPS-treated hPDLCs or transfected cells were evaluated through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1ß), were quantified using Enzyme-linked Immunosorbent Assay (ELISA). The assessment of osteogenic proteins, such as runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG), as well as noncanonical nuclear factor-kappaB (NF-κB) pathway-related proteins (p65, p-p65, IkBα, p-IkBα), was conducted using western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry assays were employed to detect cell viability. RESULTS: LPS induced an inflammatory response and hindered the osteogenic differentiation of hPDLCs (p < 0.05, p < 0.01). Furthermore, ATF1 silencing enhanced cell proliferation and suppressed apoptosis in LPS-stimulated hPDLCs (p < 0.05, p < 0.01). ATF1 silencing not only restrained the inflammatory response but also promoted the osteogenic differentiation of LPS-stimulated hPDLCs (p < 0.05, p < 0.01). Importantly, ATF1 silencing effectively blocked the LPS-induced activation of the NF-κB signaling pathway (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: ATF1 emerges as a promising treatment option, inhibiting the osteogenic differentiation of hPDLCs and mitigating the inflammatory response by preventing the phosphorylation of the NF-κB signaling pathway.

Anti-inflammatory potential via the MAPK signaling pathway of Lactobacillus spp. isolated from canine feces.

Two probiotic candidates, Lactobacillus reuteri C1 (C1) and Lactobacillus acidophilus C5 (C5), which were previously isolated from canines, were evaluated in the present study. L. reuteri and L. acidophilus have anti-oxidant, anti-inflammatory, immune-enhancing, and anti-cancer properties and exhibit various probiotic effects in humans and animals. The strains C1 and C5 demonstrated good tolerance to acid and bile salt exposure, exhibited effective adhesion to HT-29 cell monolayer, and displayed sensitivity to antibiotics, thus affirming their probiotic characteristics. Moreover, C1 and C5 exhibited the ability to downregulate the expression of inducible NO synthase (iNOS), an immunomodulatory factor, leading to a reduction in NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These strains also demonstrated potent anti-inflammatory effects in LPS-stimulated RAW 264.7 cells, achieved through the augmentation of anti-inflammatory cytokine IL-10 expression and the inhibition of pro-inflammatory cytokine IL-1ß expression. These anti-inflammatory effects of C1 and C5 were closely associated with the mitogen-activated protein kinase (MAPK) signaling pathway. The results of the present study suggest that the C1 and C5 probiotic candidates attenuate LPS-induced inflammation via the MAPK signaling pathway and the strains can be used as probiotics considering their anti-inflammatory potential.

Oxyresveratrol Improves Cognitive Impairments and Episodic-like Memory through Modulating Neuroinflammation and PI3K-Akt Signaling Pathway in LPS-Induced Mice.

Oxyresveratrol is one of the active ingredients derived from mulberry branch with strong anti-inflammatory bioactivity. In this research, we want to explore if oxyresveratrol can improve cognitive impairments and episodic-like memory and its mechanism. In LPS-induced BV-2 cells, 25 µM OXY can significantly inhibit the expression of NO and alter the M1/M2 polarization by regulating M1/M2 phenotype makers. In vivo, OXY (50, 100 mg/kg) significantly reversed cognitive impairments and alleviated neuronal injuries caused by neuroinflammation. According to network pharmacology analysis, OXY alleviated neuroinflammation via the PI3K-Akt pathway. In general, the research revealed that OXY can improve cognitive impairments and episodic-like memory through alleviating LPS-induced neuroinflammation and regulating the PI3K-Akt signaling pathway.

Propofol improves survival in a murine model of sepsis via inhibiting Rab5a-mediated intracellular trafficking of TLR4.

BACKGROUND: Propofol is a widely used anesthetic and sedative, which has been reported to exert an anti-inflammatory effect. TLR4 plays a critical role in coordinating the immuno-inflammatory response during sepsis. Whether propofol can act as an immunomodulator through regulating TLR4 is still unclear. Given its potential as a sepsis therapy, we investigated the mechanisms underlying the immunomodulatory activity of propofol. METHODS: The effects of propofol on TLR4 and Rab5a (a master regulator involved in intracellular trafficking of immune factors) were investigated in macrophage (from Rab5a-/- and WT mice) following treatment with lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) in vitro and in vivo, and peripheral blood monocyte from sepsis patients and healthy volunteers. RESULTS: We showed that propofol reduced membrane TLR4 expression on macrophages in vitro and in vivo. Rab5a participated in TLR4 intracellular trafficking and both Rab5a expression and the interaction between Rab5a and TLR4 were inhibited by propofol. We also showed Rab5a upregulation in peripheral blood monocytes of septic patients, accompanied by increased TLR4 expression on the cell surface. Propofol downregulated the expression of Rab5a and TLR4 in these cells. CONCLUSIONS: We demonstrated that Rab5a regulates intracellular trafficking of TLR4 and that propofol reduces membrane TLR4 expression on macrophages by targeting Rab5a. Our study not only reveals a novel mechanism for the immunomodulatory effect of propofol but also indicates that Rab5a may be a potential therapeutic target against sepsis.

The effect of selenium on the proliferation of bovine endometrial epithelial cells in a lipopolysaccharide-induced damage model.

BACKGROUND: Endometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism. RESULTS: In this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/ß-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1, TGFB3 and VEGFA. Se decreased the BCL-2/BAX protein ratio, promoted ß-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/ß-catenin and PI3K/AKT signaling pathways inhibited by LPS. CONCLUSIONS: In conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/ß-catenin signaling pathways.

Effects of endoplasmic reticulum stress on chondrocyte apoptosis via the PI3K/AKT signaling pathway.

Chondrocytes undergo endoplasmic reticulum stress (ERS)-induced apoptosis under abnormal stimulation. However, the underlying molecular mechanism remains unclear. We investigated the regulatory effect of the PI3K/AKT signaling pathway on ERS and its effect on chondrocyte apoptosis. In addition, we established a unilateral anterior crossbite (UAC) model in rats to induce temporomandibular joint osteoarthritis (TMJOA). Chondrocytes were isolated from the temporomandibular joints and treated with lipopolysaccharide (LPS) in vitro. Protein expression of ERS and apoptosis markers (GRP78 and CASP12) was analyzed by immunohistochemistry and western blotting. The expression of GRP78, CASP12, p-PI3K, and p-AKT significantly increased in the UAC group. LY294002, a PI3K/AKT signaling pathway inhibitor, reduced the protein expression of GRP78, ATF4, CHOP, and CASP12, whereas 740 Y-P, an activation agent, elevated the expression of proteins GRP78, ATF4, CHOP, and CASP12. In the present study, UAC and LPS stimulation induced apoptosis of chondrocytes in the ERS pathway. Inhibition of the PI3K/AKT signaling pathway reduced ERS-induced chondrocyte apoptosis.

Reyanning mixture inhibits M1 macrophage polarization through the glycogen synthesis pathway to improve lipopolysaccharide-induced acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Reyanning (RYN) mixture is a traditional Chinese medicine composed of Taraxacum, Polygonum cuspidatum, Scutellariae Barbatae and Patrinia villosa and is used for the treatment of acute respiratory system diseases with significant clinical efficacy. AIM OF THE STUDY: Acute lung injury (ALI) is a common clinical disease characterized by acute respiratory failure. This study was conducted to evaluate the therapeutic effects of RYN on ALI and to explore its mechanism of action. MATERIALS AND METHODS: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the chemical components of RYN. 7.5 mg/kg LPS was administered to induce ALI in rats. RYN was administered by gavage at doses of 2 ml/kg, 4 ml/kg or 8 ml/kg every 8 h for a total of 6 doses. Observations included lung histomorphology, lung wet/dry (W/D) weight ratio, lung permeability index (LPI), HE staining, Wright-Giemsa staining. ELISA was performed to detect the levels of TNF-α, IL-6, IL-10, Arg-1,UDPG. Immunohistochemical staining detected IL-6, F4/80 expression. ROS, MDA, SOD, GSH/GSSG were detected in liver tissues. Multiple omics techniques were used to predict the potential mechanism of action of RYN, which was verified by in vivo closure experiments. Immunofluorescence staining detected the co-expression of CD86 and CD206, CD86 and P2Y14, CD86 and UGP2 in liver tissues. qRT-PCR detected the mRNA levels of UGP2, P2Y14 and STAT1, and immunoblotting detected the protein expression of UGP2, P2Y14, STAT1, p-STAT1. RESULTS: RYN was detected to contain 1366 metabolites, some of the metabolites with high levels have anti-inflammatory, antibacterial, antiviral and antioxidant properties. RYN (2, 4, and 8 ml/kg) exerted dose-dependent therapeutic effects on the ALI rats, by reducing inflammatory cell infiltration and oxidative stress damage, inhibiting CD86 expression, decreasing TNF-α and IL-6 levels, and increasing IL-10 and Arg-1 levels. Transcriptomics and proteomics showed that glucose metabolism provided the pathway for the anti-ALI properties of RYN and that RYN inhibited lung glycogen production and distribution. Immunofluorescence co-staining showed that RYN inhibited CD86 and UGP2 expressions. In vivo blocking experiments revealed that blocking glycogen synthesis reduced UDPG content, inhibited P2Y14 and CD86 expressions, decreased P2Y14 and STAT1 mRNA and protein expressions, reduced STAT1 protein phosphorylation expression, and had the same therapeutic effect as RYN. CONCLUSION: RYN inhibits M1 macrophage polarization to alleviate ALI. Blocking glycogen synthesis and inhibiting the UDPG/P2Y14/STAT1 signaling pathway may be its molecular mechanism.

Roles of PD-L1 in human adipose-derived mesenchymal stem cells under inflammatory microenvironment.

Mesenchymal stem cells (MSCs) display unique homing and immunosuppression features which make them promising candidates for cell therapy in inflammatory disorders. It is known that C-X-C chemokine receptor type 4 (CXCR4, also known as CD184) is a critical receptor implicated in MSCs migration, and the protein programmed death ligand-1 (PD-L1) is involved in MSC's immunosuppression. However, it remains unclear how the molecular mechanisms regulate PD-L1 expression for migration and immunosuppression of MSCs under the inflammatory microenvironment. In this article, we used the human adipose-derived mesenchymal stem cells (hADMSCs) treated with lipopolysaccharide (LPS) as an in vitro inflammatory model to explore the roles of PD-L1 on the migration and immunosuppression of MSC. Our results demonstrate that in hADMSCs, LPS significantly increased PD-L1 expression, which mediated the migration of the LPS-treated hADMSCs via CXCR4. In addition, we found that the increased PD-L1 expression in the LPS-treated hADMSCs inhibited B cell proliferation and immunoglobulin G secretion through nuclear factor-κB. Our study suggests that the PD-L1 plays critical roles in the homing and immunosuppression of MSCs which are a promising cell therapy to treat inflammatory diseases.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Hydrogen ameliorates endotoxin-induced acute lung injury through AMPK-mediated bidirectional regulation of Caspase3.

Septic lung injury is characterized by uncontrollable inflammatory infiltrations and acute onset bilateral hypoxemia. Evidence has emerged of the beneficial effect of hydrogen in acute lung injury (ALI), but the underlying mechanism is unclear. In this research, the recovery action of hydrogen on lipopolysaccharide (LPS)-induced ALI in mice and A549 cells was investigated. The 7-day survival rate and body weight of mice were measured after intraperitoneal injection of LPS. Lung function was determined by a whole body plethysmography (WBP) system using the indicators respiratory rate and enhanced pause. Hematoxylin and eosin (HE) staining confirmed the signs of pulmonary edema and inflammatory ooze. Reverse transcription-polymerase chain reaction (RT-PCR) quantification was used to detect the expression of inflammatory factors. Western blotting analysis evaluated the expression levels of involved proteins in the AMP-activated protein kinase (AMPK) pathway. The experimental results confirmed that hydrogen provided an essential solution to the dissipative effects of LPS on survival rate, weight loss and lung function. The LPS-stimulated inflammatory factors, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were also suppressed by hydrogen in A549 cells. Western blot analysis showed that hydrogen significantly upregulated the levels of phosphorylated AMPK (p-AMPK) and lowered the LPS-induced increased expression of dynamin-related protein 1 (Drp1) and Caspase3. These findings prove that hydrogen attenuated LPS-treated ALI by activating the AMPK pathway, supporting the feasibility of hydrogen treatment for sepsis.

Reproducible isolation of bovine mammary macrophages for analysis of host pathogen interactions.

BACKGROUND: Macrophages residing in milk are vital during intramammary infections. This study sought to develop a method enabling the investigation of macrophage responses to pathogens. Streptococcus uberis is the predominant cause of bovine mastitis UK-wide and its pathogenesis is unusual compared to other intramammary pathogens. Previous studies utilise macrophage cell lines, isolated bovine blood derived monocytes, or macrophages from raw milk through complex or inconsistent strategies such as fluorescence activated cell sorting (FACS), centrifugation and selective adherence, and CD14 antibody-microbeads. The centrifuge steps required in the initial stages often damage cells. Thus, the aim of this study was to develop a reliable, reproducible, and cost-effective method for isolating mammary macrophages from milk in a way that allows their culture, challenge with bacteria, and measurement of their response ex-vivo. RESULTS: This method achieves an average yield of 1.27 × 107 cells per litre of milk. Whole milk with somatic cell range of 45-65 cells/µL produced excellent yields, with efficient isolations accomplished with up to 150 cells/µL. This strategy uses milk diluted in PAE buffer to enable low-speed centrifugation steps followed by seeding on tissue-culture-treated plastic. Seeding 1,000,000 milk-extracted cells onto tissue culture plates was sufficient to obtain 50,000 macrophage. Isolated macrophage remained responsive to challenge, with the highest concentration of IL-1ß measured by ELISA at 20 h after challenge with S. uberis. In this model, the optimal multiplicity of infection was found to be 50:1 bacteria:macrophage. No difference in IL-1ß production was found between macrophages challenged with live or heat-killed S. uberis. Standardisation of the production of IL-1ß to that obtained following macrophage stimulation with LPS allowed for comparisons between preparations. CONCLUSIONS: A cost-effective method, utilising low-speed centrifugation followed by adherence to plastic, was established to isolate bovine mammary macrophages from raw milk. This method was shown to be appropriate for bacterial challenge, therefore providing a cost-effective, ex-vivo, and non-invasive model of macrophage-pathogen interactions. The optimal multiplicity of infection for S. uberis challenge was demonstrated and a method for standardisation against LPS described which removes sample variation. This robust method enables, reproducible and reliable interrogation of critical pathogen-host interactions which occur in the mammary gland.