LPS Group Research Articles

Extracorporeal Shockwave Therapy Alleviates Inflammatory Pain by Down-Regulating NLRP3 Inflammasome in Experimental Chronic Prostatitis and Chronic Pelvic Pain Syndrome.

To evaluate the anti-inflammatory and antioxidative effects of extracorporeal shockwave therapy (ESWT) on prostatitis and explore the mechanism of alleviating pain. For in vitro testing, RWPE-1 cells were randomly divided into 5 groups: (1) RWPE-1 group (normal control), (2) LPS group (lipopolysaccharide inducing inflammation), (3) 0.1ESWT group (treated by 0.1 mJ/mm² energy level), (4) 0.2ESWT group (treated by 0.2 mJ/mm² energy level), and (5) 0.3ESWT group (treated by 0.3 mJ/mm² energy level). After ESWT was administered, cells and supernatant were collected for ELISA and western blot. For in vivo testing, Sprague-Dawley male rats were randomly divided into 3 groups: (1) normal group, (2) prostatitis group, and (3) ESWT group (n=12 for each). Prostatitis was induced by 17 beta-estradiol and dihydrotestosterone (DHT) administration. Four weeks after ESWT, the pain index was assessed for all groups and prostate tissues were collected for immunohistochemistry, immunofluorescence, apoptosis analysis and, western blot. Our in vitro studies showed that the optimal energy flux density of ESWT was 0.2 mJ/mm². In vivo, ESWT ameliorated discomfort in rats with prostatitis and inflammation symptoms were improved. Compared to normal rats, overexpressed NLRP3 inflammasomes triggered apoptosis in rats with prostatitis and this was improved by ESWT. TLR4-NFκB pathway was overactive after experimental prostatitis, compared to normal and ESWT groups, and prostatitis induced alterations in BAX/BAK pathway were inhibited by ESWT. ESWT improved CP/CPPS by reducing NLRP3 inflammasome and ameliorated apoptosis via inhibiting BAX/BAK pathway in a rat model. TLR4 may play a key role in bonding NLRP3 inflammasome and BAX/BAK pathways. ESWT might be a promising approach for the treatment of CP/CPPS.

α-Tocopherol Transfer Protein-Null Mice with Very Low α-Tocopherol Status Do Not Have an Enhanced Lipopolysaccharide-Induced Acute Inflammatory Response

BackgroundThe α-tocopherol transfer protein-null (Ttpa−/−) mouse model is a valuable tool for studying the molecular and functional consequences of vitamin E (α-tocopherol, αT) deficiency. Because αT has been associated with reduced oxidative stress and improved immune function, we hypothesized that depleted αT concentration would exacerbate LPS-induced acute inflammatory response in the brain and heart of Ttpa−/− mice fed a vitamin E deficient (VED) diet. ObjectivesThe objective was to investigate how extremely low αT status, followed by exposure to LPS, altered the acute inflammatory response to LPS in Ttpa−/− and wild-type (Ttpa+/+) mice. MethodsThree-week-old male Ttpa+/+ and Ttpa−/− littermates (n = 36/genotype) ingested a VED diet ad libitum for 4 wk. At week 7, mice received an intraperitoneal LPS (1 or 10 μg/mouse) or saline (control) injection and were killed 4 h postinjection. Brain and heart IL-6 protein concentrations and tissue and serum αT concentrations were measured via ELISA and HPLC with photodiode array detection, respectively. Hippocampal Il-6, Tnf, and Gpx1 gene expression were measured via reverse transcriptase-quantitative polymerase chain reaction, and blood immune cell profiles were measured via a hematology analyzer. ResultsαT accumulation in analyzed tissues and serum of Ttpa−/− mice was substantially lower than Ttpa+/+ mice. Circulating white blood cell concentration, particularly lymphocytes, were lower in all LPS groups compared with controls (P < 0.01). The 10 μg LPS groups had elevated IL-6 in the cerebellum and heart compared with controls, confirming an acute inflammatory response (P < 0.01). Hippocampal and heart Il-6 gene expression in the LPS-treated Ttpa−/− mice was upregulated in a dose-dependent manner (P < 0.05). ConclusionsThe 10 μg LPS dose enhanced inflammatory markers in the brain, heart, and serum in each genotype but the lower αT status in Ttpa−/− mice did not further impact the acute immune responses.

Evaluation of changes of apelin and apelin receptor (APJ) expression in cervix-uterus and placental axis in an LPS-induced preterm labor model.

Although preterm birth is among the preventable causes of maternal and infant death, its mechanism has not yet been clarified. When evaluated in terms of the results, the psycho-social burden of mother-infant losses and the costs of rehabilitation, care, and treatment for postpartum sequelae are high. When evaluated in terms of its causes, infection/inflammation has an important place. Therefore, it is essential to understand the role of pro- and anti-inflammatory proteins in the process. In our study, apelin and apelin receptor (APJ) expression in the cervix-uterus and placental axis were evaluated at tissue and protein levels in pregnant and non-pregnant control, sham, PBS, and LPS groups in the infection model in which LPS induction was performed by midline laparotomy, in CD-1 mice. The evaluation of this axis regarding apelin and apelin receptor in the preterm birth model is new in the literature. Apelin is expressed more intensely in uterine epithelial cells than in the cervix. In the placenta, expression is more intense in the junctional zone compared to other zones. Apelin protein levels decrease significantly in the cervix and placenta whereas it increases in the uterus. While no change was observed in the expression of the apelin receptor at the tissue and protein level in the cervix and uterus, it increased in both aspects in the placenta in the invasive procedure groups. We propose that the decrease in apelin protein due to LPS in the preterm delivery model may be related to the effort to compensate for the balance deteriorated in the pro-inflammatory direction with post-transitional modification at the tissue level. The tendency of apelin to increase with pregnancy has led to the conclusion that it is necessary for a healthy pregnancy. Although the apelin receptor does not change with inflammation, it is necessary to investigate the mechanisms associated with its stress and trauma-induced increase, since it increases in the invasive procedure group.

Effects of green tea polyphenols on inflammation and iron status.

Inflammation is an underlying problem for many disease states and has been implicated in iron deficiency (ID). This study aimed to determine whether iron status is improved by epigallocatechin-3-gallate (EGCG) through reducing inflammation. Thirty-two male Sprague-Dawley rats were fed an iron-deficient diet for 2 weeks and then randomly divided into four groups (n 8 each): positive controls, negative controls, lipopolysaccharide (LPS, 0⋅5 mg/kg body weight), and LPS + EGCG (LPS plus 600 mg EGCG/kg diet) for 3 additional weeks. The study involved testing two control groups, both treated with saline. One group (positive control) was fed a regular diet containing standard iron, while the negative control was fed an iron-deficient diet. Additionally, two treatment groups were tested. The first group was given LPS, while the second group was administered LPS and fed an EGCG diet. Iron status, hepcidin, C-reactive protein (CRP), serum amyloid A (SAA), and interleukin-6 (IL-6) were measured. There were no differences in treatment groups compared with control in CRP, hepcidin, and liver iron concentrations. Serum iron concentrations were significantly lower in the LPS (P = 0⋅02) and the LPS + EGCG (P = 0⋅01) than in the positive control group. Compared to the positive control group, spleen iron concentrations were significantly lower in the negative control (P < 0⋅001) but not with both LPS groups. SAA concentrations were significantly lower in the LPS + EGCG group compared to LPS alone group. EGCG reduced SAA concentrations but did not affect hepcidin or improve serum iron concentration or other iron markers.

PS-B09-9: ENOS-NO PATHWAY REGULATES INFLAMMASOME ACTIVATION IN DIABETIC MICE VIA MODULATION OF CEBP TRANSLOCATION

Background: Diabetic kidney disease (DKD) is one of the most common vascular complications of diabetes mellitus (DM). Endothelial dysfunction is also a common pathophysiologic mechanism of diabetic vascular complications. Nitric oxide (NO) produced in the endothelial cells by endothelial nitric oxide synthase (eNOS) is an important mediator for the maintenance of vascular homeostasis. In persons with diabetes, eNOS produces superoxide anion rather than nitric oxide, referred to as eNOS uncoupling which may contribute to endothelial dysfunction, albuminuria, and diabetic nephropathy. eNOS uncoupling can then lead to dysfunction of the eNOS-NO-pathway in diabetic glomeruli. To determine the mechanism of regulation of inflammasome activation by eNOS-NO pathway in DKD, we used eNOS deficient mice and eNOS-ASC double knockout mice. Methods: We used wild-type (WT) and eNOS-deficient mice (eNOSKO) to determine the role of the eNOS-NO pathway. Diabetes was induced by a single intraperitoneal injection of streptozocin. We then divided the mice into four groups: WT, WT/STZ, eNOSKO, and eNOS/STZ. Four weeks after the induction of DM, the mice were sacrificed, and their kidney tissues were harvested. Urinary albumin excretion was checked before the sacrifice, and glomerular damage was assessed by periodic acid-Schiff (PAS) staining. To determine the role of inflammasome on the development of glomerulopathy, we generated eNOS-ASC double knockout mice (eNOS-ASC-DKO). Also, to investigate inflammasome activation in glomeruli, the glomeruli were isolated from the kidney tissue by using Dynabeads. Results: Urinary albumin excretion was increased in WT/STZ compared with WT. These urinary albumin excretions were increased much more in eNOS/STZ than in WT/STZ, while the glomeruli were more damaged in eNOSKO/STZ than in WT/STZ. Urinary albumin excretion was lower in eNOS-ASC-DKO/STZ compared with eNOSKO/STZ, and the glomerular damage also was ameliorated in eNOS-ASC-DKO after treatment with STZ. S-Nitroso glutathione (GSNO) attenuated NLRP3 inflammasome activation after treatment with LPS-ATP. These results indicate that NO directly inhibits NLRP3-inflammasome activation. In addition, GSNO decreased the expression of inflammasome-related genes. To investigate the detailed the mechanism (epigenetic regulation), we performed Assay for Transposase-Accessible Chromatin-Sequencing (ATAC-seq) analysis. The binding region of the transcription factor CCAAT/ enhancer binding protein (C/EBP) β; was closed in the LPS+GSNO group significantly, compared with the LPS group. Conclusion: In eNOS-deficient mice, inflammasomes were activated, and glomerular injuries were exacerbated. However in eNOS-ASC-STZ mice, the glomerular damage were attenuated. Our data suggested that eNOS/NO signaling attenuates inflammasome activation via regulation of CEBP β;, leading to the suppressed development of DKD.

Detection of melatonin protective effects in sepsis via argyrophilic nucleolar regulatory region-associated protein synthesis and TLR4/NF-κB signaling pathway.

In this study, the protective effect of melatonin was investigated in lipopolysaccharide induced sepsis model. Twenty-eight rats were randomly divided: Control, Melatonin, LPS and LPS + Melatonin. After LPS application, surgically remove kidney and liver tissues. The level of malondialdehyde (MDA) an oxidative stress marker and the immunoreactivity of Toll-like receptor-4 (TLR4), tumor necrosis factor-α (TNF-α), and transcription factor NF-κB were evaluated immunohistochemically. Expression levels for TLR4, TNF-α, NF-kB, IL-1β (interleukin 1 beta), and IL-6 (interleukin 6) were evaluated. Additionally, Argyrophilic NOR staining was performed in tissues. Vacuolization and inflammation were more intense in the kidney and liver sections in the LPS group compared to the other groups. It was observed that vacuolization and inflammation were decreased in LPS + Melatonin applied groups. It was determined that glomerular damage was increased in the LPS and LPS-melatonin groups, but the damage rate LPS-Melatonin group was decrease in the LPS group. It was determined that the MDA level in tissues of the LPS group was importantly increased compared to other groups. Additionally, TAA/NA ratio statistically significant differences were discovered between the groups. This study supports the potential protective effects of 10mg/kg melatonin by modulating critical markers of local immune reaction in a model of LPS-induced sepsis.

Selenium exerts protective effects on inflammatory cardiovascular damage: molecular aspects via SIRT1/p53 and Cyt-c/Cas-3 pathways

Systemic inflammatory response could affect many systems. Cardiac dysfunction develops due to cardiovascular system damage and could be mortal. Selenium is a trace element that can be used as a dietary supplement and has antioxidant, anti-inflammatory, and anti-apoptotic properties. This study aims to evaluate the protective effects of selenium on cardiovascular damage via silenced information regulator 1 (SIRT1)/p53 and cytochrome C (Cyt-c)/ caspase-3 (Cas-3) pathways. Thirty-two rats were randomly divided into 4 groups as control, LPS (0.1mg/kg, intraperitoneally(i.p.), 2-7 days) and LPS + Selenium (LPS-0.1mg/kg, i.p., 2-7 days, selenium - 100µg/kg, i.p., 1-7 days) and selenium (100µg/kg, i.p., 1-7 days) group. On the 8th day of the experiment, rats were sacrificed. Blood samples and half of the left ventricles were collected for biochemical and genetic analysis. The remaining left ventricles and aorta were taken for histological and immunohistochemical analysis. In the LPS group myocardial hemorrhages, hyperemia, and endothelial cell loss were observed. Also, Cas-3 and vascular endothelial growth factor (VEGF) expressions; creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor-alpha (TNF-α), ischemia modified albumin (IMA), total oxidant status (TOS), oxidative stress index (OSI) levels; p53, Cyt-c, Cas-3 mRNA expressions increased while total antioxidant status (TAS) levels, glutathione peroxidase (GPx) activity, SIRT1 mRNA expression decreased. Selenium treatment reversed all these changes. Selenium showed protective effects on cardiovascular injury via regulating SIRT1/p53 and Cyt-c/Cas-3 pathways. This study enlightened the possible usage of selenium on cardiotoxicity.

WP1130 relieves septic shock in mice by inhibiting NLRP3 inflammasome activation

To investigate the mechanism by which the small molecule compound WP1130 inhibits NLRP3 inflammasome activation and alleviates septic shock. Mouse bone marrow-derived macrophages (BMDM) and human THP-1 cells were pre-treated with WP1130 before stimulation with different NLRP3 inflammasome agonists (Nigericin, ATP, MSU and intracellular LPS transfection), and AIM2 inflammasomes were activated with poly A: T. The levels of caspase-1 and IL-1β in the cell culture supernatant were determined using Western blotting and ELISA, and mitochondrial damage in the cells was observed using confocal microscopy. In the animal experiment, male C57BL/6 mice were randomized into blank control group, septic shock group (LPS group) and WP1130 treatment group (WP1130+LPS group), and the levels of IL-1β and TNF-α in the serum and peritoneal cavity were detected using ELISA. In murine BMDM and human THP-1 cells, WP1130 significantly inhibited NLRP3 agonists-induced caspase-1 and IL-1β secretion in a dose-dependent manner (P < 0.05) but did not obviously affect the secretion of such inflammatory factors as IL-6 and TNF-α that were not associated with inflammasomes (P>0.05). Treatment with WP1130 did not significantly affect poly A: T-induced activation of AIM2 inflammasomes (P>0.05) or induce obvious changes in mitochondrial damage, an upstream signal of NLRP3 inflammasome activation. In the mouse model of LPS-induced septic shock, WP1130 treatment significantly reduced the level of IL-1β (P < 0.05) without obviously affecting TNF-α level either in the serum or in the peritoneal cavity (P>0.05). WP1130 specifically inhibits NLRP3 inflammasome activation to alleviate LPS-induced septic shock in mice.

Effects of puerarin on the intervertebral disc degeneration and biological characteristics of nucleus pulposus cells

Context Intervertebral disc degeneration (IDD) is the pathological basis of spinal degenerative diseases. Puerarin (PU) is an isoflavonoid with functions and medicinal properties. Objective To explore the effect of PU on IDD and its potential mechanism of action. Materials and methods Sprague-Dawley (SD) rats were divided into sham, IDD, low PU, and high PU groups. Rat nucleus pulposus cells (NPCs) were isolated and divided into control, IL-1β, 100 and 200 μmol/mL PU, TAK-242 (TLR4 inhibitor), or 200 μmol/mL PU + LPS (TLR4 activator) groups. The water content, inflammatory factors, proliferation activity, TLR4/NF-κB pathway activity, apoptosis rate, protein expression of apoptosis, and histology of the extracellular matrix (ECM) were analysed. Results In vivo: Compared with the IDD group, disorganization of intervertebral disc tissue was significantly improved, water content (2.80 ± 0.24 mg, 3.91 ± 0.31 mg vs. 2.02 ± 0.21 mg) and expression levels of collagen II and aggrecan were significantly increased, and the levels of inflammatory factors and the expression levels of TLR4, MyD88, and p-p65 were significantly decreased in IDD rats treated with PU. In vitro: Compared with the IL-1β group, the proliferation activity of IL-1β-treated NPCs and the expression of collagen II and aggrecan were significantly increased, while the apoptosis rate, levels of inflammatory factors, and the expression levels of TLR4, MyD88, and p-p65 were significantly decreased in IL-1β-treated NPCs treated with PU. LPS reversed the biological function changes of IL-1β-treated NPCs induced by PU. Conclusions PU can delay the progression of IDD by inhibiting activation of the TLR4/NF-κB pathway.

The anti-inflammatory cytokine IL-22 exhibited a proinflammatory effect in the ovary aiding with the parturition of ovoviviparous black rockfish (Sebastes schlegelii)

IntroductionAs a unique type of immunological process, pregnancy and subsequent parturition are associated with a series of inflammatory events. As an anti-inflammatory cytokine, IL-22 has been proven to participate in the parturition process in mammals. However, less is known about ovoviviparous teleosts. To investigate the role of IL-22 in fish parturition, black rockfish, an ovoviviparous teleost, was used. This fish is unique in that it provides over 60% nutrition supply for over 50 thousand embryos at the same time though a placenta-like structure during pregnancy while delivering all fries in two hours.MethodsSequence alignment, phylogenetic tree analysis and homology modeling were performed on IL-22 and its receptor. In situ hybridization demonstrated ovarian localization of il22 and il22r1. The protein of rIL-22 was obtained through the prokaryotic expression. RNA-seq analysis was performed on black rockfish ovarian cells treated with rIL-22.ResultsIn situ hybridization results showed that both il22 and il22ra1 were localized in the perinatal follicle layer and embryonic envelope. The il22 expression level was significantly increased during parturition compared to before and after parturition (P&amp;lt;0.05). To further understand the mechanism of IL-22 in parturition, rIL-22 was obtained. The downstream variation genes in primary cultured ovarian cells in the perinatal period were analyzed according to the transcriptomic results. A total of 168 differentially expressed genes (DEGs) were identified in the rIL-22 group compared with the PBS control group (padj &amp;lt; 0.05 and |log2FoldChange| 1). Of those, 134 DEGs were identified in the LPS and rIL-22 treatment groups, including chemokine, cytokine, and PG synthesis. 34 DEGs were mainly identified in immune-related pathways and reorganization of the cytoskeleton responded only to rIL-22 stimulation but not LPS. KEGG pathways mainly included immune response, PG synthesis, cell death and angiogenesis (p value &amp;lt; 0.05).DiscussionTaken together, our results indicated that IL-22 plays an important role in the parturition of black rockfish by upregulating PG synthesis and increasing chemokine, proinflammatory factor, as well as PG synthetase levels.

Pectin supplement alleviates gut injury potentially through improving gut microbiota community in piglets.

As pectin is widely used as a food and feed additive due to its tremendous prebiotic potentials for gut health. Yet, the underlying mechanisms associated with its protective effect remain unclear. Twenty-four piglets (Yorkshire × Landrace, 6.77 ± 0.92 kg) were randomly divided into three groups with eight replicates per treatment: (1) Control group (CON), (2) Lipopolysaccharide-challenged group (LPS), (3) Pectin-LPS group (PECL). Piglets were administrated with LPS or saline on d14 and 21 of the experiment. Piglets in each group were fed with corn-soybean meal diets containing 5% citrus pectin or 5% microcrystalline cellulose. Our result showed that pectin alleviated the morphological damage features by restoring the goblet numbers which the pig induced by LPS in the cecum. Besides, compared with the LPS group, pectin supplementation elevated the mRNA expression of tight junction protein [Claudin-1, Claudin-4, and zonula occludens-1 (ZO-1)], mucin (Muc-2), and anti-inflammatory cytokines [interleukin 10 (IL-10), and IL-22]. Whereas pectin downregulated the expression of proinflammatory cytokines (IL-1β, IL-6, IL-18), tumor necrosis factor-&alpha (TNF-α), and NF-κB. What is more, pectin supplementation also significantly increased the abundance of beneficial bacteria (Lactobacillus, Clostridium_sensu_stricto_1, Blautia, and Subdoligranulum), and significantly reduced the abundance of harmful bacteria, such as Streptococcus. Additionally, pectin restored the amount of short-chain fatty acids (SCFAs) after being decreased by LPS (mainly Acetic acid, Propionic acid, and Butyric acid) to alleviate gut injury and improve gut immunity via activating relative receptors (GPR43, GPR109, AhR). Mantel test and correlation analysis also revealed associations between intestinal microbiota and intestinal morphology, and intestinal inflammation in piglets. Taken together, dietary pectin supplementation enhances the gut barrier and improves immunity to ameliorate LPS-induced injury by optimizing gut microbiota and their metabolites.

Protective effects of chicoric acid on LPS-induced endometritis in mice via inhibiting ferroptosis by Nrf2/HO-1 signal axis.

Chicoric acid (CA), a natural phenolic acid extracted from Mediterranean vegetable chicory, has anti-oxidative effect. We aimed to investigate the effects of CA on endometritis and clarify the underlying mechanism. C57BL/6 mice were divided into five groups: control group, LPS group, and LPS+CA groups. All mice except control group were infused of LPS into the uterus. The mice of LPS+CA groups were intraperitoneally injected CA 1h before LPS challenge. CA significantly alleviatedLPS-induced pathological damage, MPO activity, and inflammatory cytokine production. CA significantly suppressed ferroptosis in LPS-induced endometritis. CA also attenuated LPS-induced NF-κB activation. Furthermore, Nrf2 and HO-1 expression were increased by CA. Moreover, the inhibition of CA on LPS-induced endometritis and ferroptosis were markedly prevented in Nrf2 knockdown mice. In conclusion, the results suggested CA protected mice against LPS-induced endometritisthrough inhibiting ferroptosis via Nrf2/HO-1 signaling pathway.

Resolvin D1 attenuates sepsis induced acute kidney injury targeting mitochondria and NF-κB signaling pathway

BackgroundAcute kidney injury is a highly common and multifactorial renal disease resulting in significant morbidity and mortality, especially sepsis-induced acute kidney injury. There is no effective therapy available to treat or prevent sepsis-induced acute kidney injury. One of the specialized pro-resolving mediators, Resolvin D1 exhibits special anti-inflammatory effects in several inflammatory disease models, but there is little evidence about the effect and mechanism of Resolvin D1 in sepsis-induced acute kidney injury. MethodsWe conducted experiments to explore the effect and mechanism of Resolvin D1 in sepsis-induced acute kidney injury. In vitro, human proximal tubular epithelial cells were used to test the apoptosis ratio, cell viability and reactive oxygen species level. In vivo, C57BL/6 mice were injected with lipopolysaccharide to establish a sepsis-induced acute kidney injury model. Renal function and structure, apoptosis ratio of kidney cells, mitochondrial structure and function and related protein and gene levels were assessed. ResultsIn vitro, the resolvin D1-treated group showed higher cell viability and lower reactive oxygen species levels and apoptosis ratios than the LPS group. In vivo, Resolvin D1 can not only improve renal function and mitochondrial function but also reduce the apoptosis ratio, while mediating mitochondrial dynamics and inhibiting NF-κB pathway. ConclusionsResolvin D1 has a good renoprotective effect by maintaining mitochondrial dynamics and inhibiting the NF-κB pathway.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

A postpartum enriched environment rescues impaired cognition and oxidative markers in aged mice with gestational inflammation.

Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process. In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50μg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR. As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR. Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Abstract 13188: Co-Translational Profiling in the Cardiac Endothelium During the Development of Sepsis

Introduction: The production of functional proteins is a multistep process whereby key maturation and regulatory processes occur co-translationally by coupling mRNA translation and nascent polypeptide chain maturation on the ribosome. However, limited data exist regarding co-translational regulation in the cells of the intact cardiovascular system. To overcome this limitation, we adapted the translating ribosome affinity purification (TRAP) approach using the HA-tagged ribosomal protein L22 reporter mouse (RiboTag) crossed with an inducible endothelial-specific Cdh5creERT2 mouse ( RiboTag EC ). Hypothesis: We hypothesized this model would permit the simultaneous immunoprecipitation (IP) of mRNA and nascent proteins for RNAseq and proteomic analysis from the coronary endothelium. Methods: Mice were injected with E. coli lipopolysaccharide (LPS) (6mg/Kg, i.p. 12 hours) to induce sepsis and perturb endothelial cell function. Hearts were homogenized, a small amount (~10%) was used for RNA-Seq and proteomics input controls (IN), and the remainder was used to IP ribosomal bound polyA+ mRNA and nascent proteins. Eluted proteins were digested, labeled with isobaric tags for quantification, and analyzed by MS3 with a Thermo Scientific Fusion Lumos Orbitrap. Results: Principal components analysis of the RNAseq data showed clear separation of the IP (endothelial cells) and IN (whole heart) transcriptomes between control and LPS groups. Transcripts characteristic of endothelial cells (EC) were enriched, whereas markers characteristic of cardiomyocytes, fibroblasts, and smooth muscle cells were depleted. Overall, &gt;7000 proteins were identified, and initial analysis showed similar depletion and enrichment of proteins in the control and LPS hearts. Alignment of transcript and protein datasets was used for bioinformatic analysis. A hierarchical view of one of the top networks in which concordant expression of both EC transcripts and proteins identified interferon-gamma (IFNG) as an upstream signal in the pathogenesis of sepsis. Conclusions: Our “functional translatome” approach is a new discovery platform for a deeper predictive analysis of functionally relevant signaling networks and disease pathways in the intact cardiovascular system.

Abstract 15468: Cd81 Interacts With Sodium Chloride Cotransporter in Lps-Induced Preeclampsia Rats and in Cultured Mouse Renal Distal Convoluted Tubule Cells

Kidney regulates blood pressure mainly through salt/water reabsorption affected by sodium transport proteins along nephron. We have reported that placental CD81 plays an important role in the pathogenesis of preelampsia. In order to explore the connection between CD81 and renal sodium transport proteins in preelampsia, we studied their interactions in vivo and in vitro. On the gestation day 18 of lipopolysaccharide-induced preeclamsia model rats with hypertension and albuminuria, the renal protein abundances of NKCC2 (150±14,% of control, n=5-6/group, mean±SE, t-test, same as below), p-NKCC2(567±70%), NCC (254±41) and WNK4 (196±12) were increased together with an increase of CD81 in kidney (225±46) and urinary exosomes(396±100). Renal NCC (215±30) was elevated while NKCC2 tended to increase at 17000g pellet in LPS group. There was no change of both NKCC2 and NCC at 200,000g pellet. In kidney homogenates, CD81 was coimmunoprecipitated with NKCC2, NCC, not NHE3 while NKCC2, NCC were coimmunoprecipitated with CD81 not CD63. In renal plasma-membrane-enriched fraction NCC, not NKCC2, was also increased in preeclampsia rats. In the apical membrane of renal tubules CD81 was colocalized with NCC while minimum colocalization was found between CD81 and NKCC2. In cultured mouse distal convoluted tubule cells, CD81 was colocalized with NCC while CD81 deletion with siRNA decreased NCC and partially reversed lipopolysaccharide-induced increase of NCC. Furthermore, CD81 was detected only in the urinary exosomes from preeclampsia patients along with increases in NKCC2 (6063±811) and NCC(1085±191)relative to normal pregnant women (n=6/group, t-test). Those findings suggest that CD81 interacts with renal NCC in vivo and in vitro and the increase of renal CD81 may up-regulate NCC, possibly NKCC2 as well, and contribute to the increased blood pressure in preeclampsia.

Lipocalin-2 participates in sepsis-induced myocardial injury by mediating lipid accumulation and mitochondrial dysfunction

Sepsis-induced cardiomyopathy (SIC) is one major cause of death for sepsis but lacks timely diagnosis and specific treatment due to unclear mechanisms. Lipocalin-2 (LCN-2) is a key regulator of lipid metabolism which has been recently proved closely related to sepsis, however, the relationship between LCN-2 and septic myocardial injury remains unknown. We aim to explore the role of LCN-2 in the pathological progress of SIC based on clinical and laboratory evidence. Consecutive patients admitted to the intensive care unit (ICU) from August 2021 to April 2022 fulfilling the criteria of severe sepsis were included. The level of LCN-2 in plasma was assayed and analyzed with clinical characteristics. Biostatistical analysis was performed for further identification and pathway enrichment. Mouse model for SIC was thereafter established, in which plasma and tissue LCN-2 levels were tested. RNA sequencing was used for verification and to reveal the possible mechanism. Mitochondrial function and intracellular lipid levels were assayed to further assess the biological effects of targeting LCN-2 in cardiomyocytes with small interference RNAs (siRNAs). The level of LCN-2 in plasma was markedly higher in patients with severe sepsis and was associated with higher cardiac biomarkers and lower LVEF. In the in vivo experiment, circulating LCN-2 from plasma was found to increase in SIC mice. A higher level of LCN-2 transcription in myocardial tissue was also found in SIC and showed a clear time relationship. RNA sequencing analysis showed the level of LCN-2 was associated with several gene-sets relevant to mitochondrial function and lipid metabolism-associated pathways. The suppression of LCN-2 protected mitochondrial morphology and limited the production of ROS, as well as restored the mitochondrial membrane potential damaged by LPS. Neutral lipid staining showed prominent lipid accumulation in LPS group, which was alleviated by the treatment of siLCN2. The level of LCN-2 is significantly increased in SIC at both circulating and tissue levels, which is correlated with the severity of myocardial injury indicators, and may work as an early and great predictor of SIC. LCN-2 probably participates in the process of septic myocardial injury through mediating lipid accumulation and affecting mitochondrial function.

Proteomics analysis of Astragalus polysaccharide on TLR4-activated lung cancer cell-derived exosomes

Astragalus polysaccharide(APS), one of the main active components of Astragali Radix, plays an anti-tumor effect by regulating the inflammatory microenvironment of tumors. Exosomes are small extracellular vesicles with a diameter ranging from 50 to 200 nm and carry several biological components from parental cells such as nucleic acids and proteins. When combined with recipient cells, they play an important role in intercellular communication and immune response. In this study, exosomes released from H460 cells at the inflammatory state or with APS addition activated by Toll-like receptor 4(TLR4) were extracted by ultracentrifugation and characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. The exosomal proteins derived from H460 cells in the three groups were further analyzed by label-free proteomics, and 897, 800, and 911 proteins were identified in the three groups(Con, LPS, and APS groups), 88% of which belonged to the ExoCarta exosome protein database. Difference statistical analysis showed that the expression of 111 proteins was changed in the LPS group and the APS group(P&lt;0.05). The biological information analysis of the differential proteins was carried out. The molecular functions, biological processes, and signaling pathways related to the differential proteins mainly involved viral processes, protein binding, and bacterial invasion of proteasome and epithelial cells. Key differential proteins mainly included plasminogen activator inhibitor-1, laminin α5, laminin α1, and CD44, indicating that tumor cells underwent systemic changes in different states and were reflected in exosomes in the inflammatory microenvironment. The analysis results also suggested that APS might affect the inflammatory microenvironment through the TLR4/MyD88/NF-κB signaling pathway or the regulation of the extracellular matrix. This study is conducive to a better understanding of the mechanism of tumor development in the inflammatory state and the exploration of the anti-inflammatory effect of APS at the exosome level.

Triggering Receptor Expressed on Myeloid Cell-2 Protects PC12 Cells Injury by Inhibiting BV2 Microglial Activation.

Microglia play a crucial role in the activation of immune defense mechanism as the resident macrophages in the central nervous system (CNS). Microglia can eliminate damaged neurons, plaques, and other infectious agents. Triggering receptor expressed on myeloid cell-2 (TREM-2) speculates to be beneficial in preventing inflammation-induced bystander damage of neurons. However, the precise molecular mechanisms underlying the regulation of TREM-2 on neurons are not clarified. We cultured PC12 cells with conditioned medium which was the supernatant of LPS-treated BV2 cells and six groups of PC12 cells (control group, LPS group, TREM-2 WT + LPS group, TREM-2 over-expression + LPS group, siRNA control + LPS group, and siRNA TREM-2 + LPS group) were investigated. The mRNA levels of inflammatory mediators: Nitric oxide synthase (iNOS) and Arginase-1(Arg-1) were quantified by using RT-PCR. Assessment of apoptosis in PC12 cells mediated by BV2 microglia was analyzed using TUNEL assays. The result showed that LPS stimulation significantly enhanced inducible iNOS (M1) production in BV2 cells (P < 0.01), and increased PC12 cells apoptosis (P < 0.01), while reduced the production of Arg-1 (M2) in BV2 cells (P < 0.01). These effects were attenuated by TREM-2 over-expression, but enhanced by TREM-2 silencing. It indicated that TREM-2 inhibited LPS-mediated neuronal apoptosis by down-regulating iNOS and up-regulating the expression of Arg-1 in BV2 microglia. Therefore, our findings may provide new insights in the regulation of TREM-2 on neuronal apoptosis via BV2 microglial M1/M2 modulation.