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This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

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SUMMARY: Systemic inflammatory response syndrome (SIRS) is a potentially fatal reaction to various forms of tissue damage and infections that cause damage to various organs. Furthermore, the brain is damaged earlier than other organs, resulting in diffuse brain dysfunction. The central clinical symptom of SIRS is delirium and emotional changes are involved in disease development. Although the amygdala is known to play a major role, the mechanisms underlying emotional changes in the early stages of SIRS have not been elucidated. Therefore, changes to dopamine levels in the amygdala were observed using an in vivo model of lipopolysaccharide (LPS)- induced SIRS to clarify the biochemical mechanisms activated in the early stages of SIRS. Extracellular dopamine was collected from the amygdala of free moving rats via microdialysis and then analyzed by high-performance liquid chromatography. In addition, emotional changes were assessed with the open field and sucrose preference tests. In the LPS group, dopamine release in the amygdala increased remarkably immediately after LPS administration, peaking at 120 min. Thereafter, dopamine release temporarily decreased, but then significantly increased again after 180 min. The present results suggest that diffuse brain dysfunction in the early stages of SIRS may involve altered dopamine levels in the amygdala.

El síndrome de respuesta inflamatoria sistémica (SRIS) es una reacción potencialmente fatal a diversas formas de daño tisular e infecciones que causan injuria a varios órganos. Además, el cerebro se daña antes que otros órganos, lo que provoca una disfunción cerebral difusa. El síntoma clínico central del SIRS es el delirio y los cambios emocionales están involucrados en el desarrollo de la enfermedad. Aunque se sabe que la amígdala desempeña un papel importante, no se han dilucidado los mecanismos que subyacen a los cambios emocionales en las primeras etapas del SRIS. Por lo tanto, en el estudio se provocaron cambios en los niveles de dopamina en la amígdala utilizando un modelo in vivo de SRIS inducido por lipopolisacáridos (LPS) para dilucidar los mecanismos bioquímicos activados en las primeras etapas del SRIS. La dopamina extracelular se recogió de la amígdala de ratas en movimiento libre mediante microdiálisis y luego se analizó mediante cromatografía líquida de alta resolución. Además, se evaluaron los cambios emocionales con las pruebas de campo abierto y de preferencia de sacarosa. En el grupo de LPS, la liberación de dopamina en la amígdala aumentó de manera notable inmediatamente después de la administración de LPS, alcanzando un máximo a los 120 minutos. A partir de entonces, la liberación de dopamina disminuyó temporalmente, pero luego volvió a aumentar significativamente después de 180 min. Los resultadosactuales sugieren que la disfunción cerebral difusa en las primeras etapas del SIRS puede implicar niveles alterados de dopamina en la amígdala.

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OBJECTIVE@#To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments.@*METHODS@#A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed.@*RESULTS@#FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05).@*CONCLUSION@#FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.

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OBJECTIVE@#To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism.@*METHODS@#Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis.@*RESULTS@#DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01).@*CONCLUSIONS@#DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.

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Objective To investigate the effect of interleukin-6 (IL-6) on the phagocytosis of MH-S alveolar macrophages and its related mechanisms. Methods A mouse acute lung injury (ALI) model was constructed by instilling lipopolysaccharide (LPS) into the airway. ELISA was used to detect the content of IL-6 in bronchoalveolar lavage fluid (BALF). In vitro cultured MH-S cells, in the presence or absence of signal transducer and activator 3 of transcription(STAT3) inhibitor Stattic (5 μmol/L), IL-6 (10 ng/mL~500 ng/mL) was added to stimulate for 6 hours, and then incubated with fluorescent microspheres for 2 hours. The phagocytosis of MH-S cells was detected by flow cytometry. Western blot analysis was used to detect the expression levels of phosphorylated Janus kinase 2 (p-JAK2), phosphorylated STAT3 (p-STAT3), actin-related protein 2 (Arp2) and filamentous actin (F-actin). Results The content of IL-6 in BALF was significantly increased after the mice were injected with LPS through the airway. With the increase of IL-6 stimulation concentration, the phagocytic function of MH-S cells was enhanced, and the expression levels of Arp2 and F-actin proteins in MH-S cells were increased. The expression levels of p-JAK2 and p-STAT3 proteins increased in MH-S cells stimulated with IL-6(100 ng/mL). After blocking STAT3 signaling, the effect of IL-6 in promoting phagocytosis of MH-S cells disappeared completely, and the increased expression of Arp2 and F-actin proteins in MH-S cells induced by IL-6 was also inhibited. Conclusion IL-6 promotes the expression of Arp2 and F-actin proteins by activating the JAK2/STAT3 signaling pathway, thereby enhancing the phagocytic function of MH-S cells.

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OBJECTIVE@#The aim of this study is to explore the potential modulatory role of quercetin against Endotoxin or lipopolysaccharide (LPS) induced septic cardiac dysfunction.@*METHODS@#Specific pathogen-free chicken embryos ( n = 120) were allocated untreated control, phosphate buffer solution (PBS) vehicle, PBS with ethanol vehicle, LPS (500 ng/egg), LPS with quercetin treatment (10, 20, or 40 nmol/egg, respectively), Quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the hearts of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting.@*RESULTS@#They demonstrated that the heart presented inflammatory responses after LPS induction. The LPS-induced higher mRNA expressions of inflammation-related factors (TLR4, TNFα, MYD88, NF-κB1, IFNγ, IL-1β, IL-8, IL-6, IL-10, p38, MMP3, and MMP9) were blocked by quercetin with three dosages. Quercetin significantly decreased immunopositivity to TLR4 and MMP9 in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of TLR4, IFNγ, MMP3, and MMP9 when compared with the LPS group. Quercetin treatment prevented LPS-induced increase in the mRNA expression of Claudin 1 and ZO-1, and significantly decreased protein expression of claudin 1 when compared with the LPS group. Quercetin significantly downregulated autophagy-related gene expressions (PPARα, SGLT1, APOA4, AMPKα1, AMPKα2, ATG5, ATG7, Beclin-1, and LC3B) and programmed cell death (Fas, Bcl-2, CASP1, CASP12, CASP3, and RIPK1) after LPS induction. Quercetin significantly decreased immunopositivity to APOA4, AMPKα2, and LC3-II/LC3-I in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of AMPKα1, LC3-I, and LC3-II. Quercetin significantly decreased the protein expression to CASP1 and CASP3 by immunohistochemical investigation or Western blotting in treatment group when compared with LPS group.@*CONCLUSION@#Quercetin alleviates cardiac inflammation induced by LPS through modulating autophagy, programmed cell death, and myocardiocytes permeability.

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OBJECTIVE@#To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats.@*METHODS@#Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β 1 (TGF-β 1), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ.@*RESULTS@#The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β 1, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression.@*CONCLUSION@#VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.

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Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P<0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a "septicemia hemorrágica" (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P < 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

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Purpose: Lipopolysaccharides is well-known in the acute renal injury process. It causes widespread activation of inflammatory cascades. Tumor necrosis factor (TNF)-α and interleukin (Il)-6 are essential proinflammatory cytokines that can induce the production of other cytokines in host response. Adalimumab suppresses TNF-α, IL-1ß, and IL-6. We aimed to evaluate whether adalimumab would prevent the toxicity of lipopolysaccharide on the rat renal tissue. Methods: Adult female Wistar rats were divided into four groups. To the control group, only intraperitoneal saline injection procedure was carried out. For adalimumab group, adalimumab was injected at a dose for two days. For lipopolysaccharide group, animals were injected with lipopolysaccharide (a dose). For lipopolysaccharide-adalimumab group, animals were given adalimumab treatment before the injection of lipopolysaccharide. Histopathological changes and immunohistochemical analysis for TNF-α and IL-6 were determined. Results: The pathological changes and immunohistochemical staining for TNF-α or IL-6 were similar for control and adalimumab groups (p > 0.05). The lipopolysaccharide group had significantly higher distorted features in the renal tissues (p < 0.001), and also significantly prominent immunohistochemical staining for TNF-α or IL-6 (0.003), compared to the control group. No severe pathological feature was detected in the lipopolysaccharide-adalimumab group, but moderate necrosis was found in all cases (p = 0.003). TNF-α staining and IL-6 staining in the lipopolysaccharide group was found to significantly prominent compared to lipopolysaccharide-adalimumab group (p = 0.013). Conclusions: Because of its anti-inflammatory property, adalimumab pretreatment may have protective effects on experimental kidney injury. Adalimumab could be considered as a protective agent to acute effects of lipopolysaccharide induced renal injury.

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La alteración de la microbiota intestinal es uno de los factores claves que afecta el inicio y la progresión de esteatosis hepática, al aumentar la permeabilidad intestinal e inducir un aumento circulante de la endotoxina lipopolisacárido (LPS). El LPS puede llegar al hígado a través de la vena porta, donde desencadena procesos inflamatorios y pro-oxidantes. Por otro lado, alimentos ricos en aceites esenciales podrían tener la capacidad de contrarrestar los efectos deletéreos del LPS a nivel hepático. El objetivo de este estudio fue evaluar el efecto del aceite de rosa mosqueta (ARM) en la prevención de procesos inflamatorios y oxidantes en hepatocitos humanos HepG2 tratados con LPS. Células HepG2 fueron expuestas a diferentes dosis de ARM (1,5, 3, 4,5, 6 y 7,5 µg/mL) por 24 horas y luego estimuladas con LPS (100 ng/mL) por 4 horas. Se evaluaron los niveles de expresión de citoquinas proinflamatorias (TNF-α, IL-6, IL-1ß, CCL2) y de la proteína antioxidante hemo oxigenasa 1 (HO-1), además de los niveles de activación de NF-κB. El tratamiento con 6 µg/mL de ARM resultó en un incremento en la viabilidad celular. El pretratamiento con ARM (1,5 - 4,5 µg/mL) fue efectivo para disminuir la expresión (TNF-α, IL-6 y CCL2) y secreción (TNF-α) de citoquinas proinflamatorias. Sin embargo, dosis altas de ARM incrementaron la activación de NF- κB y disminuyeron la expresión de HO-1 en células tratadas con LPS. Estos resultados sugieren un efecto antinflamatorio y un posible rol en el proceso antioxidante para dosis bajas de ARM en hepatocitos humanos frente al daño con LPS.

Rosa mosqueta (Rosa Rubiginosa) oil decreases inflammatory markers in human liver HepG2 cells treated with lipopolysaccharide. Gut microbiota alteration is one of the key factors affecting the onset and progression of hepatic steatosis by enhancing intestinal permeability and inducing an increase in circulating endotoxin lipopolysaccharide (LPS). LPS reaches the liver through the portal vein, where triggers inflammatory and pro-oxidant processes. On the other hand, essential oils-rich foods may have the capacity to abolish the deleterious effects of LPS in the liver. The objective of this study was to evaluate the effect of rosa mosqueta oil (ARM) in the prevention of inflammatory and oxidative processes in human hepatic cells HepG2 treated with LPS. HepG2 cells were exposed to different doses of ARM (1.5, 3, 4.5, 6 y 7.5 µg/mL) for 24 hours and then stimulated with LPS (100 ng/mL) for 4 hours. NF-κB activation, proinflammatory cytokines expression level (TNF-α, IL-6, IL-1ß, CCL2), and the antioxidant protein expression of hem oxygenase 1 (HO-1) were evaluated. Treatment with 6 µg/mL increased cellular viability. ARM pretreatment was effective in decreasing the expression (TNF-α, IL-6 y CCL2) and secretion (TNF-α) of proinflammatory cytokines. On the other hand, high doses of ARM increased NF-κB activation and decreased HO-1 expression in cells treated with LPS. These results may suggest an anti-inflammatory effect and a possible role in the antioxidant process in the case of the lower doses of ARM in human hepatic cells treated with LPS.

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Sepsis is a complex syndrome caused by multiple pathogens and involves multiple organ failure, particularly spleen dysfunction. In 2017, the worldwide incidence was 48.9 million sepsis cases and 11 million sepsis-related deaths were reported (Rudd et al., 2020). Inflammation, oxidative stress, and apoptosis are the most common pathologies seen in sepsis. Liensinine (LIE) is a bisbenzylisoquinoline-type alkaloid extracted from the seed embryo of Nelumbo nucifera. Lotus seed hearts have high content of LIE which mainly has antihypertensive and antiarrhythmic pharmacological effects. It can exert anti-carcinogenic activity by regulating cell, inflammation, and apoptosis signaling pathways (Manogaran et al., 2019). However, its protective effect from sepsis-induced spleen damage is unknown. In this research, we established a mouse sepsis model induced by lipopolysaccharide (LPS) and investigated the protective effects of LIE on sepsis spleen injury in terms of inflammatory response, oxidative stress, and apoptosis.

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This study was aimed to investigate the effect of hypoxia on lipopolysaccharide (LPS)-induced CXC-chemokine ligand-10 (CXCL10) expression and the underlying mechanism. C57BL/6J mice were randomly divided into control, hypoxia, LPS, and hypoxia combined with LPS groups. The LPS group was intraperitoneally injected with 0.5 mg/kg LPS, and the hypoxia group was placed in a hypobaric hypoxia chamber (simulated altitude of 6 000 m). The serum and hippocampal tissue samples were collected after 6 h of the treatment. The levels of CXCL10 in the serum and hippocampal tissue of mice were detected by ELISA. The microglia cell line BV2 and primary microglia were stimulated with hypoxia (1% O2) and/or LPS (100 ng/mL) for 6 h. The mRNA expression level of CXCL10 and its content in culture supernatant were detected by real-time quantitative PCR and ELISA, respectively. The phosphorylation levels of nuclear factor κB (NF-κB) signaling pathway-related proteins, p65 and IκBα, were detected by Western blot. Moreover, after NF-κB signaling pathway being blocked with a small molecular compound, PDTC, CXCL10 mRNA expression level was detected in the BV2 cells. The results showed that in the LPS-induced mouse inflammatory model, hypoxia treatment could promote LPS-induced up-regulation of CXCL10 in both serum and hippocampus. Compared with the cells treated with LPS alone, the expression of CXCL10 mRNA and the content of CXCL10 in the culture supernatant of BV2 cells treated with hypoxia combined with LPS were significantly increased. The CXCL10 mRNA level of primary microglial cells treated with hypoxia combined with LPS was significantly up-regulated. Compared with the cells treated with hypoxia or LPS alone, the phosphorylation levels of p65 and IκBα in the BV2 cells treated with hypoxia combined with LPS were significantly increased. PDTC blocked the induction of CXCL10 gene expression by LPS in the BV2 cells. These results suggest that hypoxia promotes LPS-induced expression of CXCL10 in both animal and cell models, and NF-κB signaling pathway plays an important role in this process.

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OBJECTIVE@#To compare the efficiency and effect of establishing rat peri-implantitis model by traditional cotton thread ligation and local injection of Porphyromonas gingivalis lipopolysaccharide (LPS) around the implant, as well as the combination of the two methods.@*METHODS@#Left side maxillary first molars of 39 male SD rats were extracted, and titanium implants were implanted after four weeks of healing. After 4 weeks of implant osseointegration, 39 rats were randomly divided into 4 groups. Cotton thread ligation (n=12), local injection of LPS around the implant (n=12), and the two methods combined (n=12) were used to induce peri-implantitis, the rest 3 rats were untreated as control group. All procedures were conducted under 5% isoflurane inhalation anesthesia. The rats were sacrificed 2 weeks and 4 weeks after induction through carbon dioxide asphyxiation method. The maxilla of the rats in the test groups were collected and marginal bone loss was observed by micro-CT. The gingival tissues around the implants were collected for further real time quantitative PCR (RT-qPCR) analysis, specifically the expression of tumor necrosis factor-alpha (TNF-α) as well as interleukin-1β (IL-1β). The probing depth (PD), bleeding on probing (BOP) and gingival index (GI) of each rat in the experimental group were recorded before induction of inflammation and before death.@*RESULTS@#After 4 weeks of implantation, the osseointegration of implants were confirmed. All the three test groups showed red and swollen gums, obvious marginal bone loss around implants. After 2 weeks and 4 weeks of inflammation induction, PD, GI and BOP of the three test groups increased compared with those before induction, but only BOP was statistically significant among the three test groups (P < 0.05). At the end of 2 weeks of inflammation induction, marginal bone loss was observed at each site in the cotton thread ligation group and the combined group. At each site, the bone resorption in the combined group was greater than that in the cotton thread ligation group, but the difference was not statistically significant (P > 0.05), bone resorption was observed at some sites of some implants in LPS local injection group. At the end of 4 weeks of inflammation induction, marginal bone loss was observed at all sites in each group. The marginal bone loss in the cotton thread ligation group and the combined group was greater than that in the LPS local injection group, and the difference was statistically significant (P < 0.05). At the end of 2 weeks and 4 weeks of induction, the expression of TNF-α and IL-1β in the test groups were higher than those in the control group (P < 0.05).@*CONCLUSION@#Compared with local injection of LPS around the implant, cotton thread ligature and the two methods combined can induce peri-implantitis in rats better and faster.

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Sepsis-induced uncontrolled systemic inflammatory response syndrome (SIRS) is a critical cause of multiple organ failure. Acute kidney injury (AKI) is one of the most serious complications associated with an extremely high mortality rate in SIRS, and it lacked simple, safe, and effective treatment strategies. Leontopodium leontopodioides (Willd.) Beauv (LLB) is commonly used in traditional Chinese medicine for the treatment of acute and chronic nephritis. However, it remains unclear whether lipopolysaccharide (LPS) affects LPS-induced AKI. To identify the molecular mechanisms of LLB in LPS-induced HK-2 cells and mice, LLB was prepared by extraction with 70% methanol, while a lipopolysaccharide (LPS)-induced HK-2 cell model and an AKI model were established in this study. Renal histopathology staining was performed to observe the morphology changes. The cell supernatant and kidney tissues were collected for determining the levels of inflammatory factors and protein expression by ELISA, immunofluorescence, and Western blot. The results indicated that LLB significantly reduced the expression of IL-6 and TNF-α in LPS-induced HK-2 cells, as well as the secretion of IL-6, TNF-α, and IL-1β in the supernatant. The same results were observed in LPS-induced AKI serum. Further studies revealed that LLB remarkably improved oxidative stress and apoptosis based on the content of MDA, SOD, and CAT in serum and TUNEL staining results. Notably, LLB significantly reduced the mortality due to LPS infection. Renal histopathology staining results supported these results. Furthermore, immunofluorescence and Western blot results confirmed that LLB significantly reduced the expression of the protein related to the NF-κB signaling pathway and NLRP3, ASC, and Caspase-1 which were significantly increased through LPS stimulation. These findings clearly demonstrated the potential use of LLB in the treatment of AKI and the crucial role of the NF-κB/NLRP3 pathway in the process through which LLB attenuates AKI induced by LPS.

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This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1β, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.

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OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

ABSTRACT

The aim of this study was to investigate the effect and molecular mechanism of Xuebijing Injection in the treatment of sepsis-associated acute respiratory distress syndrome(ARDS) based on network pharmacology and in vitro experiment. The active components of Xuebijing Injection were screened and the targets were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The targets of sepsis-associated ARDS were searched against GeneCards, DisGeNet, OMIM, and TTD. Weishengxin platform was used to map the targets of the main active components in Xuebijing Injection and the targets of sepsis-associated ARDS, and Venn diagram was established to identify the common targets. Cytoscape 3.9.1 was used to build the "drug-active components-common targets-disease" network. The common targets were imported into STRING for the building of the protein-protein interaction(PPI) network, which was then imported into Cytoscape 3.9.1 for visualization. DAVID 6.8 was used for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment of the common targets, and then Weishe-ngxin platform was used for visualization of the enrichment results. The top 20 KEGG signaling pathways were selected and imported into Cytoscape 3.9.1 to establish the KEGG network. Finally, molecular docking and in vitro cell experiment were performed to verify the prediction results. A total of 115 active components and 217 targets of Xuebijing Injection and 360 targets of sepsis-associated ARDS were obtained, among which 63 common targets were shared by Xuebijing Injection and the disease. The core targets included interleukin-1 beta(IL-1β), IL-6, albumin(ALB), serine/threonine-protein kinase(AKT1), and vascular endothelial growth factor A(VEGFA). A total of 453 GO terms were annotated, including 361 terms of biological processes(BP), 33 terms of cellular components(CC), and 59 terms of molecular functions(MF). The terms mainly involved cellular response to lipopolysaccharide, negative regulation of apoptotic process, lipopolysaccharide-mediated signaling pathway, positive regulation of transcription from RNA polyme-rase Ⅱ promoter, response to hypoxia, and inflammatory response. The KEGG enrichment revealed 85 pathways. After diseases and generalized pathways were eliminated, hypoxia-inducible factor-1(HIF-1), tumor necrosis factor(TNF), nuclear factor-kappa B(NF-κB), Toll-like receptor, and NOD-like receptor signaling pathways were screened out. Molecular docking showed that the main active components of Xuebijing Injection had good binding activity with the core targets. The in vitro experiment confirmed that Xuebijing Injection suppressed the HIF-1, TNF, NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways, inhibited cell apoptosis and reactive oxygen species generation, and down-regulated the expression of TNF-α, IL-1β, and IL-6 in cells. In conclusion, Xuebijing Injection can regulate apoptosis and response to inflammation and oxidative stress by acting on HIF-1, TNF, NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways to treat sepsis-associated ARDS.

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Acute lung injury (ALI) is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers, resulting in high incidence and mortality rates. Currently, there is a lack of safe and effective drugs for the treatment of ALI. In a previous clinical study, we observed that Jinyinqingre oral liquid (JYQR), a Traditional Chinese Medicine formulation prepared by the Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings. However, the potential role of JYQR in ALI/acute respiratory distress syndrome (ARDS) and its anti-inflammatory mechanism remains unexplored. Thus, the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI and an in vitro RAW264.7 cell model. JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues. Additionally, JYQR administration led to a noteworthy reduction in total protein levels within the BALF, a decrease in MPAP, and attenuation of pleural thickness. These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI. Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins, namely NLRP3 and GSDMD, as well as proinflammatory cytokine levels in mice and RAW2647 cells. Consequently, JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway. JYQR exerts a protective effect against LPS-induced ALI in mice, and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway.

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Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.

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Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.