LPS-induced Oxidative Stress Research Articles

Blockade of Indoleamine 2,3-Dioxygenase attenuates lipopolysaccharide-induced kidney injury by inhibiting TLR4/NF-κB signaling

Blockade of indoleamine 2,3-dioxygenase (IDO) has been shown to alleviate lipopolysaccharide (LPS)-induced endotoxic shock and reduce sepsis mortality, but its effect on LPS-induced kidney damage has not been reported. Herein, we established a mouse kidney injury model by intraperitoneal injection of 10mg/kg LPS and established an in vitro renal tubular epithelial cell injury model by stimulating TCMK-1 cells with 10mg/L LPS. We found that pretreatment with 1-methyl tryptophan (1-MT), an IDO inhibitor, significantly improved LPS-induced mouse survival, and IDO knockout (KO) mice also had higher survival rates after LPS exposure than wild-type mice. At the same time, IDO KO or pretreatment with 1-MT not only reduced serum creatinine, blood urea nitrogen, renal tubular injury pathological score, but also inflammatory factors and oxidative stress status in serum or kidney of LPS-exposed mice. In vitro, blockade of IDO with 1-MT significantly inhibited LPS-induced apoptosis, inflammation and oxidative stress in TCMK-1 cells. In addition, blockade of IDO significantly inhibited LPS-activated TLR4/NF-κB signaling pathway in kidney of mice or in TCMK-1 cells. In conclusion, our results suggested that blockade of IDO attenuated kidney inflammation, apoptosis and oxidative stress to protect against LPS-induced septic kidney injury via inhibiting the TLR4/NF-κB signaling pathway.

Quercetin Reprograms Immunometabolism of Macrophages via the SIRT1/PGC-1α Signaling Pathway to Ameliorate Lipopolysaccharide-Induced Oxidative Damage.

The redox system is closely related to changes in cellular metabolism. Regulating immune cell metabolism and preventing abnormal activation by adding antioxidants may become an effective treatment for oxidative stress and inflammation-related diseases. Quercetin is a naturally sourced flavonoid with anti-inflammatory and antioxidant activities. However, whether quercetin can inhibit LPS-induced oxidative stress in inflammatory macrophages by affecting immunometabolism has been rarely reported. Therefore, the present study combined cell biology and molecular biology methods to investigate the antioxidant effect and mechanism of quercetin in LPS-induced inflammatory macrophages at the RNA and protein levels. Firstly, quercetin was found to attenuate the effect of LPS on macrophage proliferation and reduce LPS-induced cell proliferation and pseudopodia formation by inhibiting cell differentiation, as measured by cell activity and proliferation. Subsequently, through the detection of intracellular reactive oxygen species (ROS) levels, mRNA expression of pro-inflammatory factors and antioxidant enzyme activity, it was found that quercetin can improve the antioxidant enzyme activity of inflammatory macrophages and inhibit their ROS production and overexpression of inflammatory factors. In addition, the results of mitochondrial morphology and mitochondrial function assays showed that quercetin could upregulate the mitochondrial membrane potential, ATP production and ATP synthase content decrease induced by LPS, and reverse the mitochondrial morphology damage to a certain extent. Finally, Western blotting analysis demonstrated that quercetin significantly upregulated the protein expressions of SIRT1 and PGC-1α, that were inhibited by LPS. And the inhibitory effects of quercetin on LPS-induced ROS production in macrophages and the protective effects on mitochondrial morphology and membrane potential were significantly decreased by the addition of SIRT1 inhibitors. These results suggested that quercetin reprograms the mitochondria metabolism of macrophages through the SIRT1/PGC-1α signaling pathway, thereby exerting its effect of alleviating LPS-induced oxidative stress damage.

Serratiopeptidase Attenuates Lipopolysaccharide-Induced Vascular Inflammation by Inhibiting the Expression of Monocyte Chemoattractant Protein-1.

Lipopolysaccharide (LPS) has potent pro-inflammatory properties and acts on many cell types including vascular endothelial cells. The secretion of the cytokines MCP-1 (CCL2), interleukins, and the elevation of oxidative stress by LPS-activated vascular endothelial cells contribute substantially to the pathogenesis of vascular inflammation. However, the mechanism involving LPS-induced MCP-1, interleukins, and oxidative stress together is not well demonstrated. Serratiopeptidase (SRP) has been widely used for its anti-inflammatory effects. In this research study, our intention is to establish a potential drug candidate for vascular inflammation in cardiovascular disorder conditions. We used BALB/c mice because this is the most successful model of vascular inflammation, suggested and validated by previous research findings. Our present investigation examined the involvement of SRP in vascular inflammation caused by lipopolysaccharides (LPSs) in a BALB/c mice model. We analyzed the inflammation and changes in the aorta by H&E staining. SOD, MDA, and GPx levels were determined as per the instructions of the kit protocols. ELISA was used to measure the levels of interleukins, whereas immunohistochemistry was carried out for the evaluation of MCP-1 expression. SRP treatment significantly suppressed vascular inflammation in BALB/c mice. Mechanistic studies demonstrated that SRP significantly inhibited the LPS-induced production of proinflammatory cytokines such as IL-2, IL-1, IL-6, and TNF-α in aortic tissue. Furthermore, it also inhibited LPS-induced oxidative stress in the aortas of mice, whereas the expression and activity of monocyte chemoattractant protein-1 (MCP-1) decreased after SRP treatment. In conclusion, SRP has the ability to reduce LPS-induced vascular inflammation and damage by modulating MCP-1.

USP13 reduces septic mediated cardiomyocyte oxidative stress and inflammation by inducing Nrf2

Background: Sepsis is a common cardiovascular complication that can cause heart damage. The regulatory role of ubiquitin-specific peptidase 13 (USP13) on erythroid 2–related factor 2 (Nrf2) has been reported, but its regulatory role in septic cardiomyopathy remains unclear.Methods: The Sprague Dawley (SD) rat model of septic myocardial injury was constructed by lipopolysaccharides (LPS). The serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels were detected, the mRNA and protein expression levels of Nrf2 and USP13 in tissues were detected by real-time quantitative reverse transcription PCR (qRT-PCR) and western blot (WB), and the expression of USP13 at the treatment time of 3 h, 6 h, and 12 h was alsodetected. The cell viability and USP13, Nrf-2 and heme oxygenase-1 (HO-1) expression levels of H9C2-treated cells by LPS and the oxidative stress level and inflammatory response of H9C2 cells were detected by enzyme-linked immunosorbent assay (ELISA) and WB.Results: The results showed that USP13 was downregulated in septic myocardial injury tissues, and the Nrf2 level was increased in vitro after the cells were treated with LPS. Overexpression of USP13 further induced Nrf2 to reduce apoptosis, oxidative stress, and expression of inflammatory factors.Conclusion: In conclusion, this study demonstrated that USP13 was downregulated in septic myocardial injury tissues, and USP13 overexpression increased Nrf2 levels and reduced apoptosis. Further studies showed that USP13 reduced LPS-induced oxidative stress and inflammation by inducing Nrf2.

HC067047 Ameliorates Sepsis-associated Encephalopathy by Suppressing Endoplasmic Reticulum Stress and Oxidative Stress-Induced Pyroptosis in the Hippocampi of Mice

Sepsis-associated encephalopathy (SAE) is a common neurological complication of sepsis and is characterized by hyperneuroinflammation. NLRP3 inflammasome-mediated pyroptosis can induce an inflammatory cascade response and plays a key role in SAE. TRPV4 is involved in the hyperinflammatory response associated with inflammation; however, whether TRPV4 inhibition might alleviate SAE-related brain damage is still unknown. Therefore, we aimed to investigate the role and mechanism of HC067047, a potent inhibitor of TRPV4, in hyperneuroinflammation and blood–brain barrier (BBB) dysfunction in a lipopolysaccharide (LPS)-induced SAE mouse model. We found that HC067047 administration significantly inhibited the expression of TRPV4 and p-CamkIIα in the hippocampi of SAE mice. Furthermore, HC067047 treatment attenuated LPS-induced endoplasmic reticulum (ER) stress and oxidative stress (OS), thus remarkably preventing NLRP3 inflammasome-mediated pyroptosis, as well as the expression of proinflammatory factors (IL-1β and IL-18). Additionally, we found that HC067047 selectively prevented pyroptosis in hippocampal cells, mainly the neurons, oligodendrocytes and the resident microglia. The disruption of BBB integrity in SAE mice was also rescued by HC067047 intervention. Thus, we can conclude that the TRPV4 inhibitor HC067047 could protect against hippocampal cell pyroptosis, which might be due to the attenuation of the NLRP3 inflammasome-mediated pyroptosis pathway caused by ER stress and OS. Our findings suggest a potential preventive role for HC067047 in SAE.

Multiple Beneficial Effects of Aloesone from Aloe vera on LPS-Induced RAW264.7 Cells, Including the Inhibition of Oxidative Stress, Inflammation, M1 Polarization, and Apoptosis

Aloesone is a major metabolic compound in Aloe vera, which has been widely used as a food source and therapeutic agent in several countries. Our recent study demonstrated that aloesone has anti-epileptic effects on glutamate-induced neuronal injury by suppressing the production of reactive oxygen species (ROS). Unless ROS are naturally neutralized by the endogenous antioxidant system, they lead to the activation of inflammation, polarization, and apoptosis. This study aimed to identify the multiple beneficial effects of aloesone and explore its molecular mechanism in macrophages. Hence, the murine macrophage cell line RAW264.7 was pretreated with aloesone and then exposed to lipopolysaccharides (LPS). The results demonstrated that aloesone, within a dosage range of 0.1-100 µM, dramatically decreased the LPS-induced elevation of ROS production, reduced nitric oxide (NO) release, inhibited the M1 polarization of RAW264.7 cells, and prevented cells from entering the LPS-induced early and late phases of apoptosis in a dose-dependent manner. Simultaneously, aloesone significantly decreased the mRNA expression of inflammation-related genes (iNOS, IL-1ꞵ, TNF-α) and increased the expression of antioxidant enzymes (Gpx-1 and SOD-1). The core genes HSP90AA1, Stat3, Mapk1, mTOR, Fyn, Ptk2b, and Lck were closely related to these beneficial effects of aloesone. Furthermore, immunofluorescence staining and flow cytometry data confirmed that aloesone significantly repressed the activation of mTOR, p-mTOR, and HIF-1α induced by LPS and inhibited the protein expression of TLR4, which is the target of LPS. In conclusion, aloesone demonstrated multiple protective effects against LPS-induced oxidative stress, inflammation, M1 polarization, and apoptosis in macrophages, suggesting its potential as a prodrug.

Saroglitazar, a dual PPAR-α/γ agonist, alleviates LPS-induced hepatic and renal injury in rats.

Lipopolysaccharide (LPS), an endotoxin within gram-negative bacteria, is associated with systemic acute inflammatory response after invading living tissues and results in sepsis. The liver and kidney are both major target organs in sepsis. Septic acute hepatic-renal injury is a serious clinical condition with high risk of morbidity and mortality. Nevertheless, effective treatment is still lacking. This study highlights saroglitazar (SAR), a dual PPAR-α/γ agonist, as a proposed prophylactic drug against LPS-induced hepatic-renal injury. Rats were pretreated with SAR (2 and 4mg/kg/day) for 15days, while sepsis was induced by LPS injection (10mg/kg) on day 15 one hour following SAR oral administration. SAR pretreatment could successfully mitigate LPS-induced hepatic-renal injury, evidenced by enhancement of renal and hepatic functions and a decrease of tissue pathological injury. Meanwhile, SAR alleviated LPS-induced oxidative stress; it reduced malondialdehyde (MDA) levels and ameliorated decreased levels of superoxide dismutase (SOD) and glutathione (GSH). LPS-induced elevations in hepatic and renal nuclear factor-kappa B (NF-κB), phosphorylated inhibitor of kappa B alpha (p-IκBα), interferon-beta (IFN-β), and hepatic high mobility group box-1 (HMGB-1) contents were significantly attenuated in SAR-treated groups. SAR showed an advantageous impact against LPS-induced activation of non-canonical inflammasome and pyroptosis via a significant reduction in cysteinyl aspartate-specific proteinase-11 (Caspase-11) and gasdermin D (GSDMD) expressions. Moreover, Nucleotide-Binding Oligomerization Domain (NOD)-Like Receptor Protein 3 (NLRP3) inflammasome activation with concomitant expression and activation of caspase-1 and release of interleukin-1beta (IL-1β) were considerably diminished following SAR pretreatment. SAR could be considered a prophylactic anti-inflammatory antioxidant drug against LPS-induced liver and kidney injury.

Nuclear SPHK2/S1P induces oxidative stress and NLRP3 inflammasome activation via promoting p53 acetylation in lipopolysaccharide-induced acute lung injury

A bulk of evidence identified that macrophages, including resident alveolar macrophages and recruited macrophages from the blood, played an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). However, the molecular mechanisms of macrophages-induced acute lung injury (ALI) by facilitating oxidative stress and inflammatory responses remain unclear. Herein, we noticed that the levels of mitochondrial reactive oxygen species (mtROS), SPHK2 and activated NLRP3 inflammasome were higher in peripheral blood mononuclear cells (PBMCs) of ARDS patients than that in healthy volunteers. Similar observations were recapitulated in LPS-treated RAW264.7 and THP-1 cells. After exposure to LPS, the SPHK2 enzymatic activity, NLRP3 inflammasome activation and mtROS were significantly upregulated in macrophages. Moreover, knockdown SPHK2 via shRNA or inhibition SPHK2 could prominently decrease LPS-induced M1 macrophage polarization, oxidative stress and NLRP3 inflammasome activation. Further study indicated that upregulated SPHK2 could increase nuclear sphingosine-1-phosphate (S1P) levels and then restrict the enzyme activity of HDACs to facilitate p53 acetylation. Acetylation of p53 reinforced its binding to the specific region of the NLRP3 promoter and drove expression of NLRP3. In the in vivo experiments, it was also observed that treating with Opaganib (ABC294640), a specific SPHK2 inhibitor, could observably alleviate LPS-induced ALI, evidencing by lowered infiltration of inflammatory cells, increased M2 macrophages polarization and reduced oxidative damage in lung tissues. Besides, SPHK2 inhibition can also decrease the accumulation of acetylated p53 protein and the activation of NLRP3 inflammasome. Taken together, our results demonstrated for the first time that nuclear S1P can regulate the acetylation levels of non-histone protein through affecting HDACs enzyme activities, linking them to oxidative stress and inflammation in response to environmental signals. These data provide a theoretical basis that SPHK2 may be an effective therapeutic target of ARDS.

Amniotic fluid stem cell attenuated necrotizing enterocolitis progression by promoting Rspo3/AMPKα axis

R-spondin 3 (Rspo3) is involved in various cellular processes. The alteration of Rspo3 participates in the differentiation of intestinal epithelial cells which are the crucial effector cells during necrotizing enterocolitis (NEC) development. Amniotic fluid stem cells (AFSCs) were recently indicated as a potential approach for NEC therapy. This study aimed to illustrate the regulatory role and mechanism of Rspo3 in the pathogenesis of NEC and whether AFSCs therapy would impact NEC by mediating Rspo3. First, the alteration of Rspo3 was investigated in the serum and tissues of NEC patients, and an in vitro cell model induced by LPS. A gain-of-function assay was conducted to explore the function of Rspo3 in NEC. Through the analysis of adenosine 5‘-monophosphate-activated protein kinase α (AMPKα) activation, the mechanism of Rspo3-mediated NEC progression was demonstrated. Finally, AFSCs were used to coculture human intestinal epithelial cells (HIECs) and the impacts on NEC development were also explored. The results found that Rspo3 was dramatically depressed during NEC progression and reversing Rspo3 expression ameliorated LPS-induced injury, inflammation, oxidative stress and tight junction dysregulation in HIECs. Besides, Rspo3 overexpression reversed AMPKα inactivation induced by NEC and an AMPKα inhibitor, Compound C, blocked the effect of Rspo3 overexpression on NEC. AFSCs treatment was beneficial for NEC therapy by restoring Rspo3 expression which was counteracted by exosome inhibitor. Generally, AFSCs attenuated NEC progression by promoting the Rspo3/AMPKα axis which might exert via the secretion of exosomes. Our conclusions might be valuable for NEC diagnosis and therapy.

Velvet antler water extract protects porcine oocytes from lipopolysaccharide-induced meiotic defects.

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram-negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress.

RmMANF prevents sepsis-associated lung injury via inhibiting endoplasmic reticulum stress-induced ferroptosis in mice

Ferroptosis plays a critical role in LPS-induced acute lung injury and is modulated by endoplasmic reticulum stress (ERS). As a typical ER stress-responsive protein, recently mesencephalic astrocyte-derived neurotrophic factor (MANF) has been demonstrated to attenuate LPS-induced acute lung injury (ALI) through repressing macrophage activation. However, whether MANF exerts a preventive role on ferroptosis and excess ER stress remains unclear. Here, we first built a protein-protein interaction (PPI) network to obtain potential interacting proteins related to MANF through STRING and GeneMANIA. Then, male C57BL/6J mice were used to build a model of LPS-induced lung injury. Two days before LPS injection, the tail vein injected recombinant murine MANF (rmMANF) at 750μg/kg. Twenty-four hours after the LPS injection, the histopathological changes and damage in the lung tissues were detected and scored by HE staining and TUNEL assay, respectively. Endogenous MANF levels, oxidative stress markers (GSH, SOD, CAT, and MDA), ERS markers (GRP78, PERK, and ATF4), and the ferroptosis markers (iron, GPX4, and 4-HNE) in the lung tissues were measured by IHC, western blotting, and commercial kits. Our results showed that LPS induced significant lung injury to the increase in MPO, MDA, and 4-HNE, a decrease in GPX4 and GSH, SOD, CAT, and total iron accumulation in LPS-exposed mice. Simultaneously, GRP78/PERK/ATF4 pathway was notably activated by LPS, accompanied by the down-regulation of MANF. Furthermore, rmMANF pretreatment markedly prevented LPS-induced lung tissue injury and ferroptosis characteristics with the increased GPX4 level in sepsis mice. Finally, we found that LPS-induced oxidative stress and activation of the GRP78/PERK/ATF4 pathway were significantly restrained by rmMANF pretreatment, except for endogenous MANF level. Overall, rmMANF pretreatment can prevent sepsis-associated lung injury by inhibiting ER stress-induced ferroptosis in mice.

Knockdown of 11β-hydroxysteroid dehydrogenase type 1 alleviates LPS-induced myocardial dysfunction through the AMPK/SIRT1/PGC-1α pathway.

Sepsis-induced myocardial dysfunction is primarily accompanied by severe sepsis, which is associated with high morbidity and mortality. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), encoded by Hsd11b1, is a reductase that can convert inactive cortisone into metabolically active cortisol. The role of 11β-HSD1 in sepsis-induced myocardial dysfunction remains poorly understood. The current study aims to investigate the effects of 11β-HSD1 on a lipopolysaccharide (LPS)-induced mouse model. LPS (10 mg/kg) was administered to wild-type C57BL/6J mice and 11β-HSD1 global knockout mice. Cardiac function was assessed by echocardiography. Transmission electron microscopy and immunohistochemical staining were performed to analyze myocardial mitochondrial injury and histological changes. The levels of reactive oxygen species and biomarkers of oxidative stress were determined. Polymerase chain reaction analysis, Western blotting, and immunofluorescent staining were employed to determine the expression of related genes and proteins. To investigate the role of 11β-HSD1 in sepsis-induced myocardial dysfunction, LPS was used to induce lentivirus-infected neonatal rat ventricular cardiomyocytes. Knockdown of 11β-HSD1 alleviated LPS-induced myocardial mitochondrial injury, oxidative stress, and inflammation, along with an improved myocardial function. Furthermore, the depletion of 11β-HSD1 promoted the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and silent information regulator 1 (SIRT1) protein levels both in vivo and in vitro. The suppression of 11β-HSD1 may be a viable strategy to improve cardiac function against endotoxemia challenges.

USP13 reduces septic mediated cardiomyocyte oxidative stress and inflammation by inducing Nrf2.

Sepsis is a common cardiovascular complication that can cause heart damage. The regulatory role of ubiquitin-specific peptidase 13 (USP13) on erythroid 2-related factor 2 (Nrf2) has been reported, but its regulatory role in septic cardiomyopathy remains unclear. The Sprague Dawley (SD) rat model of septic myocardial injury was constructed by lipopolysaccharides (LPS). The serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels were detected, the mRNA and protein expression levels of Nrf2 and USP13 in tissues were detected by real-time quantitative reverse transcription PCR (qRT-PCR) and western blot (WB), and the expression of USP13 at the treatment time of 3 h, 6 h, and 12 h was also detected. The cell viability and USP13, Nrf-2 and heme oxygenase-1 (HO-1) expression levels of H9C2-treated cells by LPS and the oxidative stress level and inflammatory response of H9C2 cells were detected by enzyme-linked immunosorbent assay (ELISA) and WB. The results showed that USP13 was downregulated in septic myocardial injury tissues, and the Nrf2 level was increased in vitro after the cells were treated with LPS. Overexpression of USP13 further induced Nrf2 to reduce apoptosis, oxidative stress, and expression of inflammatory factors. In conclusion, this study demonstrated that USP13 was downregulated in septic myocardial injury tissues, and USP13 overexpression increased Nrf2 levels and reduced apoptosis. Further studies showed that USP13 reduced LPS-induced oxidative stress and inflammation by inducing Nrf2.

Decursin alleviates LPS-induced lung epithelial cell injury by inhibiting NF-κB pathway activation.

To reveal the possible effects of decursin on viability, oxidative stress, and inflammatory response in lipopolysaccharide (LPS)-treated human bronchial epithelial cells-2B (BEAS-2B) and human pulmonary artery endothelial cells (HPAEC) cells, and revealed the potential mechanisms. LPS was used to induce acute lung injury (ALI) in normal human lung epithelial cells, including BEAS-2B and HPAEC cells. Cell viability and apoptosis in response to LPS and decursin in BEAS-2B and HPAEC cells were, respectively, evaluated by MTT colorimetric and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The oxidative stress and inflammatory response in LPS-treated BEAS-2B and HPAEC cells were detected by enzyme-linked-immunosorbent serologic assay. In addition, the role of decursin in nuclear -factor-kappa B (NF-κB) activation was analyzed by immunoblot and immunofluorescence assays. Our data revealed that decursin could alleviate the viability of LPS-induced BEAS-2B and HPAEC cells. Decursin could also reduce LPS-induced oxidative stress in BEAS-2B and HPAEC cells. In addition, it could reduce LPS-induced inflammation in BEAS-2B and HPAEC cells. Mechanically, decursin suppressed the activation of NF-κB pathway. Decursin suppressed NF-κB pathway, and therefore alleviated ALI.

Selenium Speciation Analysis Reveals Improved Antioxidant Status in Finisher Pigs Fed l-Selenomethionine, Alone or Combined with Sodium Selenite, and Vitamin E

Conditions associated with selenium (Se) and/or vitamin E (VitE) deficiency are still being reported in high-yielding pigs fed the recommended amounts. Here, the dietary effects of Se source (sodium selenite, NaSe, 0.40 or 0.65 mg Se/kg; l-selenomethionine, SeMet, 0.19 or 0.44 mg Se/kg; a NaSe-SeMet mixture, SeMix, 0.44–0.46 mg Se/kg) and VitE concentration (27, 50–53 or 101 mg/kg) on the antioxidant status of finisher pigs were compared with those in pigs fed non-Se-supplemented diets (0.08–0.09 mg Se/kg). Compared to NaSe-enriched diets, SeMet-supplemented diets resulted in significantly (p < 0.0018) higher plasma concentrations of total Se (14–27%) and selenospecies (GPx3, SelP, SeAlb; 7–83%), significantly increased the total Se accumulation in skeletal muscles, myocardium, liver and brain (10–650%), and enhanced the VitE levels in plasma (15–74%) and tissues (8–33%) by the end of the 80-day trial, proving better Se distribution and retention in pigs fed organic Se. Injecting lipopolysaccharide (LPS) intravenously half-way into the trial provoked a pyrogenic response in the pigs followed by a rapid increase of inorganic Se after 5–12 h, a drastic drop of SeMet levels between 12 and 24 h that recovered by 48 h, and a small increase of SeCys by 24–48 h, together with a gradual rise of GPx3, SelP and SeAlb in plasma up to 48 h. These changes in Se speciation in plasma were particularly significant (0.0024 > p > 0.00007) in pigs receiving SeMet- (0.44 mg Se/kg, above EU-legislated limits) or SeMix-supplemented (SeMet and NaSe both at 0.2 mg Se/kg, within EU-legislated limits) diets, which demonstrates Se metabolism upregulation to counteract the LPS-induced oxidative stress and a strengthened antioxidant capacity in these pigs. Overall, a Se source combination (without exceeding EU-legislated limits) and sufficient VitE supplementation (≥ 50 mg/kg) improved the pigs’ antioxidant status, while doubling the allowed dietary organic Se increased the Se in tissues up to sixfold without compromising the animal’s health due to toxicity. This study renders valuable results for revising the current dietary SeMet limits in swine rations.Graphical

MiRNA-15/IL-10Rα axis promotes Kabasura Kudineer (Indian traditional Siddha formulation) induced immunomodulation by suppressing oxidative stress

Ethnopharmacological relevanceKabasura Kudineer (KK), the traditional Indian medicine of Siddha, effectively manages common respiratory symptoms such as flu, cold, and fever. However, there is no evidence of the immunomodulatory capacity of KK in the cultured Jurkat T-lymphocytes under the LPS insult studied. Aim of the studyAssess the effect of the traditional Indian medicine of Siddha, Kabasura Kudineer (KK) on immunomodulation by suppressing oxidative damage in cultured Jurkat T cells in vitro. The miRNA activity on anti-inflammatory gene receptors and cellular nitric oxide levels also was studied. Materials and methodsJurkat T cells were exposed to LPS treatment in the presence or absence of KK. Cell viability and nitric oxide (NO) were measured with MTT and Griess assay. Cellular antioxidant systems (glutathione and SOD) were determined using glutathione and SOD assay. Lipid peroxidation was measured using an MDA assay. MiRNA-15a-5p expression was performed using microRNA qPCR Assays. Both inflammatory and anti-inflammatory genes (IL-6, IL-1, IL-10, IL-13) were performed using a qPCR and ELISA assay. ResultsThe data showed that reduced cell proliferation and exaggerated NO production was observed in LPS treated condition compared to the control condition. Further, LPS treatment increased lipid peroxidation and reduced antioxidant enzyme activities (SOD and glutathione) in cultured Jurkat T cells. However, treatment with KK or N-acetyl cysteine (NAC; antioxidant) treatment mitigates the above effect. Mechanistically, LPS-induced oxidative stress upregulated miR- 15-5p expression and suppressed IL-10 Receptor alpha (IL-10Rα) by binding to its 3′-UTR region. The deregulated expression of IL-10Rα expression leads to increased IL-6 and IL-1β expression in LPS-induced Jurkat T cells; however, treatment with KK or NAC reversed the above effects. ConclusionCollectively, our study revealed the previously undefined mechanistic role of Kabasura Kudineer (KK) that alleviates the LPS-induced oxidative damage associated with inflammation by inhibiting the miRNA-15-5p/IL-10Rα axis.

Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice.

Acute lung injury (ALI) is a life-threatening complication during sepsis and contributes to multiple organ failure and high mortality for septic patients. The present study aims to investigate the role and molecular basis of growth differentiation factor 7 (GDF7) in sepsis-induced ALI. Mice were subcutaneously injected with recombinant mouse GDF7 Protein (rmGDF7) and then intratracheally injected with lipopolysaccharide (LPS) to generate sepsis-induced ALI. Primary peritoneal macrophages were isolated to further evaluate the role and underlying mechanism of GDF7 in vitro. GDF7 was downregulated in LPS-stimulated lung tissues, and rmGDF7 treatment significantly inhibited inflammation and oxidative stress in ALI mice, thereby preventing LPS-induced pulmonary injury and dysfunction. Mechanistically, we found that rmGDF7 activated AMP-activated protein kinase (AMPK), and AMPK inhibition significantly blocked the anti-inflammatory and antioxidant effects of rmGDF7 during LPS-induced ALI. Further findings revealed that rmGDF7 activated AMPK through a downregulated stimulator of interferon gene (STING) in vivo and in vitro. GDF7 prevents LPS-induced inflammatory response, oxidative stress, and ALI by regulating the STING/AMPK pathway. Our findings for the first time identify GDF7 as a potential agent for the treatment of sepsis-induced ALI.

Litsea salicifolia Roxb. bark ethanolic extract attenuates rheumatoid arthritis by inhibiting TLR4/NF-κB and activating SIRT1-Nrf2/HO-1 signaling axis: In-vitro and in-vivo evaluations

BackgroundLitsea salicifolia Roxb. (LS) is a pioneer medicinal plant that can be found in tropical and subtropical countries like India, China and Japan. LS is traditionally used for treating chronic pulmonary inflammatory diseases like asthma and wound healing. Also, LS contains an essential oil with powerful anti-oxidant and excite-repellency properties. However, no research findings have been reported about its protective activity on chronic inflammatory disease like rheumatoid arthritis (RA) till yet. PurposeThis study aims to investigate the anti-rheumatic potential of ethanolic extract of LS (LSE) in-vitro on RAW 264.7 cells for its anti-oxidant and anti-inflammatory activities and in-vivo CFA-induced adjuvant arthritis in the rat model. MethodsThe chemical characterization of LSE was identified by using LC-MS/MS. The in-vitro effects of LSE on RAW 264.7 cells stimulated with LPS 1 µg/ml were investigated to its potential therapeutic effects. Freund's complete adjuvant (CFA) arthritis was induced by 100 µl of CFA intradermally administered to rats. LSE 100 and 200 mg/kg were administered orally from day1 to day 28. Parameters such as paw volumes measured, X-ray radiography analysis, anti-oxidant status, Gene expression studies, western blot analysis and histological analysis were performed to check the anti-oxidant and anti-inflammatory effects of LSE. ResultsOur in-vitro results revealed that LSE at 5 μg/ml and 10 μg/ml reduced the LPS-induced oxidative stress burden evidenced by reduced ROS levels and improved endogenous anti-oxidants Nrf2 and SIRT1 in RAW 264.7 cells. Also, LSE inhibited the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, PGE2, COX-2 and NO levels induced by LPS in murine macrophage cells at 5 μg/ml and 10 μg/ml in a dose dependent manner. Our in-vivo results revealed that LSE significantly attenuated the CFA-induced inflammation by restoring body weight, arthritis index, nitrosative and lipid peroxidation assays. LSE treatment significantly reduced the pannus formation and bone erosion evidenced by histopathology of joint tissues. Additionally treatment with LSE significantly reduced inflammation-mediated protein expression levels like TLR4, NF-κB, COX-2 and iNOS levels and improved the Nrf2/HO-1/SIRT1 levels in paw tissues evidenced by immunoblotting. ConclusionLSE alleviated RA through its anti-oxidant and anti-inflammatory properties by inhibiting TLR4-mediated NF-κB nuclear translocation and by activating the Nrf2/SIRT1 signaling pathways, according to our in-vitro and in-vivo findings.

Amentoflavone Exerts Anti-Neuroinflammatory Effects by Inhibiting TLR4/MyD88/NF-κB and Activating Nrf2/HO-1 Pathway in Lipopolysaccharide-Induced BV2 Microglia.

Amentoflavone, a natural biflavone, exerts anti-inflammation, antioxidation, and antiapoptosis effects on many diseases. However, the mechanism of amentoflavone on neuroinflammation-related diseases has not been comprehensively examined clearly. BV2 microglial cells were treated with amentoflavone (10 μM), followed by lipopolysaccharide (LPS). Microglial activation and migration ability and the expression of proinflammatory cytokines and other signaling proteins were determined using immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay, and wound-healing assays. Amentoflavone restored LPS-induced microglia activation, migration, and inflammation response which depends on regulating toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway. In addition, amentoflavone also enhanced nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) levels in LPS-treated BV2 microglial cells. Amentoflavone ameliorated LPS-induced neuroinflammatory response and oxidative stress in BV2 microglia. These data provide new insight into the mechanism of amentoflavone in the treatment of neuroinflammation-related diseases. Therefore, amentoflavone may be a potential therapeutic option for neurological disorders.

Dexmedetomidine Attenuates Lipopolysaccharide-Induced Sympathetic Activation and Sepsis via Suppressing Superoxide Signaling in Paraventricular Nucleus.

Sympathetic overactivity contributes to the pathogenesis of sepsis. The selective α2-adrenergic receptor agonist dexmedetomidine (DEX) is widely used for perioperative sedation and analgesia. We aimed to determine the central roles and mechanisms of DEX in attenuating sympathetic activity and inflammation in sepsis. Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide (LPS) in rats. Effects of DEX were investigated 24 h after injection of LPS. Bilateral microinjection of DEX in the paraventricular nucleus (PVN) attenuated LPS-induced sympathetic overactivity, which was attenuated by the superoxide dismutase inhibitor DETC, cAMP analog db-cAMP or GABAA receptor antagonist gabazine. Superoxide scavenger tempol, NADPH oxidase inhibitor apocynin, adenylate cyclase inhibitor SQ22536 or PKA inhibitor Rp-cAMP caused similar effects to DEX in attenuating LPS-induced sympathetic activation. DEX inhibited LPS-induced superoxide and cAMP production, as well as NADPH oxidase, adenylate cyclase and PKA activation. The roles of DEX in reducing superoxide production and NADPH oxidase activation were attenuated by db-cAMP or gabazine. Intravenous infusion of DEX inhibited LPS-induced sympathetic overactivity, NOX activation, superoxide production, TNF-α and IL-1β upregulation in the PVN and plasma, as well as lung and renal injury, which were attenuated by the PVN microinjection of yohimbine and DETC. We conclude that activation of α2-adrenergic receptors with DEX in the PVN attenuated LPS-induced sympathetic overactivity by reducing NADPH oxidase-dependent superoxide production via both inhibiting adenylate cyclase-cAMP-PKA signaling and activating GABAA receptors. The inhibition of NADPH oxidase-dependent superoxide production in the PVN partially contributes to the roles of intravenous infusion of DEX in attenuating LPS-induced sympathetic activation, oxidative stress and inflammation.