The alleviating effect of Phillygenin on the regulation of respiratory microbiota and its metabolites in IBV-infected broilers by inhibiting the TLR7/MyD88/NF-κB axis.

Phillygenin (PHI) is an active ingredient derived from the leaf of Forsythia suspensa that has been found to alleviate inflammation and peroxidation response. Avian infectious bronchitis (IB) is a major threat to poultry industry viral respiratory tract disease that infected with infectious bronchitis virus (IBV). This study investigated the protection of PHI to CEK cell and broiler's tracheal injury triggered by avian infectious bronchitis virus (IBV). The results showed that IBV infection did not cause serious clinical symptoms and slowing-body weight in PHI-treated broilers. The expression of virus loads, pro-inflammation factors (IL-6, TNF-α, and IL-1ß) in CEK cell, and tracheas were decreased compared to the IBV group, exhibiting its potent anti-inflammation. Mechanistically, the study demonstrated that the inhibition of TLR7/MyD88/NF-κB pathway was mainly involved in the protection effect of PHI to inflammation injury. Interestingly, a higher abundance of Firmicutes and Lactobacillus in respiratory tract was observed in PHI-treated broilers than in the IBV group. Significant differences were observed between the IBV group and PHI-treated group in the Ferroptosis, Tryptophan metabolism, and Glutathione metabolism pathways. PHI exhibited potent protection effect on IBV infection and alleviated inflammation injury, mainly through inhibiting TLR7/MyD88/NF-κB pathway. The study encourages further development of PHI, paving the way to its clinical use as a new candidate drug to relieve IBV-induced respiratory symptoms.

Dihydrokaempferol attenuates LPS-induced inflammation and apoptosis in WI-38 cells.

BACKGROUND: Globally, pneumonia has been associated as a primary cause of mortality in children aged less than 5 years. Dihydrokaempferol (DHK) has been proposed for being correlated with the process of various diseases. Nevertheless, whether DHK has a role in the progression of infantile pneumonia remains unclear. This study aimed at exploring whether DHK was involved in the progression of infantile pneumonia. METHODS: Human fibroblast cells WI-38 were treated with lipopolysaccharide (LPS). The viability of WI-38 cells was measured via Cell counting kit-8. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α). Western blot analysis revealed the protein levels of IL-1ß, IL-6, TNF-α, Bax, and cleaved-caspase 3. Flow cytometry was applied for exploring the apoptosis of WI-38 cells. The concentrations of IL-1ß, IL-6, and TNF-α were assessed via enzyme-linked-immunosorbent serologic assay. RESULTS: DHK modulated the viability of WI-38 cells in infantile pneumonia. Furthermore, we identified that DHK treatment inversely changed LPS induction-mediated elevation on the levels of inflammation biomarkers. Besides, DHK counteracted LPS-induced production of reactive oxygen species (ROS) in WI-38 cells. DHK also decreased LPS-induced elevation of WI-38 cells apoptosis and mediated the levels of apoptosis-associated indexes. Moreover, modulating sirtuin-1 (SIRT1) protein level was lowered by the induction of LPS, and was reversed by DHK treatment. In addition, DHK counteracted LPS induction-mediated elevation of p-p65 and phosphorylated inhibitor of nuclear factor kappa-B kinase subunit alpha (p-IκBα) protein levels. CONCLUSION: DHK alleviated LPS-induced WI-38 cells inflammation injury in infantile pneumonia through SIRT1/NF-κB pathway. The results shed light on the implications of DHK on the prevention and treatment of infantile pneumonia.

Negative feedback of NF-κB signaling by long noncoding RNA MALAT1 controls lipopolysaccharide-induced inflammation injury in human lung fibroblasts WI-38.

The study was designed to elucidate the regulatory mechanism of long noncoding RNA human metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in lipopolysaccharides (LPS)-caused inflammation injury in human lung fibroblasts WI-38. WI-38 cells were stimulated with LPS to construct acute pneumonia cell model. MALAT1 in LPS-stimulated WI-38 cells was examined. LPS-induced inflammation injury was estimated using viability, apoptosis, and cytokine secretion including interleukin-1ß (IL-1ß) and IL-6. Furthermore, the modulatory relations of MALAT1 and nuclear factor-kappa B (NF-κB) signaling were explored. We found that the sensitivity of WI-38 cells to apoptosis was enhanced by LPS-caused inflammation. Moreover, LPS promoted MALAT1 expression which was found to alleviate LPS-caused damages. Besides, NF-κB p65 overexpression resulted in an increased expression of MALAT1, and MALAT1 was identified as a target gene of p65. Furthermore, overexpression of MALAT1 reduced NF-κB activation. Pulldown assay showed that MALAT1 could directly interact with p65. Taken together, our findings revealed that MALAT1 was upregulated in LPS-stimulated WI-38 cells. Through directly interacting with p65, MALAT1 blocked LPS-caused activation of NF-κB and repressed LPS-induced inflammation injury. MALAT1 may serve as a potential diagnostic indicator or therapeutic target for pneumonia.

GSK-3ß inhibition protects the rat heart from the lipopolysaccharide-induced inflammation injury via suppressing FOXO3A activity.

Sepsis-induced cardiac dysfunction represents a main cause of death in intensive care units. Previous studies have indicated that GSK-3ß is involved in the modulation of sepsis. However, the signalling details of GSK-3ß regulation in endotoxin lipopolysaccharide (LPS)-induced septic myocardial dysfunction are still unclear. Here, based on the rat septic myocardial injury model, we found that LPS could induce GSK-3ß phosphorylation at its active site (Y216) and up-regulate FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK-3ß inhibitors and further reversed through ß-catenin knock-down. This pharmacological inhibition of GSK-3ß attenuated the LPS-induced cell injury via mediating ß-catenin signalling, which could be abolished by FOXO3A activation. In vivo, GSK-3ß suppression consistently improved cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines in LPS-induced model was also blocked by inhibition of GSK-3ß, which curbed both ERK and NF-κB pathways, and suppressed cardiomyocyte apoptosis via activating the AMP-activated protein kinase (AMPK). Our results demonstrate that GSK-3ß inhibition attenuates myocardial injury induced by endotoxin that mediates the activation of FOXO3A, which suggests a potential target for the therapy of septic cardiac dysfunction.

Emodin Attenuates Lipopolysaccharide-Induced Injury via Down-Regulation of miR-223 in H9c2 Cells.

Emodin is a natural product extracted from Rheum palmatum. There are few recent studies on emodin in the treatment of myocarditis. This study aimed to investigate the effect of emodin on lipopolysaccharide (LPS)-induced inflammatory injury in cardiomyocytes. H9c2 cells were treated with 10 µM of LPS and different concentrations (0, 1, 5, 10, 15, and 20 µM) of emodin. The expression of miR-223 was changed by transient transfection. Thereafter, cell viability, apoptosis, the expression of CyclinD1 and Jnk-associated proteins, and the release of pro-inflammatory factors were assessed by cell Counting Kit-8, flow cytometry analysis, quantitative real-time polymerase chain reaction Western blot, and enzyme-linked immunosorbent assay respectively. The results showed that 20 µM of emodin significantly decreased H9c2 cells viability. LPS significantly damaged H9c2 cells, as cell viability was reduced, CyclinD1 was down-regulated, apoptosis was induced, the release of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-alpha were increased, and the phosphorylation of Jnk and c-Jun were promoted. Emodin protected H9c2 cells against LPS-induced inflammatory injury. miR-223 expression was significantly up-regulated by LPS exposure, while emodin lessened this up-regulation. LPS-injured H9c2 cells were attenuated by the overexpression of miR-223; emodin has protective actions on LPS-injured H9c2 cells and targets. Besides, SP600125 (an inhibitor of Jnk) eliminated miR-223-modulated inflammatory injury in H9c2 cells. These data demonstrated that emodin could attenuate LPS-induced inflammatory injury and deactivate Jnk signaling pathway through down-regulation of miR-223.

MicroRNA-106b overexpression alleviates inflammation injury of cardiac endothelial cells by targeting BLNK via the NF-κB signaling pathway.

We aim to investigate whether microRNA-106b (miR-106b) affects the inflammation injury of cardiac endothelial cells (ECs) by targeting B-cell linker (BLNK) via the NF-κB signaling pathway. Human cardiac microvascular endothelial cells (HCMECs) were assigned into the control, hypoxia/reoxygenation (H/R), negative control (NC), pyrrolidine dithiocarbamate (PDTC), miR-106b mimic, miR-106b inhibitor, and si-BLNK, and miR-106b inhibitor+si-BLNK groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were conducted for miR-106b expression and expressions of BLNK, interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, NF-κB, pIκBα, BTK, and PLC-γ2. Enzyme-linked immunosorbent assay was applied for levels of IL-6, IL-10, and TNF-α; cell counting Kit-8 assay for cell proliferation; and flow cytometry for cell cycle and ensuing apoptosis. In-vitro tube formation assay was performed for tube formation ability. Dual-luciferase reporter assay revealed that BLNK was verified as the target gene of miR-106b. Compared with the H/R and NC groups, the PDTC, miR-106b mimic, and si-BLNK groups had declined expressions of IL-6, IL-1ß, TNF-α, BTK, PLC-γ2, NF-κB p105/p50, and pIκBα as well as levels of L-6 and TNF-α, but contrarily elevated levels of NF-κB p105/p50 and IL-10. The PDTC, miR-106b mimic, and si-BLNK groups had less cell number in G0 /G1 phase but higher cell count in both S and G2 phases, decreased cell apoptosis but increased proliferation and tube formation ability, while opposite trends were observed in the miR-106b inhibitor group. Our findings indicated that the overexpression of miR-106b alleviated the inflammation injury of cardiac ECs by targeting BLNK via the NF-κB signaling pathway.

T-2 Toxin Caused Mice Testicular Inflammation Injury via ROS-Mediated NLRP3 Inflammasome Activation.

T-2 toxin treatment causes male reproduction system dysfunction, although the exact mechanism remains unclear. In this research, male Kunming mice and TM4 cells were treated with varying concentrations of the T-2 toxin for evaluating the adverse effect of T-2 toxin on male reproductive function. MCC950 or NAC was used to block NLRP3 inflammasome activation and eliminate reactive oxygen species (ROS) accumulation in the TM4 cell, respectively. The results showed that: (1) T-2 toxin caused testicular atrophy, destroyed the microstructure and ultrastructure of the testis, and caused sperm deformities; (2) T-2 toxin increased the content and gene expressions of TNF-α and IL-6 and decreased the IL-10 content and gene expression, causing testis and TM4 cell inflammatory injury; (3) T-2 toxin activated NLRP3 inflammasome in the testis and TM4 cells and caused ROS accumulation in the testis; (4) suppressing NLRP3 inflammasome activation using 20 nM MCC950 alleviated the TM4 cell inflammatory damage caused via the T-2 toxin; nevertheless, 20 nM MCC950 did not reduce ROS accumulation in TM4 cells; and (5) NAC relieved the inflammatory damage in TM4 cells by inhibiting NLRP3 inflammasome activation. Taken together, T-2 toxin caused testicular inflammation injury through ROS-mediated NLRP3 inflammasome activation, resulting in male reproductive dysfunction.

Research progress on the mechanism by which skin macrophage dysfunction mediates chronic inflammatory injury in diabetic skin.

Macrophages, the main immune cells in the skin, form an innate immune barrier. Under physiological conditions, skin maintains immune barrier function through macrophage phagocytosis and antigen presentation. Parenchymal and stromal cell regeneration plays an important role in skin injury repair and uses macrophage plasticity to influence and stabilize the skin microenvironment. Diabetic skin lesions are the most common diabetes complication and are involved in the early pathophysiology of diabetic foot. Therefore, studying the initial link in diabetic skin lesions is a research hot spot in the early pathogenesis of diabetic foot. Skin inflammation caused by hyperglycaemia, oxidative stress and other injuries is an important feature, but the specific mechanism is unknown. Recent studies have suggested that chronic inflammatory injury is widely involved in a variety of skin diseases, and whether it plays an important role in diabetic skin lesions is unclear. In this review, current research hotspots were combined with the pathogenesis of diabetic skin lesions and analysed from the perspectives of the physiological function of skin macrophages, the impairment of skin macrophages in diabetes, and the mechanism of chronic inflammatory injury in macrophages to provide a theoretical basis for early screening and evaluation of diabetic foot.

Knockdown of circ-UQCRC2 ameliorated lipopolysaccharide-induced injury in MRC-5 cells by the miR-326/PDCD4/NF-κB pathway.

BACKGROUND: Circular RNAs (circRNAs) have been shown as important modulators in the pathogenesis of pediatric pneumonia. In this paper, we focused on the molecular basis of circRNA ubiquinol-cytochrome c reductase core protein 2 (circ-UQCRC2, circ_0038467) in lipopolysaccharide (LPS)-induced cell injury. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to gauge the levels of circ-UQCRC2, microRNA (miR)-326 and programmed cell death 4 (PDCD4) mRNA. PDCD4 protein expression and the activation of the NF-κB signaling pathway were evaluated by western blot. Ribonuclease R (RNase R) assay was performed to assess the stability of circ-UQCRC2. Cell viability and apoptosis were detected by the Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 were measured by the enzyme-linked immunosorbent assay (ELISA). Targeted relationship between miR-326 and circ-UQCRC2 or PDCD4 was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. RESULTS: Our data showed the up-regulation of circ-UQCRC2 level in pneumonia serum and LPS-treated MRC-5 cells. The silencing of circ-UQCRC2 attenuated LPS-induced MRC-5 cell injury. Mechanistically, circ-UQCRC2 directly targeted miR-326, and circ-UQCRC2 regulated PDCD4 expression through miR-326. MiR-326 was a downstream effector of circ-UQCRC2 function, and PDCD4 was a functional target of miR-326 in regulating LPS-induced MRC-5 cell injury. Additionally, circ-UQCRC2 knockdown inactivated the NF-κB signaling pathway by regulating the miR-326/PDCD4 axis. CONCLUSION: Our findings demonstrated a novel regulatory network, the miR-326/PDCD4/NF-κB pathway, for the function of circ-UQCRC2 in LPS-induced cell injury in MRC-5 cells.

STIM1 mediates IAV-induced inflammation of lung epithelial cells by regulating NLRP3 and inflammasome activation via targeting miR-223.

AIMS: Influenza A virus (IAV) infection accelerates the inflammatory injury of lung epithelial cells that contributes to pulmonary lesion. Recently, stromal interaction molecule 1 (STIM1) was found to mediate cellular immune response and participated in lung tumorigenesis. Our study aimed to illustrate the function and mechanism of STIM1 in IAV-induced inflammation injury and oxidative stress of lung epithelial cells. MAIN METHODS: We evaluated the levels of STIM1 in IAV-infected patients' serum and BEAS-2B cells using RT-qPCR, Elisa and western blotting methods. MTT and Elisa were performed to measure cell viability and cytokine contents. Besides, ROS intensity, SOD contents and cell apoptosis were detected based on DCFH-DA probe, colorimetry and cell death kits. A luciferase assay and Pearson's correlation analysis evaluated the associations between target genes. KEY FINDINGS: STIM1 was dramatically up-regulated in IAV-infected patients' serum and BEAS-2B cells. Silencing STIM1 in vitro inhibited oxidative stress and inflammatory responses induced by IAV, and reversed cell viability and suppressed apoptosis. Moreover, miR-223 and NLRP3 were negatively and positively correlated with STIM1. STIM1 was found to regulate NLRP3 expression by binding the AACUGAC motif in miR-223. STIM1/miR-223/NLRP3 axis modulated IAV-induced inflammation injury of lung epithelial cells. SIGNIFICANCE: Our evidence indicated that silencing STIM1 alleviated IAV-induced inflammation injury of lung epithelial cells by inactivating NLRP3 and inflammasome via promoting miR-223 expression. These findings may contribute to understand the mechanism of IAV-induced lung injury and help for therapy of IAV infection.

MiR-215-5p inhibits the inflammation injury in septic H9c2 by regulating ILF3 and LRRFIP1.

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) playing crucial roles in sepsis-induced diseases, including myocardial inflammation. Nevertheless, the expression pattern and role of miR-215-5p in myocardial inflammation are still un-investigated up to now. The purpose of our study is to further inquire the effect of miR-215-5p on lipopolysaccharide (LPS)-activated inflammation injury in H9c2 cells and the possibly associated mechanisms. First of all, LPS-induced H9c2 cells models were constructed and affirmed via detection of pro-inflammatory factors, the viability and apoptosis. MiR-215-5p was overtly down-regulated in LPS-treated H9c2 cells and miR-215-5p overexpression could suppress the inflammation injury. LRRFIP1 was proved to be the target gene of miR-215-5p and meanwhile, miR-215-5p also targeted ILF3 that experimented to bind to and stabilize LRRFIP1. Final rescue assays confirmed that the overexpression of LRRFIP1 or ILF3 rescued the repressive effect of miR-215-5p up-regulation on the inflammation injury in septic H9c2. Totally, miR-215-5p exerted protective function in the inflammation injury in septic H9c2 via targeting ILF3 and LRRFIP1, suggesting an additional treatment method for sepsis-activated myocardial inflammation.

Effect of HBx on inflammation and mitochondrial oxidative stress in mouse hepatocytes.

Hepatitis B virus × protein (HBx) serves an important role in the pathogenesis of the hepatitis B virus infection. Previous studies have reported that the interaction between HBx and hepatocyte mitochondria is an important factor leading to liver cell injury and apoptosis, ultimately inducing the formation of liver cancer. In the present study, a mouse model expressing HBx was constructed using hydrodynamic in vivo transfection based on the interaction between HBx and cytochrome c oxidase (COX) subunit III. The specific mechanism of HBx-induced oxidative stress in mouse hepatocytes and the subsequent effect on mitochondrial function and inflammatory injury was assessed. The results demonstrated that HBx reduced the activity of COX and the expression of superoxide dismutase and upregulated the expression of malondialdehyde, NF-κB and phospho-AKT, thus increasing oxidative stress. In addition, HBx induced an increase in interleukin (IL)-6, IL-1ß and IL-18 expression levels, which created an inflammatory microenvironment in the liver, further promoting hepatocyte inflammatory injury. Therefore, it was proposed that HBx may affect hepatocyte mitochondrial respiration by reducing the activity of cytochrome c oxidase, leading to mitochondrial dysfunction and inducing hepatocyte inflammation and injury.

Hypoxic preconditioning relieved ischemic cerebral injury by promoting immunomodulation and microglia polarization after middle cerebral artery occlusion in rats.

OBJECTIVES: This study was designed to investigate whether immunomodulation and Microglia polarization is involved in the anti-inflammatory and neuroprotective effect induced by hypoxic preconditioning (HPC) in the middle cerebral artery occlusion (MCAO) brain injury model. METHODS: Longa method, (neurological disability status scale) NDSS method and TTC staining were used to evaluate the degree of cerebral infarction injury under different treatments (Sham, HPC, MCAO and co-treatment with HPC and MCAO). Western blot was used to detect expression profiles of apoptosis and related factors of neurological function. Flow cytometry was performed to analyze changes in the ratio of helper T cells, toxic T cells and NK cells in peripheral immune cells. And immunohistochemistry was used to examine the changes in microglial morphology. ELISA was used to evaluate the levels of nerve growth factors and neurogenesis conditions. Finally, RT-PCR was determined to analyze the transformation of microglia phenotype after HPC and MCAO treatment. RESULTS: MCAO dramatically induced local formation of cerebral infarction. HPC relieved MCAO-induced cerebral infarction and increased rat cognition. HPC affected activation of microglia without significantly affecting in peripheral immune cell populations. After HPC co-treatment with MCAO, the M1 phenotype of microglia was changed and there was a transformation to M2. CONCLUSION: The treatment of HPC remarkably affected the polarization of microglia cells in MCAO rats, and reduced the cerebral nerve injury and played a protective role in MCAO model.

Notoginsenoside R1 alleviates lipopolysaccharide-triggered PC-12 inflammatory damage via elevating microRNA-132.

BACKGROUND: Delayed inflammatory response is closely associated with the severity of Spinal cord injury (SCI). Herein, the function and molecular mechanism of notoginsenoside R1 (NGR1) in the in vitro model of SCI inflammation injury were explored. METHODS: PC-12 neuronal cells were subjected with LPS to construct a cell-based model of SCI inflammatory injury. NGR1 was applied in this cell model. miR-132 was silenced by transfection with miR-132 inhibitor. Cell viability and apoptosis were assessed, respectively. Then, the expression changes of pro-inflammatory cytokines and JNK pathway were examined. RESULTS: In this model, LPS was neurotoxic, with inhibiting PC-12 cell viability, inducing apoptosis, and enhancing concentrations of IL-6, IL-8 and TNF-α. However, NGR1 weakened the influence of LPS on PC-12 cells via elevating cell viability, decreasing apoptosis, decreasing pro-inflammatory cytokines expression, and suppressing activation of JNK signalling pathway. miR-132 was up-regulated by NGR1 treatment. Silence of miR-132 eliminated the influence of NGR1 on LPS-stimulated PC-12 cells. CONCLUSION: NGR1 relieved PC-12 cells from LPS-triggered inflammatory damage via elevating miR-132 and hereafter suppressing JNK pathway.

Baicalin alleviates IL-1ß-induced inflammatory injury via down-regulating miR-126 in chondrocytes.

Baicalin is a flavonoid extracted from Scutellaria baicalensis Georgi, with anti-inflammatory and anti-apoptotic activities. The objective of this study was to explore the effect and mechanism of baicalin on chondrocyte inflammatory response in OA. Different concentrations of IL-1ß (0, 0.1, 2, 5 and 10 ng/mL) were used to simulate inflammatory injury in CHON-001 cells. The expression of miR-126 was altered by transfection with miR-126 mimic. Thereafter, cells were treated with baicalin, and cell viability, apoptosis, the expressions of apoptosis-related protein and pro-inflammatory factors were respectively detected using CCK-8 assay, flow cytometry, qRT-PCR and western blot analysis. We found that IL-1ß induced a significantly inflammatory injury in CHON-001 cells. Baicalin alleviated IL-1ß-induced inflammatory injury, as it increased cell viability, decreased cell apoptosis and repressed the production of IL-6, IL-8 and TNF-α. miR-126 was up-regulated by IL-1ß treatment while was down-regulated by baicalin. More interestingly, the protective actions of baicalin on IL-1ß-injured CHON-001 cells were partially eliminated by miR-126 overexpression. Further, NF-κB signaling pathway was activated by IL-1ß, and deactivated by addition of baicalin. The deactivation of NF-κB signaling pathway induced by baicalin upon IL-1ß exposure was recovered by miR-126 overexpression. In conclusion, this study demonstrated that baicalin protected CHON-001 cells against IL-1ß-induced inflammatory injury possibly via down-regulation of miR-126 and thereby deactivation of NF-κB signaling pathway.

Neuroimmunomodulatory effects of transcranial laser therapy combined with intravenous tPA administration for acute cerebral ischemic injury.

At present, the only FDA approved treatment for ischemic strokes is intravenous administration of tissue plasminogen activator within 4.5 hours of stroke onset. Owing to this brief window only a small percentage of patients receive tissue plasminogen activator. Transcranial laser therapy has been shown to be effective in animal models of acute ischemic stroke, resulting in significant improvement in neurological score and function. NEST-1 and NEST-2 clinical trials in human patients have demonstrated the safety and positive trends in efficacy of transcranial laser therapy for the treatment of ischemic stroke when initiated close to the time of stroke onset. Combining intravenous tissue plasminogen activator treatment with transcranial laser therapy may provide better functional outcomes. Statins given within 4 weeks of stroke onset improve stroke outcomes at 90 days compared to patients not given statins, and giving statins following transcranial laser therapy may provide an effective treatment for patients not able to be given tissue plasminogen activator due to time constraints.