Nuclear factor-κB P65 Research Articles

Novel repair mechanisms in a renal ischaemia/reperfusion model: Subsequent saxagliptin treatment modulates the pro-angiogenic GLP-1/cAMP/VEGF, ANP/eNOS/NO, SDF-1α/CXCR4, and Kim-1/STAT3/HIF-1α/VEGF/eNOS pathways

The reno-protective effects of antidiabetic dipeptidyl peptidase (DPP)-4 inhibitors have been studied regarding their antioxidant and anti-inflammatory properties. However, the potential ability of saxagliptin to ameliorate renal injury by enhancing neovascularization has not been elucidated. To address this issue, saxagliptin (10 and 30 mg/kg) was administered to Wistar rats after the induction of renal ischaemia/reperfusion (I/R). Our results showed that saxagliptin operated through different axes to ameliorate I/R injury. By inhibiting DPP-4, saxagliptin maintained stromal cell-derived factor-1α expression and upregulated its chemokine receptor CXCR4 to trigger vasculogenesis through the enhanced migration of endothelial progenitor cells (EPCs). Additionally, this compound rescued the levels of glucagon-like peptide-1 and its downstream mediator cAMP to increase vascular endothelial growth factor (VEGF) and CXCR4 levels. Moreover, saxagliptin stimulated atrial natriuretic peptide/endothelial nitric oxide synthase to increase nitric oxide levels and provoke angiogenesis and renal vasodilation. In addition to inhibiting DPP-4, saxagliptin increased the renal kidney injury molecule-1/pY705-STAT3/hypoxia-inducible factor-1α/VEGF pathway to enhance angiogenesis. Similar to other gliptins, saxagliptin exerted its anti-inflammatory and antioxidant effects by suppressing the renal contents of p (S536)-nuclear factor-κB p65, tumour necrosis factor-α, monocyte chemoattractant protein-1, myeloperoxidase, and malondialdehyde while boosting the glutathione content. These events improved the histological structure and function of the kidney, as evidenced by decreased serum creatinine, blood urea nitrogen, and cystatin C and increased serum albumin. Accordingly, in addition to its anti-inflammatory and antioxidant activities, saxagliptin dose-dependently ameliorated I/R-induced renal damage by enhancing neovascularization through improved tissue perfusion and homing of bone marrow-derived EPCs to mediate repair processes.

Knockdown of Tumour Necrosis Factor-α-Induced Protein 8-Like 2 Promotes Normal and Diabetic Corneal Epithelial Wound Healing and Attenuates Corneal Apoptosis via Upregulation of Nuclear Factor-κB p50

Background: Corneal epithelial defects are frequently observed in ocular surface disorders. Diabetic keratopathy impairs wound healing after corneal injury. The expression and roles of tumour necrosis factorα-induced protein 8-like 2 (TIPE2), a novel regulator of proliferation, apoptosis, inflammation, and immunity in the cornea during normal and diabetic corneal epithelial wound healing are unknown. Here, we evaluated the roles of TIPE2 in the cornea. Methods: The expression of TIPE2, nuclear factor (NF)-κB p50, and AKT was measured in intact and healing corneal epithelia in normal and streptozotocin-induced diabetic mice. Corneal epithelial wound healing and corneal apoptosis were determined in normal, TIPE2-deficient, and diabetic mice with or without NF-κB p50 inhibitor, AKT inhibitor, or TIPE2 siRNA treatment. Findings: TIPE2 levels were upregulated after corneal epithelial injury. TIPE2 deficiency accelerated corneal epithelial wound healing, decreased corneal apoptosis, and increased NF-κB p50 and phospho-AKT levels in intact and healing corneal epithelia. AKT inhibitor treatment suppressed NF-κB p50 expression, and NF-κB p50 or AKT inhibition impaired epithelial wound healing and increased corneal apoptosis. Diabetic mice showed more dramatic TIPE2 upregulation, NF-κB p50 downregulation, and phospho-AKT suppression. TIPE2 deficiency, and TIPE2 siRNA treatment improved corneal epithelial wound healing in diabetic mice. Interpretation: TIPE2 knockdown ameliorated corneal epithelial wound healing under normal and diabetic conditions by reducing apoptosis via upregulation of NF-κB p50 through AKT signalling. TIPE2 may be a therapeutic target in normal and diabetic corneal epithelial defects. Funding Statement: This research was supported by the National Natural Science Foundation of China (81670828, 81570820) and the Innovation Project of Shandong Academy of Medical Sciences. Lingling Yang are partially supported by the Taishan Scholar Youth Professional Program (tsqn20161059). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: All animal experiments were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Ethics Committee of Shandong Eye Institute.

Diacerein Downregulates NLRP3/Caspase-1/IL-1β and IL-6/STAT3 Pathways of Inflammation and Apoptosis in a Rat Model of Cadmium Testicular Toxicity.

The potential gonadal protective effect of diacerein (DCN) and its underlying mechanisms were studied in a rat model of cadmium-induced testicular toxicity. The rats received DCN (50mg/kg/day, p.o.) for 10days and one injection of CdCl2 (2mg/kg, i.p.) on day 9. Cadmium significantly declined serum testosterone and significantly raised interleukin-1β, interleukin-6, interleukin-18, tumor necrosis factor-α, caspase-1, phosphorylated signal transducer and activator of transcription-3 (pSTAT3), nuclear factor-κB p65, Bax, and caspase-3 in rat testes. DCN significantly ameliorated the changes in the biochemical measurements observed with CdCl2 insult. Additionally, DCN preserved the normal testicular architecture, maintained spermatogenesis, and lowered the expression of NOD-like receptor family protein 3 (NLRP3) inflammasome in testes of rats that received CdCl2. It was concluded that DCN significantly protected the gonads of male rats exposed to cadmium toxicity through modulation of NLRP3/caspase-1/IL-1β and IL-6/STAT3 pathways of inflammation and apoptosis.

Protective effects of catalpol on diabetes mellitus-induced male reproductive damage via suppression of the AGEs/RAGE/Nox4 signaling pathway

AimsDiabetes mellitus (DM)-induced reproductive damage is an important cause of infertility for male DM patients, we herein evaluated the effects of catalpol on diabetic reproductive damage through the suppression of the AGEs/RAGE/Nox4 signaling pathway. MethodsKK-Ay diabetic reproductive damage mice were administered with catalpol for 8 weeks, the testis/body weight ratio, testicular histopathology, the levels of endogenous hormone and the activity of testicular marker enzymes were determined. In vitro, the GC-2 cell injury model was induced by advanced glycation end-products (AGEs) and pretreated with catalpol. Cell viability, apoptosis, and oxidative stress markers were detected and the mechanism based on the AGEs/RAGE/Nox4 pathway was explored. Key findingsCatalpol showed remarkable capacity on protecting diabetic reproductive damage by improving the histomorphology of the testes, increasing the testis/body weight ratio and activity of acid phosphatase (ACP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (γ-GT). The reduced testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in DM mice were also reversed with catalpol intervention. Moreover, catalpol showed markedly effects of anti-oxidative in vivo and in vitro, which significantly down-regulated reactive oxygen species (ROS) levels and restored superoxide dismutase (SOD) activity, meanwhile decreased GC-2 cell apoptosis and Bax/Bcl-2 ratio. Moreover, the over-expression of receptors for AGEs (RAGE), NADPH oxidase type 4 (Nox4) and phosphorylation of nuclear transcription factor-κB p65 (NF-κB p65) were suppressed by catalpol. SignificanceCatalpol could alleviate DM-induced male reproductive damage by inhibiting oxidative stress-induced apoptosis and inflammation mediated by AGEs/RAGE/Nox4 signaling pathway.

MiR-150 attenuates LPS-induced acute lung injury via targeting AKT3

Acute lung injury (ALI) and its severe manifestation of acute respiratory distress syndrome (ARDS) in human lung are induced by inflammatory cytokines and endogenous factors such as miRNAs. However, the role of miR-150 in lipopolysaccharide (LPS)-induced ALI is not clear. Here, we found miR-150 expression was significantly reduced in the serum of patients with ARDS, and negatively associated with the disease severity and 28-day survival of ARDS. In vivo, miR-150 decreased total cell and neutrophil counts, and production of inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) as well as levels of total protein, albumin and IgM in the bronchoalveolar lavage (BAL) fluid in LPS-induced ALI mice. Meanwhile, miR-150 improved the 72 h survival rate of LPS-induced ALI mice. In-vitro assays demonstrated that miR-150 alleviated LPS-induced A549 cell apoptosis, autophagy, and release of inflammatory cytokines. Further, AKT3 was a direct target of miR-150. Silencing of AKT3 partially reversed LPS-induced A549 cell injury, and enhanced the protective effects of miR-150. In addition, miR-150 or si-AKT3 effectively inhibited the phosphorylation levels of c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) (p65 and IκBα). In conclusion, miR-150 alleviated LPS-induced acute lung injury via directly targeting AKT3 expression or regulating JNK and NF-κB pathways, which may be a promising therapeutic strategy to treat ALI/ARDS.

The interleukin-33-mediated inhibition of expression of two key genes implicated in atherosclerosis in human macrophages requires MAP kinase, phosphoinositide 3-kinase and nuclear factor-\u03baB signaling pathways

Atherosclerosis, a chronic inflammatory disorder of the walls of arteries, causes more deaths worldwide than any other disease. Cytokines, which are present at high levels in atherosclerotic plaques, play important roles in regulating the initiation and the progression of the disease. Previous studies using animal and cell culture model systems revealed protective, anti-atherogenic effects of the cytokine interleukin-33 (IL-33). The action of this cytokine involves both the induction and suppression of expression of many genes. Unfortunately, the signaling pathways that are responsible for the inhibition of gene expression by this cytokine are poorly understood. Further studies are required given the important roles of genes whose expression is inhibited by IL-33 in key cellular processes associated with atherosclerosis such as monocyte recruitment, foam cell formation and lipoprotein metabolism. We have investigated here the roles of various known IL-33 activated signaling pathways in such inhibitory actions using RNA interference-mediated knockdown assays and monocyte chemotactic protein-1 and intercellular adhesion molecule-1 as model genes. Key roles were identified for extracellular signal-regulated kinase-1/2, p38α kinase, c-Jun N-terminal kinase-1/2, phosphoinositide 3-kinase-γ, and p50 and p65 nuclear factor-κB in such inhibitory action of IL-33. These studies provide new insights on the signaling pathways through which IL-33 inhibits the macrophage expression of key atherosclerosis-associated genes.

GEN-27 exhibits anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway.

Prolonged inflammation and deregulated cytokine production are associated with diversified inflammatory diseases. Genistein (GEN), the active and predominant isoflavonoid in dietary soybean, possesses anti-inflammatory activity. Our study aimed to assess the anti-inflammatory effects of GEN-27, a derivative of GEN, as well as explore the potential molecular mechanisms using lipopolysaccharide (LPS)-induced RAW264.7 cells. In our study, we demonstrated that GEN-27 administration (1, 5, or 10 μM) dose-dependently inhibited nitrite and nitric oxide (NO) levels in LPS-stimulated RAW264.7 cells. Also, GEN-27 suppressed the release of LPS-induced pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-18. Moreover, GEN-27 attenuated LPS-induced inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) expressions at messenger RNA and protein levels, and reversed the promoter activity of iNOS in RAW264.7 cells. Mechanistically, GEN-27 abated LPS-induced reactive oxygen species production, as well as mitigated LPS-induced increase of caspase 1 activity and the protein levels of NOD-like receptor 3 (NLRP3), anti-apoptosis-associated speck-like protein-containing a CRAD (ASC), and caspase 1 in RAW264.7 cells in a dose-dependent manner. Similarly, GEN-27 dose-dependently weakened adenosine triphosphate-induced NLRP3 and IL-1β in RAW264.7 cells. In addition, GEN-27 treatment significantly suppressed LPS-induced phosphorylation of nuclear factor-κB (NF-κB) p65 and alleviated LPS-induced increase of transcriptional activity of NF-κB in RAW264.7 cells. In summary, these results revealed that GEN-27 exhibited anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway, suggesting that GEN-27 may be served as a promising therapeutic agent for the prevention and therapy of inflammatory-associated diseases.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38− progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

Activation of Liver X Receptors by GW3965 Attenuated Deoxycorticosterone Acetate-Salt Hypertension-Induced Cardiac Functional and Structural Changes.

In this study, the effect of liver X receptor (LXR) activation on hypertension-induced cardiac structural and functional alterations was investigated. Hypertension was induced by deoxycorticosterone acetate (DOCA)-salt administration in uninephrectomized rats for 6 weeks. LXR agonist GW3965 (3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid was given for the past week. Rhythmic activity and contractions of the isolated heart tissues were recorded. Biochemical parameters were assessed in ventricular tissue and plasma samples. Cardiac expressions of various proteins were examined, and histopathological evaluation was performed in the left ventricle and liver. GW3965 reduced systolic blood pressure and enhanced noradrenaline-stimulated papillary muscle contraction induced by DOCA-salt + uninephrectomy. Plasma and tissue total antioxidant capacity (TAC) increased and tissue 4-hydroxynonenal (4-HNE) levels decreased in the DOCA-salt group. GW3965 elevated plasma and tissue TAC levels in both of groups. Glucose-regulated protein-78 (GRP78), phospho-dsRNA-activated-protein kinase-like ER kinase (p-PERK), matrix metalloproteinase-2 (MMP-2), and nuclear factor-κB p65 (NF-κB p65) expression was augmented, and inhibitor-κB-α (IκB-α) expression was reduced in hypertensive hearts. The altered levels of all these markers were reversed by GW3965. Also, GW3965 ameliorated DOCA-salt + uninephrectomy-induced cardiac and hepatic inflammation and fibrosis. However, GW3965 unchanged the plasma lipid levels and hepatic balloon degeneration score. These results demonstrated that LXR activation may improve hypertension-induced cardiac changes without undesired effects.

Quercetin reduces TNF-α-induced mesangial cell proliferation and inhibits PTX3 production: Involvement of NF-κB signaling pathway.

Lupus nephritis (LN) is an autoimmune disease caused by systemic lupus erythematosus. Excessive proliferation of mesangial cells is one of the most serious pathological manifestations of LN. In addition, the expression of PTX3 is elevated in the serum of patients with LN. Quercetin has good anti-inflammatory effects and immunomodulatory activities. In this study, the result of MTT indicated that quercetin treatment alleviated the excessive proliferation of mesangial cells. ELISA and immunofluorescence experiments showed that quercetin treatment inhibited the expression of PTX3. Three doses of quercetin (20, 40, and 80μM) were selected for the experiment. It is noteworthy that the efficacy of quercetin at 80μM was significantly better than that of other dose groups. And the effect in inhibiting PTX3 expression was comparable with that of the PDTC (80μM) positive control. Western blot and qRT-PCR analysis revealed that quercetin treatment reduced the expression of nuclear factor-κB p65 and IKKβ, increased the expression of IκBα, and inhibited the expression of PTX3. In conclusion, through inhibiting the activation of nuclear factor-κB signaling pathway, quercetin treatment could reduce the expression of PTX3 and inhibit the excessive proliferation of mesangial cells, suggesting that quercetin is a potential therapeutic drug for LN.

Amelioration of Enterotoxigenic Escherichia coli-Induced Intestinal Barrier Disruption by Low-Molecular-Weight Chitosan in Weaned Pigs is Related to Suppressed Intestinal Inflammation and Apoptosis.

Enterotoxigenic Escherichia coli (ETEC) infection destroys the intestinal barrier integrity, in turn, disrupting intestinal homoeostasis. Low-molecular-weight chitosan (LMWC) is a water-soluble chitosan derivative with versatile biological properties. Herein, we examined whether LMWC could relieve ETEC-induced intestinal barrier damage in weaned pigs. Twenty-four weaned pigs were allotted to three treatments: (1) non-infected control; (2) ETEC-infected control; and (3) ETEC infection + LMWC supplementation (100 mg/kg). On day 12, pigs in the infected groups were administered 100 mL of ETEC at 2.6 × 109 colony-forming units/mL to induce intestinal barrier injury. Three days later, serum samples were obtained from all pigs, which were then slaughtered to collect intestinal samples. We evidenced that LMWC not only increased (P < 0.05) the occludin protein abundance but also decreased (P < 0.05) the interleukin-6, tumour necrosis factor-α and mast cell tryptase contents, and the apoptotic epithelial cell percentages, in the small intestine of ETEC-infected pigs. Furthermore, LMWC down-regulated (P < 0.05) the small intestinal expression levels of critical inflammatory- and apoptotic-related genes, such as Toll-like receptor 4 (TLR4) and tumour necrosis factor receptor 1 (TNFR1), as well as the intra-nuclear nuclear factor-κB (NF-κB) p65 protein abundance, in the ETEC-infected pigs. Our study indicated a protective effect of LMWC on ETEC-triggered intestinal barrier disruption in weaned pigs, which involves the repression of intestinal inflammatory responses via blocking the TLR4/NF-κB signalling pathway and the depression of epithelial cell death via TNFR1-dependent apoptosis.

Anti-Apoptotic Effect of G-Protein-Coupled Receptor 40 Activation on Tumor Necrosis Factor-α-Induced Injury of Rat Proximal Tubular Cells

G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic β-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway.

Tofacitinib inhibits ox-LDL-induced adhesion of THP-1 monocytes to endothelial cells

Atherosclerosis is a chronic inflammatory disease of the blood vasculature. Endothelial dysfunction is an early event in the development of atherosclerosis and the endothelium plays an important role in the innate immune defense in the pathology of cardiovascular diseases. New therapies are being developed based on the involvement of the immune system in atherosclerosis. In this study, we demonstrate that a commonly used anti-rheumatic drug, tofacitinib, possesses vascular protective properties in cultured primary human aortic endothelial cells (HAECs). Tofacitinib ameliorates oxidized low-density lipoprotein (ox-LDL)-induced adhesion of THP-1 cells to HAECs, suppresses the expression of vascular adhesion molecules and production of cytokines, including vascular cell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Moreover, tofacitinib inhibits elevation of endothelial lectin-like ox-LDL receptor-1 (LOX-1) and production of reactive oxygen species (ROS) triggered by ox-LDL. As a result, the presence of tofacitinib reduces ox-LDL-induced cytotoxicity and improves endothelial viability. Mechanistically, we demonstrate that tofacitinib suppresses ox-LDL-mediated activation of NF-κB inhibitor α (IκB-α), accumulation of nuclear p65 and activation of nuclear factor κB (NF-κB) promoter, indicating that tofacitinib inhibits NF-κB activation. Collectively, our data support that tofacitinib possesses a novel protective function in endothelial cells, implying that tofacitinib could have the therapeutic potential to modulate inflammation in cardiovascular diseases.

The therapeutic effects of Jaceosidin on lipopolysaccharide-induced acute lung injury in mice

Acute lung injury (ALI) results from various factors including uncontrolled pulmonary inflammation, oxidative damage and the over-activated complement with high mortality rates. Jaceosidin was a flavonoid compound with significant anti-complement activity. We aimed to investigate the therapeutic effects of Jaceosidin on ALI induced by lipopolysaccharide (LPS). Mice were orally administrated with Jaceosidin (15, 30 and 60 mg/kg) after LPS challenge. 24 h after LPS challenge, Jaceosidin could significantly decrease the lung wet-to-dry weight (W/D) ratio and the protein concentration in bronchoalveolar lavage fluid (BALF). Jaceosidin could down-regulate the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), together with up-regulation the levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) in BALF. Jaceosidin could significantly decrease the levels of myeloperoxidase (MPO), cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB), COX-2 mRNA and NF-κB p65 mRNA together with increasing the activity of catalase (CAT). Additionally, Jaceosidin attenuated lung histopathological changes, inhibited the expressions of COX-2 and NF-κB p65 and reduced complement deposition with decreasing the levels of complement 3 (C3) and complement 3c (C3c) in serum. These data suggest that Jaceocidin may dampen the inflammatory response and decrease the levels of complement together with the antioxidant activity following LPS-induced ALI.

Protective effects of microRNA-181b on aged rats with sepsis-induced hippocampus injury in vivo

To investigate the protective effect of microRNA-181b (miR-181b) on aged rats with sepsis-induced hippocampus injury in vivo. Seventy-five male healthy old Sprague-Dawley (SD) rats were randomly divided into five groups (n = 15) using a random number table: sham operation group (Sham group), sepsis group [cecal ligation and puncture (CLP) group], miR-181b Agomir+CLP group (Ag+CLP group), miR-181b Antagomir+CLP group (An+CLP group) and normal saline (NS) control group (NS+CLP group). Rats sepsis model was reproduced by CLP, and in Sham group, the cecum of rats was separated only after abdominal operation without ligation or perforation. The rats in Ag+CLP group were given miR-181b Agomir 10 μL via lateral ventricle at 24 hours before CLP, the rats in An+CLP group were given 10 μL miR-181b Antagomir, and those in NS+CLP group were given 10 μL NS. At 6, 12, 24 hours after CLP, 5 rats of each group were sacrificed randomly, and hippocampus were harvested. The expression of miR-181b in hippocampus was determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression of nuclear factor-ΚB p65 (NF-ΚB p65) was determined by Western Blot. The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Compared with Sham group, the expression of miR-181b in hippocampus of CLP group was significantly decreased at 6 hours after CLP (2-ΔΔCT: 0.70±0.12 vs. 0.98±0.06, P < 0.05), and the expressions of NF-ΚB p65, IL-1β and TNF-α were significantly increased [NF-ΚB p65/Histone H3: 0.30±0.03 vs. 0.07±0.01, IL-1β (ng/L): 120.39±8.02 vs. 50.55±11.12, TNF-α (ng/L): 59.48±4.60 vs. 40.31±3.96, all P < 0.05], this trend was continued till 24 hours, and these results indicated that there was obvious inflammation in hippocampus of sepsis rats. There was no statistical difference in the expression of miR-181b, NF-ΚB p65, IL-1β or TNF-α in hippocampus between NS+CLP group and CLP group, which indicated that injection of NS into the rat lateral ventricle, had not aggravated the damage degree of hippocampus. Compared with CLP group, the expression of miR-181b in hippocampus of Ag+CLP group was significantly increased at 6 hours after CLP (2-ΔΔCT: 1.87±0.25 vs. 0.70±0.12, P < 0.05), and the expressions of NF-ΚB p65, IL-1β and TNF-α were significantly lowered [NF-ΚB p65/Histone H3: 0.16±0.03 vs. 0.30±0.03, IL-1β (ng/L): 73.76±8.17 vs. 120.39±8.02, TNF-α (ng/L): 49.52±4.77 vs. 59.48±4.60, all P < 0.05]. There was no statistical difference in the expression of miR-181b in hippocampus between An+CLP group and CLP group (2-ΔΔCT: 0.80±0.08 vs. 0.70±0.12 at 6 hours, 0.48±0.03 vs. 0.46±0.05 at 12 hours, 0.61±0.09 vs. 0.63±0.07 at 24 hours, all P > 0.05), but the expressions of NF-ΚB p65, IL-1β and TNF-α in hippocampus at 6 hours after CLP of An+CLP group were significantly higher than those of CLP group [NF-ΚB p65/Histone H3: 0.44±0.02 vs. 0.30±0.03, IL-1β (ng/L): 134.21±5.78 vs. 120.39±8.02, TNF-α (ng/L): 67.62±5.86 vs. 59.48±4.60, all P < 0.05], this trend was continued till 24 hours after CLP. The above results showed that overexpression of miR-181b might attenuate the inflammation of hippocampus through down-regulation of NF-ΚB, IL-1β and TNF-α. The expression of hippocampal miR-181b was significantly decreased in septic rats. Up-regulation of miR-181b could inhibit the activation of NF-ΚB signal pathway and the release of the inflammatory cytokine IL-1β and TNF-α stimulated by sepsis, and alleviate the inflammatory reaction and hippocampus injury in rat with sepsis.

Dexmedetomidine ameliorates LPS induced acute lung injury via GSK-3β/STAT3-NF-κB signaling pathway in rats

Acute lung injury (ALI) is a serious complication of sepsis and an important cause of death in intensive care. Studies have shown that DEX can inhibit inflammation. However, the anti-inflammatory effect and protective mechanism of DEX in lipopolysaccharide (LPS) induced ALI are still unclear. ALI model was established by intraperitoneal injection of LPS (10 mg/kg) in Sprague-Dawley (SD) male rats. Firstly, at 4, 6, 8, 12 and 24 h after LPS treatment, lung injury including pathologic histology, lung edema, and inflammation were detected. The optimal time point for lung injury was determined to be 12 h, at which time DEX was added to further test. Furthermore, STAT3 inhibitor (NSC74859) and GSK-3β inhibitor (SB216763) were added to verify the role of STAT3, GSK-3β and NF-κB in ameliorated ALI. Our results show that DEX pretreatment significantly decreased lung Wet-to-Dry weight (W/D) ratio and MPO activity and ameliorated LPS induced lung histopathological alterations. In addition, we confirmed that DEX can increased the phosphorylation of STAT3 and GSK-3β, and inhibit the phosphorylation of nuclear factor-κB (NF-κB) p65 in the inflammatory response induced by LPS. What's more, NSC74859 inhibited the phosphorylation of STAT3 and reversed the protect effect of DEX on LPS. SB216763 inhibited the phosphorylation of NF-κB and reversed the damage effect of LPS and plays the same anti-inflammatory effect as DEX. In summary, our data demonstrated that DEX can ameliorate ALI induced by LPS through GSK-3β/STAT3-NF-κB pathway.

Protective effect of Cordyceps sinensis extract on lipopolysaccharide-induced acute lung injury in mice.

Background: To study the protective effect of Cordyceps sinensis extract (Dong Chong Xia Cao in Chinese [DCXC]) on experimental acute lung injury (ALI) mice.Methods and results: ALI model was induced by intratracheal-instilled lipopolysaccharide (LPS, 2.4 mg/kg) in BALB/c male mice. The mice were administrated DCXC (ig, 10, 30, 60 mg/kg) in 4 and 8 h after receiving LPS. Histopathological section, wet/dry lung weight ratio and myeloperoxidase activity were detected. Bronchoalveolar lavage fluid (BALF) was collected for cell count, the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and nitric oxide (NO) in BALF was detected by ELISA, the protein and mRNA expression of nuclear factor-κB p65 (NF-κB p65), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung tissue was detected by Western blot and RT-PCR. The result showed that DCXC could reduce the degree of histopathological injury, wet/dry weight ratio (W/D ratio) and myeloperoxidase activity (P<0.05) with a dose-dependent manner. The increased number of total cells, neutrophils and macrophages in BALF were significantly inhibited by DCXC treatment (P<0.05). The increased levels of TNF-α, IL-1β, IL-6 and NO in BALF after LPS administration was significantly reduced by DCXC (P<0.05). In addition, the increased protein and mRNA levels of iNOS, COX-2 and NF-κB p65 DNA binding ability in LPS group were dose-dependently reduced by DCXC treatment (P<0.05).Conclusion: DCXC could play an anti-inflammatory and antioxidant effect on LPS-induced ALI through inhibiting NF-κB p65 phosphorylation, and the expression of COX-2 and iNOS in lung. The result showed that DCXC has a potential protective effect on the ALI.

GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1β and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.

Ethanol Extract of Illicium henryi Attenuates LPS-Induced Acute Kidney Injury in Mice via Regulating Inflammation and Oxidative Stress.

The root bark of Illicium henryi has been used in traditional Chinese medicine to treat various diseases. Its ethanol extract (EEIH) was found to contain a large number of phenols and possess in vitro antioxidant activities. The present study aimed to investigate its protective effect against lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice. BALB/c mice were intraperitoneally pretreated with EEIH for five days, and then LPS injection was applied to induce AKI. Blood samples and kidney tissues were collected and used for histopathology, biochemical assay, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analyses. EEIH not only significantly dose-dependently attenuated histological damage and reduced renal myeloperoxidase (MPO) activity (from 9.77 ± 0.73 to 0.84 ± 0.30 U/g tissue) but also decreased serum creatinine (from 55.60 ± 2.70 to 27.20 ± 2.39 µmol/L) and blood urea nitrogen (BUN) (from 29.95 ± 1.96 to 16.12 ± 1.24 mmol/L) levels in LPS-treated mice. EEIH also markedly dose-dependently inhibited mRNA expression and production of TNF-α (from 140.40 ± 5.15 to 84.74 ± 5.65 pg/mg), IL-1β (from 135.54 ± 8.20 to 77.15 ± 5.34 pg/mg), IL-6 (from 168.74 ± 7.23 to 119.16 ± 9.35 pg/mg), and COX-2 in renal tissue of LPS-treated mice via downregulating mRNA and protein expressions of toll-like receptor 4 (TLR4) and phosphorylation of nuclear factor-κB (NF-κB) p65. Moreover, EEIH significantly dose-dependently reduced malondialdehyde (MDA) (from 5.43 ± 0.43 to 2.80 ± 0.25 nmol/mg prot) and NO (from 1.01 ± 0.05 to 0.24 ± 0.05 µmol/g prot) levels and increased superoxide dismutase (SOD) (from 22.32 ± 2.92 to 47.59 ± 3.79 U/mg prot) and glutathione (GSH) (from 6.57 ± 0.53 to 16.89 ± 0.68 µmol/g prot) levels in renal tissue induced by LPS through upregulating mRNA expression of nuclear factor erythroid 2 related factor 2 (Nrf2). Furthermore, EEIH inhibited LPS-induced intracellular reactive oxygen species (ROS) production from RAW264.7 cells in a concentration-dependent manner. These results suggest that EEIH has protective effects against AKI in mice through regulating inflammation and oxidative stress.

BIO alleviates inflammation through inhibition of GSK-3β in a rat model of intracerebral hemorrhage.

Inflammation plays a key role in secondary brain damage following intracerebral hemorrhage (ICH). Glycogen synthase kinase-3β (GSK-3β) plays a strong proinflammatory role in many CNS diseases, including stroke. The present study was undertaken to examine the effects of 6-bromoindirubin-3'-oxime (BIO), a specific inhibitor of GSK-3β, on inflammation in ICH rats. An ICH rat model was induced by autologous whole-blood injection into the striatum. First, 10, 20, 40, 60, 80, or 100 μg/kg BIO was applied to ICH animals to determine an optimal dosage for producing sufficient GSK-3β inhibition in rat ipsilateral hippocampus by Western blotting. Second, 40 μg/kg BIO was applied to ICH rats for 1, 3, 7, or 14 days, respectively, to determine a suitable intervention time course of BIO by Western blotting analysis on GSK-3β. Third, Western blotting and enzyme-linked immunosorbent assay were used for quantification of inflammation-related factors upstream or downstream of GSK-3β in rat ipsilateral hippocampus. Then, immunohistochemical staining was applied to detect activated microglia and apoptotic cells in rat ipsilateral hippocampus. Last, neurobehavioral tests were performed to assess the sensorimotor impairments in the ICH rats. The results show that BIO 1) blocked GSK-3βTyr216 phosphorylation/activation, thus stabilizing β-catenin, increasing upstream brain-derived neurotrophic factor and downstream heat shock protein 70 levels, and decreasing the levels of nuclear factor-κB p65 and cyclooxygenase 2; 2) decreased the levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-1β and IL-6 and elevated the level of antiinflammatory cytokine IL-10; 3) inhibited microglia activation and cell apoptosis; and 4) improved the sensorimotor deficits of ICH rats. BIO posttreatment inhibited microglia activation, prevented inflammation and hippocampal cell death, and ameliorated functional and morphological outcomes in a rat ICH model through inactivation of GSK-3β.