α7nAChR is involved in anti-inflammation of physiological concentrations of glucocorticoids

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR) in anti-inflammation of glucocorticoids (GCs) at physiological concentrations. METHODS: MTT assay was used to measure the viability of BV-2 cells, which were processed by hydrocortisone at different concentrations. On the basis of inflammatory model induced by LPS in BV-2 cells, experimental groups were divided as follows: (1) control; (2) LPS; (3) GCs＋LPS; (4) methyllycaconitine (MLA)＋GCs＋LPS. The levels of TNF-α and IL-1β in the cell supernatants were detected by ELISA. RESULTS: Hydrocortisone at concentrations of 2 000 and 1 000 nmol/L decreased the cell viability to (76.9±5.5)% and (90.8±7.3)%, respectively, indicating the cellular injury by GCs at over-physiological doses. LPS significantly induced the releases of TNF-α and IL-1β in a time- and dose-dependent manner in BV-2 cells. Hydrocortisone at physiological concentrations (500 and 250 nmol/L) reduced the releases of TNF-α and IL-1β in BV-2 cells stimulated by LPS, and MLA at concentration of 10 nmol/L antagonized the anti-inflammatory effect of GCs. CONCLUSION: α7nAChR is involved in the anti-inflammatory effect of the physiological concentrations of GCs.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

Activation of macrophage peroxisome proliferator-activated receptor α inhibits macrophage inflammation-induced cardiac fibroblast activation and migration

AIM: To investigate the effect of macrophage peroxisome proliferator-activated receptor α (PPARα) activation on macrophage inflammation-induced activation and migration of cardiac fibroblasts. METHODS: Mouse bone marrow-derived macrophages were treated with vehicle, PPARα agonist WY14643 (10 μmol/L), angiotensin Ⅱ (Ang II; 1 μmol/L) or Ang II+WY14643 for 24 h, and the supernatants were collected as conditioned medium (CM) to stimulate cardiac fibroblasts for additional 24 h. The mRNA levels of PPARα, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the macrophages as well as fibrotic markers collagen type Ⅰ alpha 2 chain (Col1a2), collagen alpha 1 chain (Col3a1) and actin alpha 2 (Acta2) in the cardiac fibroblasts were detected by RT-qPCR. The protein levels of IL-6 and IL-1β in the macrophages as well as collagen I, collagen III and α-smooth muscle actin (α-SMA; encoded by Acta2 gene) in the cardiac fibroblasts were determined by Western blot. Wound-healing assay was applied to eva-luate the migration ability of cardiac fibroblasts. RESULTS: Ang II significantly increased the mRNA levels of pro-inflammatory factors, such as IL-6, IL-1α and TNF-α, but decreased the mRNA level of PPARα in the macrophages. Administration of PPARα agonist WY14643 dramatically decreased Ang II-induced mRNA levels of IL-6, IL-1β and TNF-α in the macrophages, and significantly decreased Ang II-induced protein expression of IL-6 and pro-IL-1β in the macrophages. The CM from Ang II-treated macrophages significantly up-regulated the mRNA levels of Col1a2, Col3a1 and Acta2 in the cardiac fibroblasts, which were inhibited by the CM from WY14643-treated macrophages. The same results were observed in the protein levels of collagen I, collagen III and α-SMA in the cardiac fibroblasts. Moreover, the CM from Ang II-treated macrophages significantly promoted cardiac fibroblast migration, whereas the CM from WY14643-treated macrophages markedly inhibited macrophage inflammation-induced cardiac fibroblast migration. CONCLUSION: WY14643-activated PPARα inhibits activation and migration of cardiac fibroblasts by attenuating Ang II-induced macrophage inflammatory response.     

Curcumin reduces neuroinflammation stimulated by Aβ25-35 in primary rat microglial cells

AIM: To investigate the effects of curcumin (Cur) on the expression of High mobility group box 1 protein (HMGB1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) in amyloid-β (Aβ)-induced primary rat microglial cells. METHODS: Microglia were derived from the cerebral cortices of postnatal rat brains. The cells were identified by immunocytochemistry using mouse anti rat Iba-1 monoclonal antibody. A cell model using primary rat microglial cells incubated with Aβ_25-35 as an inflammation model of Alzheimer's disease (AD) was set up. The morphological characters of primary rat microglial cells were observed. The concentration of Aβ_25-35 and the treatment concentration of curcumin were selected by CCK-8 assay. Cultured primary rat microglial cells were divided into 5 groups:normal cell group, Aβ_25-35 group, Cur group, Aβ_25-35+Cur group and Aβ_25-35+DMSO group. The expression of HMGB1, NF-κB, and receptor for advanced glycation end products (RAGE) was detected by Western blot. The levels of HMGB1, IL-1β, and TNF-α in the culture supernatant were measured by ELISA. RESULTS: The purity of primary microglias determined by Iba-1 immunofluorescence was more than 95%. The protein levels of HMGB1, RAGE and NF-κB were significantly increased after Aβ_25-35 stimulation. After treatment with Cur, the protein levels of HMGB1, RAGE and NF-κB were significantly decreased (P<0.05). The levels of HMGB1, IL-1β and TNF-α in the supernatant were significantly increased after Aβ_25-35 stimulation. Cur significantly decreased the level of HMGB1, IL-1β and TNF-α in the supernatant. CONCLUSION: Curcumin significantly inhibits neuroinflammation stimulated by Aβ_25-35 in primary rat microglial cells.     

Adipolin/CTRP12 protects against LPS-induced ARDS by up-regulating alveolar epithelial sodium channel in mice

AIM: To investigate the effect of adipolin/CTRP12 in LPS-induced acute respiratory distress syndrome(ARDS) and its potential regulation on alveolar epithelial sodium channel(ENaC) in mice. METHODS: C57BL/6J mice(n=40) were randomly divided into control group, LPS group, adipolin group and wortmannin(PI3K inhibitor) group with 10 mice in each group using random number table. The pathological changes of the lung tissues were evaluated by HE staining. The alveolar fluid clearance(AFC) was measured by Evans blue-marked albumin, and the concentrations of total protein in bronchoalveolar lavage fluid(BALF) were assessed by bicinchoninic acid(BCA) method. In BALF, the levels of IL-1β and TNF-α were determined by ELISA, and the activity of myeloperoxidase(MPO) was detected by an MPO assay kit. The total cell counts and polymorphonuclear neutrophil(PMN) counts in the BALF were analyzed by Giemsa staining. The mRNA levels of α-ENaC were assessed by qPCR, while the protein levels of α-ENaC and p-Akt were determined by Western blot. RESULTS: Compared with control group, the classic ARDS pathological changes were observed in the mice in LPS group, manifesting by severe pathological lung injury(P<0.05), increases in W/D weight ratio, total protein levels, cell counts, MPO activitiy, and IL-1β and TNF-α levels in the BALF, and decrease in AFC(P<0.05), accompanied by down-regulated levels of α-ENaC and p-Akt in the lung tissues(P<0.05). The deteriorating effects triggered by LPS were significantly reversed by administration of adipolin. However, PI3K inhibitor wortmannin canceled the beneficial effects of adipolin on LPS-induced ARDS, as evidenced by aggravated lung injury, increased levels of W/D weight ratio, protein levels, cell counts, MPO activity, and IL-1β and TNF-α levels in the BALF(P<0.05), and decreased levels of AFC, α-ENaC and p-Akt in the lung tissues. CONCLUSION: Adipolin protects against LPS-induced ARDS in the mice by up-regulating α-ENaC and enhancing AFC via PI3K/Akt signal pathway.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

CB2 receptor agonist JWH133 exerts protective effects on rat model of paraquat-induced acute lung injury

AIM: To study the protective effects of cannabinoid CB2 receptor agonist JWH133 on rat acute lung injury induced by paraquat (PQ).METHODS: Male Sprague-Dawley rats (n=72) were randomly divided into 4 groups. PQ group: PQ was administered intraperitoneally at the dose of 20 mg/kg; Low-dose JWH133 pretreatment group (L-JWH133 group): JWH133 (5 mg/kg, ip) was administered 1 h before PQ exposure; high-dose JWH133 pretreatment group (H-JWH133 group): JWH133 (20 mg/kg, ip) was administered 1 h before PQ exposure; control group: 1 mL saline was administered intraperitoneally. Arterial blood, bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 8 h, 1 d and 3 d after PQ exposure. PaO2 and the levels of TNF-α and IL-1β in BALF were measured via blood gas analyzer and ELISA, respectively. The pathological changes and lung injury scores were assessed at 3 d after PQ exposure. NF-κB and AP-1 protein levels were also determined by Western blotting.RESULTS: The decrease in PaO2, structural injury of the lung tissues, interstitial pulmonary edema, and the increase in IL-1β and TNF-α in BALF were observed in PQ-treated rats compared with control group. JWH133 pretreatment reduced the degree of lung tissue injury, decreased the levels of IL-1β and TNF-α in BALF and the NF-κB and AP-1 protein expression in the lung tissue compared with PQ group, especially in H-JWH133 group. CONCLUSION: CB2 receptor agonist JWH133 inhibits NF-κB and AP-1 protein expression in the lung tissues, and reduces the secretion of IL-1β and TNF-α in BALF after paraquat exposure, thus attenuating paraquat-induced acute lung injury.     

Resveratrol attenuates inflammatory pain induced by complete Freund's adjuvant via NF-κB signaling pathway in a mouse model

AIM: To investigate the anti-inflammatory action of resveratrol (Res) and its correlation with nuclear factor-κB (NF-κB) signaling pathway in a mouse model of inflammatory pain.METHODS: BALB/c mice (n=60) were randomly divided into 6 groups:normal control group, inflammatory pain model group, positive control (dexamethasone, 0.5 mg/kg) group and resveratrol (100, 50 and 25 mg/kg) groups (10 mice in each group). In order to observe the anti-inflammatory pain effects of reseratrol on mice, the paw withdrawal mechanical threshold, paw withdrawal thermal latency and cold withdrawal times were detected. In order to analyze the mechanism of analgesic effect of resveratrol, the expression levels of NF-κB, inhibitor of NF-κB (IκB) α, inhibitor of NF-κB kinase (IKK) β, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the spinal cord tissues (L4~L6) of the mice were determined by RT-PCR and Western blot.RESULTS: The resveratrol at 100 and 50 mg/kg increased the paw withdrawal mechanical threshold, prolonged the paw withdrawal thermal latency, and decreased the cold withdrawal times in the inflammatory pain mice (P<0.05 or P<0.01). The resveratrol at 100 mg/kg down-regulated the mRNA and protein expression levels of NF-κB, IκBα, IKKβ, TNF-α and IL-1β in the spinal cord tissues (L4~L6) of inflammatory pain mice (P<0.05 or P<0.01).CONCLUSION: Resveratrol ameliorates the inflammatory pain of the mice induced by complete Freund's adjuvant. The mechanism is related to the inhibition of NF-κB signaling pathway.     

Effects of icariin preconditioning on inflammatory responses in myocar-dial ischemia-reperfusion injury

AIM:To observe the effects of icariin on myocardial ischemia-reperfusion injury. METHODS:The left anterior descending coronary artery was ligated for 30 min and then loosened for 2 h to establish the rat model of myocardial ischemia-reperfusion injury. Forty-eight healthy adult male SD rats weighing 250~300 g were randomly divided into sham group, model group, low-, middle-and high-dose icariin groups, and aspirin group. The morphological changes of the myocardium were observed by HE staining. The protein expression of NF-κB p65 in the myocardial nucleus was determined by the method of immunohistochemistry. The content of tumor necrosis factor α (TNF-α) in the myocardial tissues was detected by Western blotting. The level of interleukin 1β (IL-1β) in the serum was measured by ELISA. The activity of myeloperoxidase (MPO) in the myocardial tissues was assayed by colorimetry. RESULTS:Compared with sham group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in all other groups increased. Compared with model group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in low-, middle- and high-dose icariin groups and aspirin group all decreased. No significant difference of the above parameters between high-dose icariin group and aspirin group was observed. CONCLUSION: Icariin preconditioning reduces inflammatory responses in the process of myocardial ischemia-reperfusion injury in a dose-dependent manner.     

Pretreatment with external trigeminal nerve electrostimulation inhibits seizures and decreases expression of IL-1β and TNF-α in hippocampus with status epilepticus induced by pentylenetetrazol in rats

AIM:To observe the effect of pretreatment with external trigeminal nerve electrostimulation (eTNS) on behavioral changes and the expression of interleukin-1β (IL-1β) and 　tumor necrosis factor α (TNF-α) in hippocampus of pentylenetetrazol (PTZ)-treated rats. METHODS:The rats were randomly divided into control group, PTZ group and eTNS group, and kindled by PTZ after administered 7 d, 14 d and 28 d of consecutive fake electrostimulation or eTNS. Subsequently, the severity and duration of seizure were quantitatively evaluated. The concentrations of IL-1β and TNF-α in hippocampus were detected by the methods of ELISA and immunohistochemisty. RESULTS:Compared with PTZ group, treatment with eTNS significantly inhibited the severity and duration of seizure (P<0.05), and significantly reduced the content of IL-1β and TNF-α in the hippocampus after status epilepticus (P<0.05 or P<0.01). CONCLUSION:Pretreatment with eTNS may provide a new approach for prevention and treatment of epileptogenesis.     

Effects of renal denervation on inflammatory factors in a rabbit model of early atherosclerosis

AIM: To evaluate the effects of renal denervation (RDN) on the expression of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-6 (IL-6) in a rabbit model of early atherosclerosis. METHODS: New Zealand male rabbits were divided into control group, RDN+ high-fat diet (HFD) group (RDN group), sham+HFD group (sham group) and HFD group. The rabbits in later 3 groups were fed with 2% cholesterol for 8 weeks to establish an early atherosclerosis model. The blood samples were collected to test the levels of lipids, norepinephrine (NE), TNF-α, IL-1α and IL-6. The protein expression of angiotensin Ⅱ (Ang Ⅱ) was detected by the method of immunohistochemistry. The levels of nuclear factor-κB (NF-κB) and Ang II 1 type receptor (AT1R) were evaluated by Western blot. The mRNA expression of TNF-α, IL-1α and IL-6 was determined by real-time PCR. RESULTS: After 1 d of RDN procedure, the NE level was lower in RDN group than that in sham group (P<0.01). After 8 weeks, the NE level was lower in RDN group than that in sham group and HFD group (P<0.05), and triglyceride (TG) was lower in RDN group than that in HFD group (P<0.05). The protein expression of Ang II was decreased in RDN group compared with sham group and HFD group (P<0.01). The protein expression of NF-κB was lower in RDN group than that in sham group (P<0.05). The plasma levels of TNF-α and IL-1α were reduced in RDN group compared with sham group and HFD group (P<0.05). The mRNA expression of TNF-α, IL-1α and IL-6 was reduced in RDN group compared with sham group (P<0.05). CONCLUSION: RDN inhibits sympathetic activity, decreases the plasma level of TG, and alleviates inflammatory reactions in the rabbits with atherosclerosis.     

Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

Protective effect of N-acetylcysteine on mice with acute lung injury induced by H9N2 swine influenza virus

AIM: To investigate the effects of N-acetylcysteine (NAC) on acute lung injury induced by H9N2 swine influenza virus (SIV) in mice. METHODS: BALB/c mice were used to establish the animal model of acute lung injury by nasal inoculation of H9N2 SIV. The mice were divided into control group (without SIV infection), H9N2 SIV group (inoculation of H9N2 SIV) and NAC group (inoculation of H9N2 SIV plus pretreatment with NAC). The pulmonary edema was evaluated by determining the lung wet weight/dry weight (W/D) ratio. The pathological changes of the lung tissues were observed. The concontrations of TNF-α, IL-1β and IL-6 in bronchoalveolar lavage fluid (BALF) were measured.The virus titer, T-SOD activity, MPO activity and MDA content in the homogenate of the lung tissues were detected. RESULTS: Treatment with NAC decreased the morality of infected mice, and significantly prolonged the survival time of infected mice. The pathological changes of the lung tissues, the lung W/D ratio and the lung index were relieved when SIV infected the mice treated with NAC. Treatment with NAC significantly decreased the infiltration of inflammatory cells including macrophages, lymphocytes and neutrophils in the BALF. The levels of TNF-α, IL-6, IL-1β and MDA and the activity of MPO were also decreased. Treatment with NAC also significantly increased the T-SOD activity. CONCLUSION: The protective effect of NAC on the acute lung injury mouse model is related to suppression of the oxidative stress and inflammatory responses.     

Inhibitory effects of right cervical sympathetic trunk transection on inflammatory response after acute myocardial infarction in rats and its influence on HMGB1/TLR4/NF-κB signaling pathway

AIM: To observe the effects of transection of right cervical sympathetic trunk (TCST) on inflammatory response and expression of high mobility group box 1 (HMGB1) and TLR4/NF-κB signaling pathway in the rats after acute myocardial infarction (AMI). METHODS: AMI model was established by ligation of left anterior descending coronary artery in SD rats, then the model rats were randomly divided into MI group and MI+TCST group. MI+TCST model was performed by transection of right cervical sympathetic trunk after left anterior descending coronary artery ligation. The rats in MI group and MI+TCST group were divided into 1, 3, 7, 14 and 28 d subgroups, and another sham operation group threading without ligation, with 8 rats in above each group. After modeling for 4 weeks, the cardiac function was measured by echocardiography. All rats were killed to harvest the hearts for mesuring cardiac hypertrophy index. The myocardial tissue close to infarction was observed with HE staining. The relative mRNA expression levels of HMGB1, tumor necrosis factor α(TNF-α) and interleukin (IL)-6 at different time points were detected by real-time PCR. The protein expression of HMGB1 and TLR4 at different time points after AMI was determined by Western blot. The effect of transection of right cervical sympathetic trunk on the expressions of HMGB1 and TLR4/NF-κB signaling pathway was also analyzed.RESULTS: Compared with the MI group, left ventricular ejection fraction (LVEF) and left ventricular shorterning fraction (LVFS) were significantly higher (P<0.05), left ventricular end-diastole dimension (LVEDd), left ventricular end-systole dimension (LVESd) and cardiac hypertrophy index were significantly lower (P<0.05), and the mRNA levels of HMGB1, TNF-α and IL-6 decreased significantly in MI+TCST group (P<0.05). Western blot results revealed that the protein expression level of HMGB1 increased in the infarct border zone at 3 d, and reached its peak at 7 d, then gradually decreased, and at 28 d after MI in MI group was still significantly higher than that in sham group (P<0.05). The protein expression of TLR4 was consistent with that of HMGB1. Transection of right cervical sympathetic trunk reduced protein expression of HMGB1 and TLR4/NF-κB signaling pathway-related proteins (P<0.05).CONCLUSION: Transection of right cervical sympathetic trunk improves ventricular remodeling and maintaining cardiac function. The mechanism may be related to inhibiting HMGB1/TLR4/NF-κB signaling pathway to reduce inflammatory response.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

Bone marrow-derived mesenchymal stem cell-conditioned medium atte-nuates LPS-induced acute lung injury in mice

AIM:To explore the effects of bone marrow-derived mesenchymal stem cells-conditioned medium (MSCs CdM) on lipopolysaccharide (LPS)-induced acute lung injury. METHODS:Lung injury was induced in mice by intraperitoneal injection of LPS. The mice were given a tail vein injection of MSCs CdM or normal saline 1 h after LPS administration. The mice were killed by an intraperitoneal injection of pentobarbital 6 h after LPS injection for either bronchoalveolar lavage fluid (BALF) and serum collection or lung histological analysis. RESULTS:Compared with control group, the BALF levels of protein, interleukin-10 (IL-10) and keratinocyte growth factor (KGF), the serum levels of tumor necrosis factor α (TNF-α) and IL-6, and the myeloperoxidase (MOP) activity in the lung tissues were significantly higher in LPS group, and severe pathological damages in the lung tissues were also observed. Treatment with MSCs CdM significantly reduced the BALF prtein level, the seum TNF-α and IL-6　levels and the lung MPO activity, and attenuated the lung pathological damages, but further increased the levels of IL-10 and KGF in the BALF. CONCLUSION:Treatment with MSCs CdM attenuates the lung injuries induced by LPS, which may be via regulating the expression of TNF-α, IL-6, IL-10 and KGF.