Role of CD163/TWEAK pathway in atherosclerosis

AIM:To investigate the effect of CD163/tumor necrosis factor-like weak inducer of apoptosis (TWEAK) pathway on atherosclerosis in mice. METHODS:APOE^-/- mice and wild-type (WT) C57BL/6 mice were divided into 4 groups (8~10 weeks, n=10):APOE^-/- +normal diet (ND) group, APOE^-/- +western diet (WD) group, WT+ND group, and WT+WD group. Detection of blood lipid levels and oil red O staining of aorta artery were performed to confirm whether the atherosclerotic model was well established in 16 weeks after feeding. The aortic tissues were harvested to measure CD163 and TWEAK protein levels by Western blot, and immunohistochemical staining was also performed to localize CD163 and TWEAK protein expression on atherosclerotic plaque in each group. The cell experiments were conducted to study whether CD163 regulated TWEAK expression in M1 macrophages and foam cells, and the possible downstream pathway was investigated. RESULTS:The blood lipid levels and aorta oil red O staining showed that the animal model of atherosclerosis was successfully established in APOE^-/- +ND group and APOE^-/- +WD group. The protein level of CD163 was significantly increased in the aortic tissue in APOE^-/- mice (P<0.05) as compared with C57BL/6 mice (P<0.05). Consistently, the protein level of TWEAK was also markedly higher in APOE^-/- +ND group and APOE^-/- +WD group than that in WT+ND group and WT+WD group (P<0.05). Immunohistochemical staining showed that CD163 was mainly expressed in the parts away from the lipid core, and TWEAK was found in all parts of the atherosclerotic plaque. CD163 significantly inhibited the protein expression of TEWAK in the M1 macrophages, and also significantly down-regulated the level of nuclear factor-κΒ (NF-κB) in the M1 macrophages and foam cells (P<0.05). CONCLUSION:The protein levels of CD163 and its ligand TWEAK are significantly increased in atherosclerotic mice. The CD163 positive macrophages are mainly located at the site far away from the lipid core, and CD163 may play an anti-atherosclerotic effect by inhibiting TWEAK/NF-κB pathway.     

Adeno-associated virus type 9-mediated p65 silencing inhibits angiotensin II-induced apoptosis of H9c2 cells

AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10^-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×10⁶ vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ.     

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.     

Effects of Tongxinluo on activation of platelet in rabbit model of atherosclerosis

AIM: To study the effects of Tongxinluo on the activation of platelets in a rabbit model of atherosclerosis. METHODS: New Zealand rabbits were randomly divided into 7 groups: normal group, model group, the groups treated with high, medium and low doses of Tongxinluo micropowder (0.15, 0.3 and 0.6 g·kg^-1·d^-1), atorvastatin group (2.5 mg·kg^-1·d^-1), and aspirin group (12.5 mg·kg^-1·d^-1). The rabbits in normal group was fed with common diet for 12 weeks, and the rabbits in model group were fed with high-fat diet for 12 weeks to establish atherosclerosis model. The rabbits in the rest groups were treated with the corresponding drugs, at the same time to give high-fat diet. Fasting for 12 h after the last treatment, whole blood was collected to perform the blood routine test, and to measure serum and plasma levels of lipids, platelet factor 4 (PF4) and soluble CD62P (sCD62P). Flow cytometry was used to analyze platelet calcium ion concentration. Electron microscopy was used for platelet superfine observations, and light microscopy for observing the pathological changes. RESULTS: Compared with normal group, the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), platelet counts, and mean platelet volume in model group were significantly elevated, and the levels of PF4, sCD62P and calcium were also significantly increased (P < 0.05). Compared with model group, except aspirin group, the levels of TG, TC and LDL-C in high, medium and low doses of Tongxinluo groups and atorvastatin group were effectively decreased. The platelet counts and mean platelet volume in all treatment groups were markedly decreased, and the serum levels of PF4, sCD62P and Ca²⁺ in platelet (P < 0.05) were reduced. In electron microscopic observation, the shape of platelet was regular and organelles distributed uniform in normal group. However, in model group, the shape of platelet was irregular, pseudopodia forming was obviously observed, and α particles and dense granules decreased, indicating that the platelet was activated. To a different extent, the platelet shape, increase in the number of α particles and dense granules were improved in treatment groups and the damage of the cytoplasm was attenuated. Through histopathological observation, the intimal was smooth and complete in normal group. In the model group, the intimal thickness markedly increased, foam cell aggregated, and plaque was formed. Compared with model group, the intimal thickening and the number of foam cells were significantly decreased, and plaque formation was not obvious in atorvastatin group and high dose of Tongxinluo group. The pathological damages in the other treatment groups were alleviated in different degrees. CONCLUSION: Tongxinluo significantly inhibits the activation of platelets in the process of atherosclerosis, and has important clinical value to delay the atherosclerotic thrombosis.     

Effects of NF-κB inhibitor on nerve injury in rats after intracerebral hemorrhage

AIM: To investigate the regulatory effect of nuclear factor-κB (NF-κB) inhibitor, pyrrolidine dithiocarbamate (PDTC), on nerve function and neural cell apoptosis in rats after intracerebral hemorrhage (ICH). METHODS: SPF Sprague-Dawley rats were randomly divided into 4 groups with 6 rats in each group:sham group, ICH group, PDTC at low concentration (P_low) group and PDTC at high concentration (P_high) group. Autologous blood injection was used to establish ICH model. After 2 h of surgery, the rats in P_low group and P_high group were intraperitoneal injected with PDTC at 100 mg/kg and 200 mg/kg, respectively, while rats in sham group and ICH group were injected with the same volume of saline. The neurological function score was classified with modified Longa grading method. TUNEL assay was used to detected the neural cell apoptosis, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured. Furthermore, the protein levels of p-P65 and cleaved caspase-3 in brain tissues were determined by Western blot. RESULTS: Compared with sham group, the rats in ICH group had higher neurological function score (P<0.05). After treatment with PDTC, the neurological function score was decreased (P<0.05), but no significant difference between P_low group and P_high group was observed. Compared with sham group, the number of apoptotic cells in ICH group was increased (P<0.05). After treatment with PDTC, the neural cell apoptosis was restrained, and the number of apoptotic cells in P_high group was lower than that in P_low group (P<0.05). Compared with sham group, the content of MDA was increased and the activity of SOD was decreased in ICH group (P<0.05). After treatment with PDTC, the content of MDA was decreased while the activity of SOD was increased, and the variation trend was more obvious in P_high group (P<0.05). Compared with sham group, the protein levels of p-P65 and cleaved caspase-3 in ICH group were increased (P<0.05). After treatment with PDTC, the protein levels of p-P65 and cleaved caspase-3 were decreased, and those in P_high group were lower than those in P_low group. CONCLUSION: NF-κB inhibitor PDTC plays a role in the se-condary brain injury after ICH, and the protective effect increases at the higher dose. The mechanism may be related to reducing MDA content and increasing SOD activity, and further inhibiting neural cell apoptosis.     

Effect of genistein on ammonia-induced NF-κB activation in astrocytes

AIM: To explore the effect of genistein on ammonia-induced nuclear factor-κB (NF-κB) activation and the underlying mechanism.METHODS: Primary astrocyte cultures were prepared and challenged with NH₄Cl to establish a hyperammonemic model. The activation of ERK, Akt and NF-κB was examined by Western blot.RESULTS: AG1478 and genistein significantly inhibited ammonia-induced activation of ERK and Akt. Ammonia-induced NF-κB nuclear translocation was significantly inhibited by the pretreatment of LY294002, genistein and AG1478.CONCLUSION: Genistein significantly inhibited ammonia-induced ERK activation and Akt-mediated NF-κB activation, which might represent the important mechanism by which this naturally occurring substance exerts its swelling-inhibiting effect.     

Effects of vinpocetine on inflammation of brain in rats with intracerebral hemorrhage

AIM: To investigate the effects of vinpocetine on inflammation of brain in intracerebral hemorrhage (ICH) rats and to explore the underlying mechanisms. METHODS: All rats were randomly divided into sham group, ICH group, ICH with low dose of vinpocetine treatment group, ICH with medium dose of vinpocetine treatment group, and ICH with high dose of vinpocetine treatment group. Except sham group, the rats in other groups were injected with type VⅡ collagenase to establish ICH model, and then the rats in vinpocetine treatment groups were received vinpocetine at 0.5, 1.0 or 1.5 mg/kg by intraperitoneal injection once a day for 7 days. After corresponding treatment, the impairment of neurological function in the rats was scored and the water content of the brain tissues was measured. Moreover, the activity of myeloperoxidase (MPO) was determined by ELISA, and the protein expression of Toll-like receptors 4 (TLR4), nuclear factor-κB(NF-κB), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molcule-1 (VCAM-1) in the brain tissues was determined by Western blot. RESULTS: Compared with ICH group, vinpocetine treatment significantly decreased the scores of the impairment of neurological function and the water content of the brain tissues. Moreover, the activity of MPO and the protein expression of TLR4, NF-κB, ICAM-1 and VCAM-1 were also reduced after vinpocetine treatment (P<0.05). CONCLUSION: Vinpocetine improves neurological function in ICH rats via suppression of inflammation by inhibiting NF-κB signaling and expression of ICAM-1 and VCAM-1.     

Renoprotective effect of L-carnitine on streptozotocin-induced diabetic nephropathy in rats

AIM: To investigate whether L-carnitine (LC) treatment confers renoprotection in a rat model of streptozotocin (STZ)-induced diabetic nephropathy (DN). METHODS: Diabetic animal model was established by intraperitoneal injection of STZ (65 mg/kg) in Sprague-Dawley rats. Diabetic rats were treated with LC (50 mg·kg^-1·d^-1 or 200 mg·kg^-1·d^-1 intravenously) daily for 12 weeks. The effects of LC on STZ-induced DN were evaluated by assessing renal function, urinary protein excretion, histopathological changes, macrophage infiltration, the expression of proinflammatory and prosclerotic cytokines, and the expression of nuclear factor-κB (NF-κB) and apoptosis-related gene. RESULTS: LC administration significantly decreased glomerulosclerosis, preserved the number of podocytes, and reduced macrophage infiltration. These changes were accompanied by improvements in urinary protein excretion and renal dysfunction. LC treatment suppressed the expression of proinflammatory and prosclerotic cytokines, and these changes were paralleled by significant attenuation of NF-κB and apoptosis-related gene expression. CONCLUSION: LC has a renoprotective effect against STZ-induced DN in rats.     

Effect of renal sympathetic denervation on cardiac hypertrophy and fibrosis

AIM:To investigate the effect of renal sympathetic denervation (RDN) on cardiac hypertrophy and fibrosis and to explore the underlying mechanisms. METHODS:Male SD rats were randomly divided into 4 groups: sham, sham plus RDN, aortic constriction (AC) and AC plus RDN group (n=15 for each group). After the intervention for 8 weeks, the haemodynamic data and cardiac function were measured by a physiological recorder. The histological structure of the heart was evaluated by HE and picro-sirius red staining. The plasma concentration of norepinephrine (NE), renin activity, angiotensin II (Ang II) concentration and cardiac Ang II level were determined by radioimmunoassay. RESULTS:Compared with AC group, RDN improved cardiac diastolic function ［left ventricular end-diastolic pressure: (8.03±1.66) mmHg vs(15.77±2.14) mmHg; -dp/dt: (7 793±587) mmHg/s vs(6 353±475) mmHg/s; both P<0.01］, inhibited cardiac hypertrophy ［left ventricular index: 3.340±0.121 vs4.244±0.102; cardiomyocyte area: (332.9±28.9) μm2 vs(401.6±33.2) μm2; both P<0.01］ and attenuated cardiac fibrosis (collagen volume fraction: 7.76%±0.85% vs12.48%±1.82%; P<0.01) in aortic constricted rats. However, RDN didn’t cause significant reduction of blood pressure in aortic constricted rats (P>0.05). RDN prevented the AC-induced increase in the plasma NE concentration, renin activity, Ang II concentration and cardiac Ang II level. CONCLUSION: RDN may directly prevent cardiac hypertrophy and fibrosis and improve cardiac function via regulating the sympathomimetic activity and renin-angiotensin system.     

Effects of baicalin on glial fibrillary acidic protein and nuclear factor-κB expression in juvenile rat hippocampus after status convulsion

AIM: To study the effects of baicalin (BC) on glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) expression and neuronal apoptosis in juvenile rat hippocampus after status convulsion (SC). METHODS: One hundred and ninety five juvenile male Sprague-Dawley rats were randomly divided into 3 groups: normal saline pretreatment group (NS group), SC group and SC with BC pretreatment group (BC group). Each of these 3 groups would be subdivided into 5 subgroups sacrificed at 4 h, 12 h, 24 h, 48 h and 72 h after SC. The rat SC model was prepared by lithium-pilocarpine chemical method. The protein expression of GFAP and NF-κB was detected by the method of immunohistochemistry. The mRNA expression of GFAP was detected by RT-PCR. The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: Compared with NS group, the GFAP positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of GFAP was significantly reduced in BC group (P<0.05). Compared with NS group, the NF-κB positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of NF-κB was significantly reduced in BC group. RT-PCR showed that the expression trend of GFAP mRNA was similar to that of the protein. Compared with NS group, the TUNEL positive cells in the hippocampus CA1 area in SC group increased significantly 12 h after SC (P<0.01), and reached a peak at 48 h. After the intervention with BC, the TUNEL positive cells decreased significantly between 12~48 h after SC (P<0.05 or P<0.01), but the number of TUNEL positive cells remained significantly greater than that in NS group (P<0.05). CONCLUSION: The expression of GFAP and NF-κB in the hippocampus increased after SC in rats. Baicalin decreases the expression of GFAP and NF-κB in hippocampus of rats with pilocarpine-induced seizures, and reduces the number of neuronal apoptosis, suggesting that baicalin may protect against the brain damage caused by status convulsion.     

Expression of MMP-2 and NF-κB in myocardium of mice with viral myocarditis

AIM: To observe the effects of TNF-α/nuclear factor-κB(NF-κB)/matrix metalloproteinase-2(MMP-2) pathway on the expression of MMP-2 in the mice with viral myocarditis. METHODS: Six-week-old inbred male mice were randomly assigned to control and myocarditis group. The mice in myocarditis group and control group were intraperitoneally inoculated with 0.1 mL 10^-5.69 TCID50/mL coxsackievirus B3 and vehicle (PBS), respectively. Ten mice were sacrificed at the 4th and 10th days after injection. The blood and heart specimens were harvested. The serum content of TNF-α was measured by ELISA. The myocardial levels of MMP-2, NF-κB p65 and IκBα were determined by Western blot. RESULTS: Compared with control group, the protein expression of MMP-2 and NF-κB p65 in the myocardium and the serum content of TNF-α were significantly increased in myocarditis group (P<0.05). The protein expression of IκBα was lower in myocarditis group than that in control group (P<0.05).CONCLUSION: TNF-α, NF-κB p65 and MMP-2 were higher in the mice with acute viral myocarditis. The increased expression of them might be involved in the pathogenesis of viral myocarditis.     

Effect of Fufang Zhenzhu Tiaozhi capsule on serum lipids and inflammatory factors in rabbit abdominal aortic restenosis model

AIM: To investigate the effect of Fufang Zhenzhu Tiaozhi capsule (FTZ) on serum lipids and inflammatory factors in rabbits with abdorminal aortic restenosis after balloon angioplasty.METHODS: New Zealand white rabbits (n=30) were divided into 5 groups. Except blank control group, the rabbits in other groups were used to establish abdominal aortic endothelium exfoliative vascular stenosis model. After 4 weeks of high-fat diet feeding, the animals in restenosis model group and drug treatment groups underwent percutaneous balloon dilatation in the stenosis. The angiographic stenosis was analyzed by a two-dimensional quantitative coronary angiography workstation with a digital subtraction X-ray machine. Blood samples were taken during angiography and the profiles of serum lipids and cytokines were measured. The expression of nuclear factor-κB (NF-κB) in the blood vessels was determined by immunohistochemistry.RESULTS: Angiography confirmed that the rates of area stenosis and diameter stenosis were significantly decreased in treatment groups compared with restenosis model group (P<0.01). Compared with restenosis model group, the serum lipid profiles and cytokine concentrations in drug treatment groups were significantly decreased (P<0.05). Immunohistochemistry showed the expression of NF-κB in restenosis model group was significantly higher than that in blank control group and drug treatment groups (P<0.05).CONCLUSION: FTZ significantly reduces the blood lipids and inflammatory factors in abdominal aortic restenosis model, and the anti-inflammatory effect may be related to the regulation of NF-κB pathway to inhibit the production of various inflammatory factors.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling 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.     

Effect of oleanolic acid on expression of TNF-α and collagen in silicotic rats in vivo

AIM: To observe the effect of oleanolic acid (OA) on the expression of Tumor necrosis factor-α (TNF-α) and collagen in silicotic rats in vivo and its possible mechanism. METHODS: Male Wistar rats were divided into 4 groups according to the randomized block design: control group, model group, OA group and solvent control group (20 rats in each group). Except control group, the rats in other groups were induced by intratracheal instillation of silicon di-oxide (SiO₂; 250 mg/kg). The rats in OA group were intragastrically administered with OA (60 mg/kg) from the second day of giving SiO₂. The rats in solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose solution (10 mL/kg). The rats in control group were given normal saline under the same condition for 56 consecutive days. All rats were killed at the 7th, 14th, 28th and 56th days. The lung coefficient was detected and the morphological changes were observed. The serum contents of TNF-α were detected by ELISA. The content of total collagen in the lung tissue was measured. The protein level of nuclear factor-κB (NF-κB) in the lung tissue was determined by immunohistochemical method. RESULTS: (1) According to the morphological changes, the silicosis model was successfully established. Compared with control group, the lung coefficient and total collagen increased obviously in model group and solvent control group. The lung coefficient and total collagen content in OA group at each time point reduced compared with those in model group and solvent group, and increased compared with those in control group at the corresponding time points. (2) The serum contents of TNF-α in model group and solvent control group significantly increased, peaking at the 14th day, slightly decreasing afterward, and showing statistically significant difference at each time point compared with those in control group. No significant difference between model group and solvent group at different time points was observed. OA had inhibitory effect on the contents of TNF-α compared with model group and solvent group at the corresponding time points. (3) NF-κB in model group and solvent control group significantly increased, peaking at the 28th day, and showing statistically significant difference at each time point compared with those in control group. The NF-κB expression in OA group was similar to model group, but significantly decreased compared with control group at each time point. CONCLUSION: OA inhibits the expression of TNF-α and collagen and attenuates the silicosis fibrosis, which may be related to the NF-κB pathway.     

EZH2-mediated regulation of NF-κB target gene expression in gastric cancer

AIM: To explore the mechanism by which over-expression of enhancer of zeste homolog 2 (EZH2) in a panel of gastric cancer cell lines is involved in tumorigenesis of gastric cancer. METHODS: Real-time PCR and Western blot were employed to examine the mRNA and protein levels of EZH2, respectively. MTS assay, cell migration and soft agar assay were performed to investigate the role of EZH2 in the regulation of stomach cancer behaviors. The effect of EZH2 on NF-κB target gene expression was determined by Luciferase reporter and real-time PCR. Co-immunoprecipitation was used to analyze the interaction of EZH2 and p65 in HEK293T cells. RESULTS: The expression levels of EZH2 were significantly increased in the gastric cancer cells compared with normal gastric epithelial cells. Pharmacological inhibition by DZNep or knockdown of EZH2 significantly compromised AGS and SNU-16 cell activity, cell migration and anchorage-independent cell growth. Moreover, siRNA knockdown of EZH2 impaired NF-κB downstream targets, such as IL-8, CXCL5 and CCL20. In addition, the interaction of EZH2 and p65 was detected. CONCLUSION: EZH2 mediates the growth of gastric cancer cells through the regulation of NF-κB downstream gene expression.     

Effect of recombinant rat CC16 protein on LPS-induced expression of TNF-α, IL-6 and IL-8 in rat tracheal epithelial cells

AIM: To explore the effect of recombinamt rat CC16 protein (rCC16) on LPS-induced expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-8 in the rat tracheal epithelial (RTE) cells.METHODS: The RTE cells were incubated with rCC16 at concentrations of 0.5, 1.0 and 2.0 mg/L in serum-free media for 2 h prior to LPS (0.1 mg/L) treatment for further 24 h. The cells were harvested for assessing the mRNA levels of TNF-α, IL-6 and IL-8 by RT-qPCR. The cell culture supernatants were collected for analyzing the protein levels of TNF-α, IL-6 and IL-8 by ELISA. In addition, the nuclear translocation of nuclear factor-κB (NF-κB) p65 was tested by Western blot.RESULTS: rCC16 inhibited LPS-induced IL-6 and IL-8 expression at both mRNA and protein levels in the RTE cells in a concentration-dependent (0~2 mg/L) manner, as demonstrated by RT-qPCR and ELISA. However, no concentration-dependent manner between the dose of rCC16 and TNF-α expression was observed, and rCC16 inhibited LPS-induced TNF-α expression at lower concentration (0.5 mg/L). rCC16 concentration-dependently inhibited the effects of LPS on the level of nuclear translocation of NF-κB p65.CONCLUSION: rCC16 suppresses LPS-mediated TNF-α, IL-6 and IL-8 production through inactivation of NF-κB activity in RTE cells.[KEY WORDS] CC16 protein; Airway inflammation; LPS; Inflammatory mediators; Nuclear factor-κB     

Anti-inflammatory effect of nitric oxide and atherosclerosis

Atherosclerosis has been considered as one of inflammatory disease. Besides its action on vasomotor tone regulation, nitric oxide (NO) is recognized to be an anti-inflammatory molecule. The anti-inflammatory effects of NO are attributable to inhibition of nuclear factor kappa B activation.     

Effects of metformin combined with Ge Xia Zhu Yu decoction on TLR4/NF-κB signaling pathway in rats with polycystic ovary and insulin resis-tance

AIM: To study the effect of metformin (Met) combinated with Ge Xia Zhu Yu decoction on Toll-like receptor-4 (TLR-4)/nuclear factor-κB (NF-κB) signaling pathway in the rats with polycystic ovary syndrome (PCOS) and insulin resistance induced by dehydroepiandrosterone, and to explore the mechanisms. METHODS: PCOS rats (after induced by dehydroepiandrosterone, n=110) were randomly divided into 3 groups:model group (30 rats), Met treatment group (40 rats) and Met combinated with Ge Xia Zhu Yu decoction treatment (combination) group (40 rats). The rats in model group were given the same volume of 0.9% sodium chloride daily by gavage. The rats in Met group were given Met (270 mg·kg^-1·d^-1) by gavage. The rats in combination group were given Met (270 mg·kg^-1·d^-1) and Ge Xia Zhu Yu decoction (34.5 mg·kg^-1·d^-1) by gavage. All rats were continuously intervened for 28 d. After the intervention, blood glucose[fasting plasma glucose (FPG) and 2-hour postprandial blood glucose (2hPBG)] was measured. The mRNA expression levels of follicular epithelial NF-κB, TLR-4 and oxidized low-density lipoprotein (ox-LDL) were detected by RT-PCR. The serum levels of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) were also detected by ELISA. RESULTS: After the intervention, FPG, 2hPBG, and serum levels of IL-6, TNF-α and CRP in Met group and combination group were lower than those in model group (P<0.05), and those in combination group were lower than those in Met group (P<0.05). Meanwhile, the mRNA expression levels of follicular epithelial NF-κB, TLR-4 and ox-LDL in Met group and combination group were lower than those in model group (P<0.05), and those in combination group were lower than those in Met group (P<0.05). CONCLUSION: Treatment with Met combined with Ge Xia Zhu Yu decoction improves insulin resistance in PCOS rats by decreasing the levels of inflammatory factors in serum and epithelial cells of ovary and inhibiting the expression of NF-κB, TLR-4 and ox-LDL in epithelial tissue of ovary.     

Role of TLR4-TRPC6 in LPS-induced NF-κB P65 expression and nuclear translocation in airway epithelial 16HBE cells

AIM: To investigate the role of Toll-like receptor 4 (TLR4) and transient receptor potential channel 6 (TRPC6) signaling pathway in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) P65 expression and nuclear translocation in airway epithelial cells (16HBE) for supplementing the mechanism for airway inflammation. METHODS: After stimulating the 16HBE cells with LPS at 1 mg/L for 0, 0.5, 2, 6, 12 and 24 h, the expression of NF-κB P65 at mRNA and protein levels in the 16HBE cells were determined by RT-PCR and Western blot respectively, and the nuclear translocation of NF-κB P65 was detected by immunocytochemical staining method. The effects of TLR4 inhibitor CLI-095 at 5 μmol/L and TRPC6 agonist Hyp9 at 10 μmol/L on LPS (1 mg/L)-induced NF-κB P65 expression and nuclear translocation in the 16HBE cells were determined by RT-PCR, Western blot and immunocytochemical staining. RESULTS: LPS increased the mRNA and protein expression of NF-κB P65 and nuclear translocation in the 16HBE cells(P<0.05). TLR4 inhibitor CLI-095 reduced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS, while Hyp9 enhanced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS in the 16HBE cells(P<0.05). CONCLUSION: LPS induces the expression and nuclear translocation of NF-κB P65 in the 16HBE cells via TLR4-TRPC6 signaling pathway.