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.     

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 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 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.     

Eeffect of HMGB1 on expression of NF-κB in BV-2 cells stimulated with Aβ25-35

AIM: To investigate the effect of high mobility group box-1 protein (HMGB1) on the expression of nuclear factor-κB (NF-κB) in BV-2 cells stimulated with amyloid β-protein (Aβ)_25-35. METHODS: Cultured BV-2 cells in logarithmic growth phase were divided into 4 groups:normal cell group (without any treatment), model group (treated with Aβ_25-35 at 40 μmol/L), RNA interference (RNAi) group (conducted with HMGB1-siRNA followed by Aβ_25-35 stimulation) and solvent control group (treated with 0.1% DMSO). After treatment with Aβ_25-35 for 24 h, the protein levels of HMGB1 and NF-κB in BV-2 cells were determined by Western blot. RESULTS: Aβ_25-35 at 40 μmol/L was used to stimulate BV-2 cells. The GFP fluorescence-tagged HMGB1-siRNA (30 nmol/L) was used to transfect BV-2 cells and its transfection efficiency was about 80%~90%. The results of Western blot showed that the protein level of HMGB1 was significantly decreased after the interference of siRNA fragment (P<0.05). The protein levels of HMGB1 and nucleic NF-κB p65 were dramatically increased in BV-2 cells stimulated with Aβ_25-35 (P<0.05). After RNA interference with HMGB1, the expression of HMGB1 and nucleic NF-κB p65 were significantly decreased in BV-2 cells stimulated with Aβ_25-35 (P<0.05). CONCLUSION: RNA interference with HMGB1 reduces the expression of nucleic NF-κB in BV-2 cells stimulated with Aβ_25-35.     

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.     

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.     

Effect of wogonoside on inflammatory response in mice with viral myocarditis induced by Coxsackie virus B3

AIM: To investigate the effect of wogonoside on the inflammatory response of mice with Coxsackie virus B3 (CVB3)-induced myocarditis and its possible regulatory mechanism. METHODS: A mouse model of viral myocarditis was constructed by infecting BALB/c mice with CVB3. BALB/c mice (n=40) were randomized into 4 groups: normal group, CVB3-induced viral myocarditis group, CVB3-induced viral myocarditis combined with wogonoside treatment group and CVB3-induced viral myocarditis combined with wogonoside plus AKT agonist treatment group. All the mice were sacrificed 7 days after treatment. In the first 3 groups, HE staining was applied to detect the infiltration of inflammatory cells in the myocardium, ELISA was applied to detect the serum levels of interleukin-1β (IL-1β) and IL-6, while Western blot was applied to detect the protein expression of inflammatory factors and the activation of AKT/NF-κB pathway. Inaddition, the activation of AKT/NF-κB pathway in the 4 groups was detected by Western blot analysis. RESULTS: HE staining showed that there was a large amount of inflammatory cell infiltration in the myocardium of CVB3-induced viral myocarditis mice, as compared with the normal group, which was significantly reduced by wogonoside treatment (P<0.05). The serum levels of IL-1β and IL-6 in the mice after CVB3 infection were significantly higher than those in normal group (P<0.05), which was also significantly reduced by wogonoside treatment (P<0.05). Western blot analysis indicated that wogonoside treatment significantly reduced the expression of inflammatory factors IL-1β and IL-6, and the phosphorylation of AKT/NF-κB pathway-related proteins in the myocardial tissue (P<0.05). After administration of AKT agonist, the inhibitory effect of wogonoside on NF-κB phosphorylation and inflammatory factors expression was significantly eliminated (P<0.05). CONCLUSION: Wogonoside attenuates the inflammatory response of mice with viral myocarditis by inhibiting the AKT/NF-κB pathway.     

Expression and significance of TLR4/NF-κB in pulmonary vascular remodeling in smoking rats

ATM: To observe the expression of Toll-like receptor 4 (TLR4), nuclear factor-κB subunit P65 protein (NF-κB P65) and proliferating cell nuclear antigen (PCNA) in the pulmonary vascular tissues of the rats exposed to smoke, and to explore the possible mechanism of TLR4/NF-κB signaling pathway in pulmonary vascular remodeling. METHODS: SPF male healthy rats (n=48) were randomly divided into control group, smoke exposure for 4 weeks group (S4 group), smoke exposure for 8 weeks group (S8 group) and smoke exposure for 12 weeks group (S12 group), with 12 rats in each group. HE staining was used to observe the morphological changes of pulmonary vessels, and then the pulmonary vascular wall area/total vascular area (WA%) and vascular wall thickness/vascular external diameter (WT%) were measured by the medical image analysis system. The expression of TLR4, NF-κB P65 and PCNA in the pulmonary vascular tissues was detected by immunohistochemical staining. The protein content was expressed by the average integral absorbance. The mRNA expression of TLR4 in the pulmonary vessels was detected by RT-qPCR. The relationships between WA%, WT%,TLR4 protein, TLR4 mRNA, P65 protein, PCNA protein and pulmonary vascular remodeling, and another relationships between WA%, WT%, P65 protein, PCNA protein and TLR4 protein were analyzed.RESULTS: The WA% and WT% in smoke exposure groups significantly increased compared with control group, and the ratio was proportional to the time of smoke exposure. The protein expression of TLR4, p65 and PCNA, and the mRNA expression of TLR4 in smoke exposure groups also increased significantly compared with control group. CONCLUSION: The extent of pulmonary vascular remodeling in the rats increases when the protein expression of TLR4 is up-regulated. There is a positive correlation between pulmonary vascular remodeling and the protein expression of TLR4 and NF-κB P65. Pulmonary vascular remodeling may be related to the activation of TLR4/NF-κB signaling pathway.     

Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

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.     

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway

AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.     

Role of NF-κB in endothelial cell damage and apoptosis induced by septic plasma

AIM：To explore the involvement of nuclear factor κB (NF-κB) in septic plasma (SP)-induced endothelial cell damage and apoptosis. METHODS：Human umbilical vein endothelial cells (HUVECs) were treated with SP from sepsis patients or normal plasma (NP) from healthy controls. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay were used to examine the phosphorylation and activity of NF-κB, and the cell apoptosis was analyzed by flow cytometry. RESULTS：The levels of tumor necrosis factor α (TNF-α) and von Willebrand factor (vWF) in SP were significantly higher than those in NP. Treatment of HUVECs with 10%, 20%, and 30% SP resulted in increased production of vWF from HUVECs in a dose-dependent manner within certain time window. Pyrrolidine dithiocarbamate (PDTC, 100 μmol/L), a selective inhibitor of NF-κB activation, inhibited the induction of vWF production from HUVECs activated by SP. SP increased phosphorylation and activity of NF-κB, and induced apoptosis of HUVECs, which was enhanced by PDTC.CONCLUSION: NF-κB is involved in endothelial cell damage induced by septic plasma and inhibits the apoptosis.     

Role of Akt/NF-κB pathway in immune-complexes-induced MCP-1 and CSF-1 expression in murine glomerular mesangial cells

AIM: To explore the role of Akt/NF-κB pathway in immune-complexes-induced monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor-1 (CSF-1) expression in Mesangial Cells. METHODS: Primary murine glomerular mesangial cells were cultured in vitro and divided into control group, stimulation group and antisense, sense and mismatched oligodeoxynucleotide group. In control group, the cells were stimulated with monomeric IgG after treatment with 0.5% lipofectin for 8 h. In stimulation group, the cells, which had been treated with 0.5% lipofectin for 8 h, were stimulated with aggregated IgG. In antisense, sense and mismatched oligodeoxynucleotide group, being transduced antisense, sense and mismatched oligodeoxynucleotide respectively with 0.5% lipofectin 8 h, the cells were stimulated with AIgG. MCP-1 and CSF-1 in supernatant were deteced with ELISA. In addition, RT-PCR was used to determine MCP-1 and CSF-1 mRNA expression, and EMSA to investigated the activation of NF-κB. RESULTS: Mesangial cells cultured in vitro had a low level NF-κB activation and a low level constitutive expression of MCP-1 and CSF-1. Stimulated with AIgG, activation of NF-κB was markedly increased(0.35±0.06 vs 0.75±0.16, P＜0.01), expression of MCP-1 and CSF-1 mRNA (0.48±0.03 vs 0.72±0.02, P＜0.05; 0.44±0.01 vs 0.59±0.02, P＜0.05), MCP-1 and CSF-1 levels in supernatant(15.52±1.81 vs 43.05±3.18, P＜0.05; 389.06±13.75 vs 764.22±31.78, P＜0.05) were markedly increased. Akt1 antisense oligodeoxynucleotide markedly inhibited immune-complexes-induced NF-κB activation, MCP-1 and CSF-1 mRNA and protein expression. CONCLUSION: Akt/NF-κB pathway mediates immune-complexes-induced MCP-1 and CSF-1 expression in mesangial cells. It suggests that Akt/NF-κB pathway may be a new therapy target for macrophage recruitment and activation in immune complexes nephritis.     

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.     

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.     

Opening of ATP-sensitive K+ channels protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting TLR4/NF-κB pathway

AIM: To investigate whether the opening of ATP-sensitive K⁺(K_ATP) channels protects H9c2 cardiac cells against high glucose(HG)-induced injury and inflammation by inhibiting the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) pathway. METHODS: The protein levels of TLR4 and NF-κB p65 were determined by Western blot. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The cell viability was measured by CCK-8 assay. Mitochondrial membrane potential(MMP) was examined by rhodamine 123(Rh 123) staining followed by photofluorography. The intracellular levels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein- diacetate(DCFH-DA) staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG(35 mmol/L glucose) for 24 h, the protein levels of TLR4 and phosphorylated NF-κB p65(p-NF-κB p65) were significantly increased. Pretreatment of the cells with 100 μmol/L diazoxide(DZ, a K_ATP channel opener) for 30 min before exposure to HG considerably blocked the up-regulation of the TLR4 and p-NF-κB protein levels induced by HG. Moreover, co-treatment of the cells with 30 μmol/L TAK-242(an inhibitor of TLR4) obviously inhibited the HG-induced up-regulation of the p-NF-κB p65 protein level. On the other hand, pretreatment of the cells with 100 μmol/L DZ had a clear myocardial protection effect, which attenuated the HG-induced cytotoxicity, inflammatory response, mitochondrial damage, oxidative stress and apoptosis, evidenced by an increase in the cell viability, and decreases in the levels of IL-1β and TNF-α, MMP loss, ROS generation and the number of apoptotic cells. Similarly, co-treatment of H9c2 cardiac cells with 30 μmol/L TAK-242 or 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries and inflammation induced by HG.CONCLUSION: The opening of K_ATP channels protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the TLR4/NF-κB pathway.     

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.