Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

Effects of simvastatin on proliferation of esophageal cancer cells through NF-κB p65 pathway

AIM To investigate the effects of simvastatin （SIM） on the proliferation of esophageal cancer Eca109 cells through NF-κB p65 pathway. METHODS Esophageal cancer Eca109 cells were cultured in vitro and treated with SIM at different concentrations （2 , 4, 8, 16 and 32 μmol/L）. The proliferation of Eca109 cells was measured by MTT assay and plate colony formation assay. The proliferation-related protein levels of cyclin D1, c-Myc, NF-κB p65 （p65） and IκB-α in the esophageal cancer Eca109 cells were determined by Western blot. ELISA was used to detect the levels of tumor necrosis factor α （TNF-α） and interleukin-6 （IL-6） in the culture supernatant of Eca109 cells. RESULTS Compared with control group, the inhibitory effects of SIM at 2 , 4 , 8 and 16 μmol/L on the viability of Eca109 cells were increased in a concentration-dependent and time-dependent manners （P<0.05）. Simultaneously, the inhibitory rates of SIM at 16 and 32 μmol/L on the viability of Eca109 cells showed no significant difference （P>0.05）. The inhibitory rates of SIM at 16 μmol/L on the viability of esophageal cancer cells was 50.61% at 48 h, which was closed to half of the inhibitory dose IC₅₀, and it was used as the optimum concentration and time for follow-up experiments. Compared with control group, the plate colony formation rate of Eca109 cells, the protein levels of cyclinD1, c-Myc, nuclear p65, TNF-α and IL-6 in 8 and 16 μmol/L groups were decreased, while the levels of cytosolic p65 and IκB-α proteins were increased （P<0.05）. No significant difference of plate colony formation rate in Eca109 cells, and the protein levels of cyclin D1, c-Myc, nuclear and cytoplasmic p65, IκB-α, TNF-α and IL-6 between 16 μmol/L SIM group and 32 μmol/L SIM group was observed （P>0.05）. CONCLUSION Simvastatin inhibits the proliferation of esophageal cancer Eca109 cells, and its mechanism may be related to up-regulation of IκB-α, down-regulation of cyclinD1 and c-myc, inhibition of p65 nuclear translocation, and the expression of TNF-α and IL-6 downstream of NF-κB p65 pathway.     

Geniposide attenuates cognitive dysfunction in sleep-deprived rats by inhibiting TLR4/NF-κB signaling pathway

AIM To investigate the effects of geniposide （Gen） on Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and cognitive dysfunction in sleep deprived rats. METHODS Wistar rats （n=120） were randomly divided into normal control （NC） group, model （M） group, low-dose （5 g·kg^-1·d^-1） Gen （Gen-L） group, medium-dose （10 g·kg^-1·d^-1） Gen （Gen-M） group, high-dose （20 g·kg^-1·d^-1） Gen （Gen-H） group and Gen-H+LPS （0.4 mg·kg^-1·d^-1, tail vein injection） group. After 7 days of intervention, the sleep deprivation model of rats in M group, Gen-L, Gen-M, Gen-H and Gen-H+LPS group was established by improved small platform water environment. The escape latency of Morris water maze experiment and the behavior correct rate of Y maze experiment were measured. The serum levels of S100B and neuron-specific enolase （NSE）, and the levels of interleukin-1β （IL-1β）, IL-6 and tumor necrosis factor-α （TNF-α） in hippocampus were detected by ELISA. The mRNA levels of TLR4 and NF-κB p65 were detected by RT-qPCR, and the protein levels of TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with NC group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the mRNA and protein expression of TLR4 and NF-κB p65 were increased significantly in M group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. Compared with M group, the escape latency, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were decreased significantly in Gen-L, Gen-M and Gen-H groups （P<0.01）, and the behavior correct rate was increased in turn （P<0.01）. Compared with Gen-H group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were increased significantly in Gen-H+LPS group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. CONCLUSION Geniposide may inhibit the TLR4/NF-κB p65 signaling pathway to effectively improve cognitive function in sleep-deprived rats and reduce hippocampus inflammation.     

RIP1-mediated necroptosis regulates inflammatory response of renal tubular epithelial cells via NF-κB signaling pathway

AIM To investigate the effect of receptor-interacting protein 1 (RIP1)-mediated necroptosis on human kidney proximal tubular cell inflammation and its related mechanisms. METHODS Human kidney proximal tubular HK-2 cells were cultured in vitro, and stimulated with tumot tumor necrosis factor-α (TNF-α) and Z-VAD-FMK for 24 h. Lactate dehydrogenase (LDH) cytotoxicity assay was used to detect the percentage of necrosis. Western blot was used to detect the protein expression of RIP1, IKK-α and NF-κB p65. The protein levels of interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were determined by Western blot and ELISA. Real-time PCR was used to detect the mRNA expression level of NF-κB p65. Furthermore, the RIP1 inhibitor necrostatin-1 (Nec-1) and the NF-κB specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) were used, and the above indicators were also detected. RESULTS Compared with control group, the protein level of RIP1 was increased in TNF-α combined with Z-VAD-FMK stimulation group (T/Z group). The protein levels of IKK-α and NF-κB p65 were obviously increased, and the release of LDH was increased (P<0.01). Western blot and ELISA showed that the expression levels of IL-1β and MCP-1 were significantly increased (P<0.01). Real-time PCR showed that the mRNA expression level of NF-κB p65 was also obviously increased. After Nec-1 or PDTC stimulation (T/Z+N group or T/Z+P group), the release of LDH, and the expression levels of inflammation-related indicators IL-1β and MCP-1 were significantly decreased. The protein expression levels of IL-1β and MCP-1 were further reduced after treatment with the above 2 stimulati (T/Z+P/N group). CONCLUSION Under T/Z condition, RIP1-mediated necroptosis plays an important role in renal tubular inflammatory response, which may be partly achieved by regulating the activation of NF-κB signaling pathway.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

Nontypeable Hemophilus influenzae induces inflammatory response of bronchial epithelial cells via NF-κB activation and histone deacetylase activity reduction

AIM: To investigate the effect of nontypeable Hemophilus influenzae (NTHi) on the inflammatory response of bronchial epithelial cells, and to preliminarily explore its mechanism. METHODS: The human normal bronchial epithelial BEAS-2B cells were cultured, and the expression levels of interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant were measured by ELISA and RT-qPCR after infection with NTHi at MOI=10. The levels of IκBα and the phosphoacetylation of histones were determined by Western blot, and then the expression of histone deacetylase (HDAC) and the enzyme activity of HDAC were detected. The binding activity of NF-κB and IL-8 was measured by electrophoretic mobility shift assay and chromatin immunoprecipitation. Finally, the expression levels of GM-CSF and IL-8 were measured after pretreatment with NF-κB and HDAC inhibitors. RESULTS: The expression levels of IL-8 and GM-CSF in the culture supernatant of BEAS-2B cells were significantly increased (P<0.05) after infection with NTHi at MOI=10. In addition, infection with NTHi significantly down-regulated the expression of cytoplasmic IκBα and enhanced the DNA-binding activity of NF-κB. The phosphoacetylation of histones H3 and H4 and the binding of IL-8 and RNA polymerase II were also significantly increased after infection with NTHi. The expression level and enzyme activity of intracellular HDAC were also significantly reduced (P<0.05) after infection with NTHi. The expression levels of GM-CSF and IL-8 were significantly reduced after pretreatment with NF-κB inhibitor (P<0.05), while the secretion of IL-8 was significantly increased after pretreatment with HDAC inhibitor (P<0.05). CONCLUSION: NTHi inhibits the expression and activity of HDAC by activating the NF-κB signaling pathway, and promotes the secretion of IL-8 and GM-CSF in bronchial epithelial cells, thereby aggravating the inflammatory response.     

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.     

Klebsiella pneumoniae capsular polysaccharide induces secretion of cytokines in bronchial epithelial cells via EGFR signaling pathway

AIM: To investigate the role of epidermal growth factor receptor (EGFR) in the secretion of inflammatory cytokines in human bronchial epithelial BEAS-2B cells induced by Klebsiella pneumoniae (KP) capsular polysaccharide (CPS). METHODS: KP was cultured in vitro, and the CPS was extracted. The BEAS-2B cells were stimulated with CPS at different concentrations, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in the supernatant were measured by ELISA. The phosphorylation level of EGFR was detected by Western blot at different time points after stimulation. After pretreatment of the BEAS-2B cells with EGFR inhibitor AG1478, the phosphorylation level of ERK was detected by Western blot, the nuclear translocation of P65 was detected by indirect immunofluorescence, and the levels of TNF-α and IL-8 in the supernatant of the cells were measured. Finally, the levels of TNF-α and IL-8 in the culture supernatant of CPS-stimulated cells were detected by ELISA after pretreated with ERK inhibitor PD98059 and NF-κB inhibitor PDTC. RESULTS: Exposure to CPS at 10 mg/L for 12 h significantly induced the BEAS-2B cells to secret TNF-α and IL-8. The phosphorylation levels of EGFR and ERK and the nuclear translocation of p65 in the BEAS-2B cells were significantly increased after CPS stimulation (P<0.05). The phosphorylation level of ERK and the nuclear translocation of p65 were significantly reduced in the cells pretreated with EGFR inhibitor AG1478. Furthermore, the levels of TNF-α and IL-8 in the supernatant were significantly decreased after pretreated with the inhibitors of EGFR, ERK and NF-κB. CONCLUSION: Klebsiella pneumonia capsular polysaccharide activates the ERK and NF-κB signaling pathways via EGFR, and then induced the secretion of inflammatory cytokines TNF-α and IL-8 in the bronchial epithelial cells, indicating that EGFR may be a key factor in the inflammatory response induced by KP infection.     

uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Aβ1-40 induces inflammation and phenotypic switching via activation of MAPKs signaling pathway in vascular smooth muscle cells

AIM To investigate the effects of amyolid β-protein 1-40 （Aβ1-40） on inflammation, viability, migration and phenotypic switching in vascular smooth muscle cells （VSMCs）, and to analyze the underlying mechanisms. METHODS The VSMCs were treated with Aβ1-40 at different concentration gradients for appropriate time. CCK-8 and Transwell assays were performed to evaluate the viability and migration ability of VSMCs. The levels of inflammatory factors including interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）, phenotypic switching-related proteins including α?smooth muscle actin （α?SMA）, osteopontin （OPN） and Krüppel?like factor 4 （KLF4）, and mitogen-activated protein kinases （MAPKs） signaling pathway-related proteins including p-p38 MAPK, p-ERK1/2 and p-JNK were determined by Western bolt. RESULTS After Aβ1-40 treatment, the levels of inflammatory factors IL-1β and TNF-α in the VSMCs were significantly increased （P<0.05）, and the expression of phenotypic switching-related proteins was altered, as indicated by down-regulation of α?SMA and up-regulation of OPN and KLF4 （P<0.05）. Treatment with Aβ1-40 within a certain concentration range promoted the viability and migration of the VSMCs. In addition, the protein levels of p-p38 MAPK, p-ERK1/2 and p-JNK were significantly increased by Aβ1-40 treatment （P<0.05）. Furthermore, pretreatment with specific inhibitors of MAPKs pathway significantly reduced the levels of IL-1β and TNF-α （P<0.05）, and inhibited the phenotypic switching, as indicated by up-regulation of α?SMA and down-regulation of OPN and KLF4 （P<0.05）. CONCLUSION Treatment with Aβ1-40 induces the inflammation and phenotypic switching in VSMCs via activation of MAPKs signaling pathway.     

Paeonol affects viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway

AIM To investigate the effect of paeonol on the viability and migration ability of hepatocellular carcinoma cells and its molecular mechanism. METHODS Human hepatocellular carcinoma Hep3B cells was treated with paeonol at different concentrations (50, 100, 200 and 400 mg/L). The cell viability was measured by CCK-8 assay to determine the optimal drug concentration. The Hep3B cells were divided into normal control (NC) group, paeonol group, miR-NC group, miR-424-3p group, paeonol+anti-miR-NC and paeonol+anti-miR-424-3p group. The expression level of miR-424-3p was detected by RT-qPCR. The migration ability was detected by Transwell assay. The protein levels of cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9 and PI3K/AKT signaling pathway-related molecules were determined by Western blot. RESULTS Paeonol intervention inhibited the viability of Hep3B cells in a concentration-dependent manner (P<0.05). The concentration of paeonol at 200 mg/L was selected for the following study. Paeonol intervention inhibited the protein expression of MMP2 and MMP9 in the Hep3B cells, and inhibited the migration ability of the Hep3B cells. Paeonol intervention promoted the expression of miR-424-3p in the Hep3B cells (P<0.05). Over-expression of miR-424-3p inhibited the expression of cyclin D1, MMP2 and MMP9 in the Hep3B cells and inhibited cell viability and migration ability (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on the viability and migration ability of the Hep3B cells (P<0.05). Paeonol inhibited phosphorylation levels of PI3K and AKT in the Hep3B cells and inhibited the activation of PI3K/AKT signaling pathway (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on PI3K/AKT signaling pathway in the Hep3B cells (P<0.05). CONCLUSION Paeonol inhibits the viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Bortezomib attenuates LPS-induced inflammatory response of human monocytes through regulating miR-223/NLRP3 signaling pathway

AIM To investigate the effects of bortezomib (BTZ) on microRNA-223 (miR-223)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway and lipopolysaccharide (LPS)-induced inflammatory response of human monocytes. METHODS Monocytes were isolated and purified from peripheral blood of rheumatodid arthritis (RA) patients. The levels of interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in supernatants of the monocytes were determined by ELISA, and the optimal induction time of LPS and the optimal treatment concentration of BTZ were selected according to the levels of IL-6, IL-1β and TNF-α. The monocytes were divided into control group, LPS induced group and BTZ group. The level of miR-223 in the monocytes was measured by RT-qPCR, and the protein levels of NLRP3, caspase-1, suppressor of cytokine signaling 1 (SOCS1) and SH2 domain-containing inositol phosphatase-1 (SHIP-1) in the monocytes were determined by Western blot. RESULTS The monocytes successfully isolated and purified from the peripheral blood of RA patients were spherical, evenly distributed and regular in shape.And after LPS induction for 24 h, the cells were mostly spindle-shaped and aggregated. According to the levels of IL-6, IL-1β and TNF-α, the optimal induction time of LPS was 24 h, and the optimal concentration of BTZ was 50 nmol/L. Compared with control group, the levels of miR-223, SOCS1 and SHIP-1 in LPS induction group were significantly decreased (P<0.05), and the levels of NLRP3 and caspase-1 were significantly increased (P<0.05). Compared with LPS induction group, the levels of miR-223, SOCS1 and SHIP-1 in BTZ group were significantly increased (P< 0.05), and the levels of NLRP3 and caspase-1 were significantly lowered (P<0.05). CONCLUSION Bortezomib may block the activation of miR-223/NLRP3 signaling pathway, thus reducing the secretion of inflammatory factors in LPS-induced human monocytes.     

Protective effect of gabexate mesilate on blood-brain barrier in rats with cerebral ischemia-reperfusion

AIM To investigate the protective effects of gabexate mesilate （GM） on blood-brain barrier （BBB） permeability in rat model with cerebral ischemia-reperfusion （I/R）. METHODS Adult male SD rats （n=180） were randomly divided into sham group, I/R group, nimodipine （NMP; 2 mg·kg^-1·d^-1） group and GM （5, 10 and 20 mg·kg^-1·d^-1） groups （n=30 in each group）. The rat model of cerebral I/R was established by blocking the middle cerebral artery with thread plug for 2 h. Ten min before modeling, the drugs were given intraperitoneally. The nerve function was detected by Longa scoring method. The permeability of BBB was measured by Evans blue permeation method, and the brain water content was measured by dry-wet weight method. The activity of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）, and the content of malondialdehyde （MDA） in brain tissue were determined by biochemical analysis. The content of tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β） and IL-6 was measured by ELISA. The mRNA expression of matrix metalloproteinase-2 （MMP-2）, MMP-9 and nuclear factor-κB （NF-κB） was detected by RT-PCR. The protein levels of MMP-2, MMP-9 and NF-κB were determined by Western blot. RESULTS Compared with I/R group, the Longa score, permeability of Evans blue and brain water content of the rats in GM （10 and 20 mg·kg^-1·d^-1） and NMP （2 mg·kg^-1·d^-1） groups were decreased. The activity of SOD and GSH-Px was increased, while the content of MDA was decreased. The content of TNF-α, IL-1β and IL-6 was decreased, and the mRNA and protein expression levels of MMP-2, MMP-9 and NF-κB were significantly down-regulated. Compared with NMP （2 mg·kg^-1·d^-1） group, the Longa score and permeability of Evans blue were decreased in GM （20 mg·kg^-1·d^-1） group, the activity of SOD was increased, and the content of MDA and TNF-α was decreased. The mRNA and protein expression levels of MMP-2, MMP-9 and NF-κB were down-regulated. All of the differences were significant （P<0.05 or P<0.01）. CONCLUSION GM has protective effect on BBB in the rats with cerebral I/R. Its mechanism may be related to inhibition of oxidative stress and inflammation, and down-regulation of MMP-2, MMP-9 and NF-κB expression.     

Resveratrol protects cortical neurons in infant rats with bacterial meningitis through miR-223-3p/NLRP3 pathway

AIM To investigate the protective effect of resveratrol (Res) on cortical neurons in rat bacterial meningitis (BM) model. METHODS Group B hemolytic Streptococcus was injected via the posterior cistern to establish a BM model. Resveratrol was administered intranasally and microRNA-223-3p (miR-223-3p) antagomir was administered by intracerebroventricular injection. HE staining was used to observe the pathological changes of the brain tissue. Loeffler scoring method was used to evaluate the neurobehavioral functions. TUNEL staining was used to detect neuronal apoptosis. The expression of interleukin-1β (IL-1β), IL-18, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) was detected by immunofluorescence staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）, cleaved caspase-1, IL-1β and IL-18 were determined by Western blot. The expression level of miR-223-3p was detected by RT-qPCR. Online software TargetScan was used to search for the complementary nucleotide sequences between miR-223-3p and NLRP3 mRNA. RESULTS Compared with sham group, the thickness of meninges in BM model was increased, the neurological score was decreased (P<0.05), and the number of TUNEL positive neurons was increased significantly (P<0.05). Astrocytes and microglia were activated, the fluorescence intensity of IL-1β and IL-18 was increased (P<0.05), and the expression levels of NLRP3, cleaved caspase-1, IL-1β, IL-18 and miR-223-3p were increased (P<0.05). Compared with BM group, after treatment with resveratrol, the neurological score was increased (P<0.05), the number of TUNEL positive neurons was decreased significantly (P<0.05), and the inflammatory response of astrocytes and microglia was suppressed. The fluorescence intensity of IL-1β and IL-18 was decreased (P<0.05), the protein levels of NLRP3, cleaved caspase-1, IL-1β and IL-18 were decreased (P<0.05), and the expression level of miR-223-3p was increased (P<0.05). A nucleotide sequence in the 3'-UTR of NLRP3 mRNA might be targeted by miR-223-3p. In the brain of rat BM model, compared with antagomir control group, the expression of NLRP3 was increased in miR-223-3p antagomir group with resveratrol treatment (P<0.05). CONCLUSION Resveratrol may reduce the inflammatory death of cortical neurons in BM model of infant rats through miR-223-3p/NLRP3 pathway, thus playing a protective role for the neurons.     

Effect of miR-133 targeting NLRP3 on inflammatory activation of mouse Kupffer cells

AIMTo explore the effect of microRNA-133 (miR-133) targeting nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) on the inflammatory activation of Kupffer cells (KCs). METHODSThe KCs were isolated from mouse liver and identified. After successful identification, 1 mg/L lipopolysaccharide (LPS) was used to induce the KCs transfected with miR-133 inhibitor or miR-133 mimic. The mRNA expression levels of miR-133 and NLRP3 were detected by RT-qPCR. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cell culture medium were measured by ELISA. The protein levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 were determined by Western blot. TargetScan was used to find the binding site of miR-133 and 3'UTR of NLRP3 mRNA, and the target relationship was identified by dual-luciferase reporter detection kit. RESULTSThe volume of KCs at 72 h was larger than that at 24 h, with clear boundary and stable shape. The result of carbon ink experiment showed that a large number of black particles were observed in the cells, which proved that the cells had strong phagocytic capacity and were KCs. After the KCs was induced by LPS at 1 mg/L, the level of miR-133 was decreased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were increased (P<0.05). After transfection with miR-133 inhibitor, the level of miR-133 in the cells was decreased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were increased (P<0.05). After transfection with miR-133 mimic, the level of miR-133 in the cells was increased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were decreased (P<0.05). TargetScan analysis showed that the 3'UTR of NLRP3 mRNA contained the conservative bases of miR-133 sequence. Relative activity of luciferase in the cells transfected with miR-133 mimic was decreased (P<0.05). CONCLUSION miR-133 attenuates inflammation of mouse KCs by targeting NLRP3, thus protecting the KCs.     

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

Protective effect of curcumin on MRL/lpr mice with lupus nephritis and its mechanism

AIM To observe the effect of curcumin （Cur） on lupus nephritis （LN） and its possible mechanism. METHODS Thirty 10-week-old MRL/lpr lupus mice were randomly divided into MRL/lpr group， Cur-L and Cur-H group with 10 mice in each group， and C57BL/6 mice （n=10） served as normal control （NC） group. The mice in Cur-L group and Cur-H group were given intragastric administration of Cur at 100 and 200 mg·kg^-1·d^-1 for 12 weeks， respectively， and the same volume of normal saline was given to the mice in NC group and MRL/lpr group. The urine protein was detected， and the morphological changes of the renal tissue were observed by HE staining after treatment. The levels of serum creatinine （SCr）， blood urea nitrogen （BUN） and serum anti-double-stranded DNA （dsDNA）， and interleukin-1β （IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） levels in serum and renal tissues were detected. The protein levels of p-IκB， NF-κB， NLRP3 and caspase-1 in the renal tissues were determined by Western blot. RESULTS Compared with MRL/lpr group， the content of urine protein in Cur groups was significantly reduced， and the renal injury was relieved. The SCr， BUN， serum anti-dsDNA， and the serum and renal levels of IL-1， IL-6 and TNF-α were all significantly reduced， and the protein levels of p-IκB， NF-κB， NLRP and caspase-1 in the renal tissue were significantly decreased （P<0.05）. CONCLUSION Cur has a certain protective effect on the kidney of MRL/lpr mice， and its mechanism may be related to the inhibition of NF-κB and NLRP3 signaling pathways.     

Niflumic acid inhibits the proliferation,migration and invasion of glioma U87 cells

AIM To investigate the effect of niflumic acid （NFA） on human glioma U87 cells and to clarify the potential mechanism. METHODS The U87 cells were cultured in vitro and divided into blank control group, and 50, 100 and 200 μmol/L NFA groups. MTT assay was performed to determine the viability of cells in various groups. Migration and invasion abilities were measured by real-time cell analysis （RTCA）. RESULTS The results of MTT assay showed that compared with blank control group, the viability of U87 cells was increased after treatment with NFA for 12 h （P<0.05 or P<0.01）, while the viability was significantly decreased after treatment with NFA for 24 and 48 h （P<0.05 or P<0.01） in a concentration-dependent manner. The results of RTCA showed that compared with control group, the cell migration and invasion abilities were inhibited in 100 and 200 μmol/L NFA groups （P<0.05 or P<0.01） and the inhibitory effects were more obvious in 200 μmol/L NFA group （P<0.01）. CONCLUSION NFA inhibits the viability, migration and invasion of human glioma U87 cells.     

Activation of NF-κB-iNOS/COX-2 signaling pathways is involved in impaired endothelium-dependent relaxation under high glucose condition

AIM To investigate the role of nuclear factor-κB （NF-κB）-inducible nitric oxide synthase （iNOS）/cyclooxygenase-2 （COX-2） signaling pathways in impaired endothelium-dependent relaxation under high glucose （HG） condition. METHODS The endothelium-dependent relaxation induced by acetylcholine （ACh） in phenylephrine-precontracted rat thoracic aortic ring was performed in the absence or presence of different inhibitors under HG （55 mmol/L glucose） condition, and then the maximal relaxation effect of ACh （E_max） and the negative logarithm of ACh concentration for inducing 50% of E_max （pD₂） were calculated. The structure of aorta was observed by HE staining and electron microscopy. The mRNA and protein expression was detected by RT-qPCR and Western blot, respectively. RESULTS The structure of endothelial cells was interrupted by HG. Meanwhile, ACh-induced vasodilatation was also impaired, in which the E_max and pD₂ were both decreased significantly （P<0.01）, accompanied by the up-regulation of NF-κB p65, iNOS and COX-2 expression at mRNA and protein levels （P<0.01）. The NF-κB inhibitor PDTC, iNOS inhibitor SMT, and COX-2 inhibitor meloxicam restored the ACh-induced vasodilatation under HG condition, in which the E_max and pD₂ were increased significantly （P<0.01）. Moreover, PDTC attenuated the pathological damage of endothelial structure, and down-regulated the expression levels of NF-κB p65, iNOS and COX-2 induced by HG （P<0.01）. CONCLUSION The activation of NF-κB-iNOS/COX-2 signaling pathways is involved in the impaired endothelium-dependent relaxation under HG condition.