Objective:To explore the regulation and mechanism of human placental extract(HPE)on lipopolysaccharide(LPS)-induced BV2 microglial cell inflammation.Methods:Microglia cell lines(BV2)were cultured in vitro and divided into PBS group,HPE group,LPS group and LPS+HPE group.BV2 cell viability was measured by CCK-8 analysis.A fluorescent probe targeting reactive oxygen species(ROS)was to detect the level of intracellular ROS.The mRNA levels of TNF-α,IL-1β and IL-6 were detected by RT-qPCR.The supernatants of different treatment groups were collected.The content of nitric oxide(NO)was detected by the Griess method,and the secretion levels of TNF-α,IL-1β and IL-6 were detected by the flow magnetic bead microarray(CBA)method.The indirect contact co-culture system between BV2 and mouse hippocampal neurons cell line HT22 cells was established to evaluate the neurotoxicity of HPE by the assessment of the cell viability and apoptosis of HT22 cells using CCK-8 or flow cytometry.The potential signaling molecules of NF-κB signaling pathway was detected by flow cytometry.Results:Compared with the PBS group,1.2 μg/ml LPS and 50 ng/ml HPE significantly inhibited the activity of BV2 cells.Compared with the PBS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells of the LPS group were significantly increased(P<0.05),and the secretion levels of NO,TNF-α,IL-1βand IL-6 in the supernatant were also significantly upregulated(P<0.05).The expressions of related signaling pathway molecules Toll-like receptor 4(TLR4),pIκBα and pNF-κB p65 were significantly increased(P<0.05).Compared with the LPS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells,the secretion levels of NO,TNF-α,IL-1β and IL-6,the neurotoxicity and neuronal apoptosis induced by microglial conditional medium,and the expressions of TLR4,pIκBα and pNF-κB p65 in the LPS+HPE group were significantly downregulate(P<0.05).Conclusion:HPE may alleviate the microglial inflammation,possibly through the TLR4/NF-κB signaling pathway.

Objective:To explore the regulation and mechanism of human placental extract(HPE)on lipopolysaccharide(LPS)-induced BV2 microglial cell inflammation.Methods:Microglia cell lines(BV2)were cultured in vitro and divided into PBS group,HPE group,LPS group and LPS+HPE group.BV2 cell viability was measured by CCK-8 analysis.A fluorescent probe targeting reactive oxygen species(ROS)was to detect the level of intracellular ROS.The mRNA levels of TNF-α,IL-1β and IL-6 were detected by RT-qPCR.The supernatants of different treatment groups were collected.The content of nitric oxide(NO)was detected by the Griess method,and the secretion levels of TNF-α,IL-1β and IL-6 were detected by the flow magnetic bead microarray(CBA)method.The indirect contact co-culture system between BV2 and mouse hippocampal neurons cell line HT22 cells was established to evaluate the neurotoxicity of HPE by the assessment of the cell viability and apoptosis of HT22 cells using CCK-8 or flow cytometry.The potential signaling molecules of NF-κB signaling pathway was detected by flow cytometry.Results:Compared with the PBS group,1.2 μg/ml LPS and 50 ng/ml HPE significantly inhibited the activity of BV2 cells.Compared with the PBS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells of the LPS group were significantly increased(P<0.05),and the secretion levels of NO,TNF-α,IL-1βand IL-6 in the supernatant were also significantly upregulated(P<0.05).The expressions of related signaling pathway molecules Toll-like receptor 4(TLR4),pIκBα and pNF-κB p65 were significantly increased(P<0.05).Compared with the LPS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells,the secretion levels of NO,TNF-α,IL-1β and IL-6,the neurotoxicity and neuronal apoptosis induced by microglial conditional medium,and the expressions of TLR4,pIκBα and pNF-κB p65 in the LPS+HPE group were significantly downregulate(P<0.05).Conclusion:HPE may alleviate the microglial inflammation,possibly through the TLR4/NF-κB signaling pathway.

Objective:To investigate the effects and mechanisms of exogenous calcium-binding protein S100A4 on prolifera-tion,survival and migration functions of glioma cells.Methods:Pan-cancer dataset was downloaded from UCSC database for the analysis of S100A4 expression and prognosis in pan-cancer,and transcriptome and clinical data of glioma patients were downloaded from CGGA database.Prognosis and progression of S100A4 expression in glioma patients were analyzed by R software.S100A4 protein network interaction of glioma patients were addressed by online analytical tools GEPIA and STRING.Human glioma cell lines(U87 and U251)were cultured in vitro,and the experiment was divided into 3 groups:PBS group,S100A4 group and S100A4+TAK242 group[Resa-torvid(TAK242)was a specific inhibitor of Toll-like receptors 4(TLR4)].Flow cytometry was used to detect proliferation and apopto-sis of glioma cells.Wound healing assay,Transwell assay and clone formation assay were used to detect migration and proliferation ability of U87 and U251 cells,and Western blot was used to detect levels of TLR4 protein and NF-κB related signaling proteins.Results:Bioinformatics results showed that S100A4 was significantly upregulated in multiple tumours(P<0.05),which included glio-blastoma(GBM),lower grade glioma(LGG),stomach adenocarcinoma(STAD),liver hepatocellular carcinoma(LIHC),etc,with poorer prognosis in GBM and LGG.Compared with glioma patients with low expression of S1004,high expression S100A4 in glioma patients had shorter survival and higher degree of WHO classification.In addition,S100A4 protein in glioma patients may interact with Annexin A2(ANXA2),TLR4 and advanced glycosylation end product-specific receptor(AGER)proteins.In cellular experiments,U87 and U251 cells showed enhanced proliferation and migration,and significantly up-regulated levels of TLR4,p-ERK1/2,p-p38 and p-p65 proteins after exogenous S100A4 treatment compared to PBS group(P<0.05),while glioma cells in S100A4+TAK242 group showed weaker proliferation and migration,and lower TLR4,p-p38 and p-p65 protein levels than those in S100A4 group,TLR4,p-ERK1/2,p-p38,p-p65 protein levels were significantly down-regulated(P<0.05).Conclusion:S100A4 may regulate func-tion of glioma proliferation,migration and survival through TLR4/NF-κB signaling pathway.

Objective:To investigate potential mechanisms of Niclosamide,a calcium-binding protein S100A4 inhibitor,in regulating inflammatory response of bronchial epithelial cells.Methods:Human bronchial epithelial cells BEAS-2B were cultured in vitro and stimulated by LPS or Niclosamide-pretreatment.Inflammatory cytokines and potential signaling molecules expressions were determined by RT-qPCR and Western blot.Intracellular ROS level was quantified by fluorescent probe.Results:Increased ROS level was observed in LPS-stimulated and Niclosamide-pretreatment cells(P<0.05).Compared with control group,mRNA and protein expressions of S100A4 were increased(P<0.05),TLR4/STAT3,MAPK1/3/SIRT1,NF-κB/IKKβ/p65 mRNA expressions were increased(P<0.05),inflammatory cytokines IL-1β and IL-6,tight junction Occludin and ZO-1 mRNA expressions were increased after LPS-treatment(P<0.05),whereas these genetic expressions were downregulated by Niclosamide-pretreatment(P<0.05),except for TLR4/MAPK1 and NF-κB/IKKβ/p65(P>0.05).Western blot showed that compared with control group,LPS-stimulation promoted protein expressions of S100A4,STAT3,MAPK3/SIRT1,IL-1β and TNF-α(P<0.05),while downregulated protein expression of Occludin.Compared with LPS group,niclosamide-pretreatment downregulated protein expressions of S100A4,STAT3,MAPK3/SIRT1,IL-1β and TNF-α(P<0.05),while restored protein expression of Occludin(P<0.05).Conclusion:Calcium-binding protein S100A4 inhibitor Niclosamide can alleviate S100A4 expression and inflammatory response of bronchial epithelial cells,which is poten-tially related to STAT3 or MAPK3/SIRT1 signaling.