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Objective To study the polarization of macrophages induced bytoxoplasma gondiiWh3 strain. Methods Macrophages THP-1 were infected withtoxoplasma gondiiWh3 strain, RH strain and ME49 strain. After 6 hours and 24 hours of infection, the supernatant and macrophages were collected. The concentrations of IL-6, IL-10, TNF-α and TGF-β of the supernatant were detected by ELISA. In macrophages, the mRNA transcription levels of M1/M2 related genes iNOS, arginase-1 and TNF-α, IL-12, TGF-β, IL-10 were detected by qRT-PCR, and the expression levels of iNOS and arginase-1 were analyzed by Western blot. Results The results of ELISA showed that the contents of IL-6 and TNF-α were the highest after infection by ME49 strain 6 hours and 24 hours(P<0.01), and the highest of IL-10 and TGF-β were detected after infected with RH strain(P<0.01). However, the content of cytokines in supernatant infection by Wh3 strain was between RH strain and ME49 strain. Some inflammatory factors and cytokines mRNA in macrophages were detected after 6 hours and 24 hours of infection, and iNOS, TNF-α and IL-12 significantly increased after infected withtoxoplasma gondiiME49 strain(P<0.05), arginase-1, IL-10 and TGF-β significantly increased after RH strain infection. It was also found that the levels of mRNA in macrophages infected with Wh3 strain was between RH strain and ME49 strain. After 24 hours of infection, the expression of iNOS protein increased with wh3 and me49 strains oftoxoplasma gondii(P<0.000 1), while the expression of arginase-1 increased with RH strain(P<0.001). Conclusion The polarization direction of macrophages was between M1 and M2 after infection withtoxoplasma gondiiWh3 strain, and it was closer to M2.

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Objective To investigate the effect of RO or combined cholesterol(CH) on keratinocytes cell proliferation and cyclin expression. Methods The KCs were cultured with different concentrations of RO for different times,the proliferation of KCs were analyzed by MTS.Cell cycle of KCs were detected by flow cytometry after using RO with different times or combined treated with CH,and Western blot was used to reveal the expression of CyclinB1 and CyclinE. Results The RO inhibited the proliferation of KCs in a quantitative and time-dependent manner.Combined use of CH reduced the proliferation inhibition of RO on KCs.RO could significantly increase the proportion of KCs cell cycle in G1 phase,while combined use of CH reduced G1 phase growth rate of KCs cell cycle.Western blot demonstrated that use of RO could reduced the CyclinB1 and CyclinE content in the KCs in a time dependence manner.Combined use of CH antagonism RO down-regulated Cyclin E,but had no significant effect on the expression of CyclinB1. Conclusion RO inhibits the proliferation of KCs by down-regulating the expression of CyclinB1 and CyclinE which blocks the G1 phase of KCs cell cycle.

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Objective To explore the differential expression of TM6 SF2 in hepatocellular carcinoma and its effect on the biological behavior of hepatocellular carcinoma cells. Methods Immunohistochemistry was used to detect TM6 SF2 expression in 15 cases of hepatocellular carcinoma and adjacent tissues. Western blot was used to detect the expression of TM6 SF2 in hepatocellular carcinoma cell line Hep G2 and normal liver cell line L-02. TM6 SF2 expression and survival curve in hepatocellular carcinoma were analyzed based on TCGA database. A TM6 SF2 overexpression vector was constructed based on the PCDNA 3.1 plasmid. Hep G2 was infected by lentiviral packaging, and stable cell lines were screened. Western blot was used to detect the TM6 SF2 over-expression efficiency. For the blank group, control group and TM6 SF2 overexpression group, CCK-8 and cell cloning method were used to detect cell proliferation activity, Annexin V-FITC/PI double labeling was used to detect cell apoptosis, and Transwell was used to detect cell invasion ability. Based on the clinical samples of hepatocellular carcinoma tissues, q-PCR was used to detect the expression levels of TM6 SF2 and SLC27 A5, and the expression correlation was analyzed. Results TM6 SF2 expression was significantly reduced in hepatocellular carcinoma tissues and cells. The 5-year survival of hepatocellular carcinoma patients with TM6 SF2 low expression was shorter. After successful construction of TM6 SF2 overexpressing stable cell line Hep G2, TM6 SF2 overexpression could significantly reduce Hep G2 cell proliferation activity, invasion ability, and promote apoptosis. There was a positive correlation between TM6 SF2 and SLC27 A5 expression in hepatocellular carcinoma tissues. Conclution TM6 SF2 shows strong potential of tumor suppressor genes and has certain clinical significance. It provides new ideas and theoretical basis for future clinical diagnosis, prognosis judgment and molecular targeted therapy of hepatocellular carcinoma based on TM6 SF2.

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Objective To study the effect of miR-34 b-5 p Up-regulation on the invasion of diffuse large B cell lymphoma and the RhoA/ROCK signaling pathway. Methods SU-DHL-4 cells were selected for study in diffuse large B lymphoma lines. A blank control group, an empty vector transfection group, and a miR-34 b-5 p transfection group were established. The expression of miR-34 b-5 p was detected by qRT-PCR. Cell proliferation, apoptosis and invasive were tested by colony formation assay, flow cytometry and Transwell test respectively. Western blot was used to detect the proteins expression levels of E-cadherin, N-cadherin Vimentin, RhoA and ROCK. Results miR-34 b-5 p expression was significantly up-regulated in cells transfected with miR-34 b-5 p. miR-34 b-5 p overexpression inhibited SU-DHL-4 cells proliferation, enhanced SU-DHL-4 cells apoptosis, reduced SU-DHL-4 cells invasion, and inhibited the RhoA/ROCK signaling pathway. Conclusion miR-34 b-5 p overexpression inhibits the growth and motility of diffuse large B cell lymphoma and the expression of RhoA/ROCK signaling pathway.

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Objective To identify two clinical isolate strains ofEggerthella lenta. Methods Two strains isolated from positive blood anaerobic culture were selected.The isolates were identified by MALDI-TOF MS and gram staining.Drug sensitivity test was performed by agar dilution method.At the same time,genomic DNA of the strains was extracted,16 S rRNA universal primer was used for PCR amplification,and the product was sequenced after recovery and purification,which was homologous with the sequences of the known bacteria in GeneBank database. Results The isolate was identified asEggerthella lentaby MALDI-TOF MS and 16 S rRNA gene assay.Drug sensitivity tests showed that the bacteria was sensitive to penicillin,piperacillin-tazobactam,ampicillin-sulbactam,imipenem,meropenem,metronidazole and clindamycin. Conclusion MALDI-TOF MS and 16 S rRNA detection can be used for identification of rare bacteria such asEggerthella lenta.

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Objective To explore the pharmacological mechanism of Qishen Yiqi dropping pills in the treatment of myocardial infarction(MI) by network pharmacology. Methods The Chinese medicine system pharmacology database and analysis platform(TCMSP) was used to search for all active compound components and targets of the four herbs in the Chinese herbal compound of Qishen Yiqi dropping pills. The targets of myocardial infarction were collected by OMIM, TTD, PharmGKB, DisGinNET and DrugBank databases. Then disease targets were merged with the therapeutic targets of the drug to obtain the targets of the drug for the disease. The drug components-disease targets network was constructed by Cytoscape software, and the core targets of the drug for treating myocardial infarction were filtrated by analyzing the network. The DAVID database performed KEGG enrichment analysis, and the Omicshare database performed GO secondary classification enrichment and visualized the results of KEGG enrichment. Results Oral bioavailability(OB)≥30%, drug-likeness(DL)≥0.18, Caco-2 permeability(Caco-2)≥-0.4 and half-life(HL)≥4 h were chosen as the active compounds ADME screening conditions. A total of 82 active components and 610 component targets were obtained. Five disease databases were used to search for myocardial infarction targets and a total of 1 713 related targets were obtained. Then 103 targets were mapped to the drug targets successfully. DAVID enrichment analysis was performed on 27 core targets greater than the average degree, and ADRB2, CHRM3, CHRM2, NOS3, NOS2, ADRA1 D, CALM1, PIK3 CG, DRD1, PDE3 A, F2, GSK3 B, COX2, ESR2, ACHE, SCN5 A, COX1, PTGS2,18 genes were the core targets for myocardial infarction, which were enriched in 19 pathways related to myocardial infarction, such as calcium signaling pathway, cAMP signaling pathway, and cGMP-PKG signaling pathway. Conclusion The theory and method of systemic pharmacology confirm the multi-components, multi-targets and multi-pathways treatment characteristics of Qishen Yiqi dropping pills, and predict its possible mechanism for treating MI, which provide reference and theory for the basic research of the disease simultaneously.

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Objective To investigate the effect of Naringin(NAR) on bleomycin-induced pulmonary fibrosis in mice and its possible mechanism. Methods Sixty male ICR mice were randomly divided into Control group,Model group,dexamethasone group(DXM,3 mg/kg),low-dose naringin group(L-NAR,25 mg/kg) and high-dose naringin group(H-NAR,100 mg/kg),with 12 mice in each group.Mouse pulmonary fibrosis model was established by intratracheal one-time infusion of bleomycin(5 mg/kg),and mice were sacrificed after 4 weeks of drug intervention.Lung mass was taken and lung index was calculated.Levels of hydroxyproline(HYP) in lung tissue were detected by alkaline hydrolysis.The pathological changes of lung tissues were observed and scored by HE staining.The degree of pulmonary fibrosis was observed and scored by Masson staining.The levels of IL-1β,IL-18,TGF-β1 in lung tissue were detected by ELISA.The expression levels of NLRP3,ASC and caspase-1 mRNA in lung tissues were detected by RT-PCR.The protein expression levels of NLRP3,ASC,caspase-1,Collagen I,α-SMA,E-cadherin and Vimentin in lung tissues were detected by Western blot. Results Compared with the Control group,the Model group showed significant lung tissue damage and severe degree of fibrosis,the lung index and lung tissue contents of HYP,IL-1β,IL-18 and TGF-β1 increased.The mRNA and protein expression levels of NLRP3,ASC,caspase-1,as the well as the protein expression levels of Collagen I,α-SMA and Vimentin increased,while the expression levels of E-cadherin significantly decreased,and the difference was statistically significant(P<0.05).Compared with the Model group,the degree of lung injury and fibrosis were significantly improved in the DXM group and the H-NAR group.The lung index and lung tissue contents of HYP,IL-1β,IL-18 and TGF-β1 decreased.The mRNA and protein expression levels of NLRP3,ASC,caspase-1,as the well as the protein expression levels of Collagen I,α-SMA and Vimentin decreased,while the expression levels of E-cadherin increased,and the difference was statistically significant(P<0.05).However,there was no significant difference in relevant indicators between the L-NAR group and the Model group. Conclusion NAR can effectively inhibit bleomycin-induced pulmonary fibrosis in mice,and the mechanism may be related to the inhibition of NLRP3 inflammatory corpuscles activation.

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Objective To establish animal models of Echinococcosis multicocularis in 3 different regions in western of China, analyze and compare the infectivity of them. Methods Forty BALB/c mice were divided into 4 groups:normal control group(n=10), EM-1 group(Qinghai strain, n=10), EM-2 group(Xinjiang strain, n=10) and EM-3 group(Sichuan strain, n=10). The infection of abdominal cavity was established. The mice were weighed 180 days after inoculation, the infection rate was calculated, the protoscolex was isolated, the livers were fixed with 4% paraformaldehyde, HE staining was also used. The average number of protoscolex was calculated and blood was taken from the angular vein at 7 days,1 month,3 months,and 6 months,respectively,and the contents of transferase( AST),alanine transferase( ALT),alkaline phosphatase( ALP),eosinophils( EOS) and leukocytes( WBC),red blood cells( RBC),platelets( PLT) were tested. Results In this study,three mouse models of abdominal cavity infection from different regions were successfully established. There were no differences in blood routine and liver function index of BALB/c mice from 3 different regions. Among them,EM-2( Xinjiang strain) group had the most protoscolex. EM-3( Sichuan strain) had the least number of protoscolex. The EM-2 group had the highest infection rate( 77. 8%),and the EM-3 infection rate was the lowest( 62. 5%). After HE staining for the liver tissues of 3 groups of mice,vesicles of varying sizes could be seen in the liver tissues of the experimental groups under low power microscope. The Xinjiang strain infected mice could be observed serious liver cell necrosis,and the area of liver necrosis was large. Conclusion The animal models of infection in abdominal cavity of different regions have been successfully established. Pathogenicity of Echinococcosis multicocularis in different regions of mice are different. The Xinjiang strain has the most serious infectivity and the Sichuan strain has the least infectivity.

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Objective To investigate the effects of placenta derived exosomes on the proliferation, invasion, migration and tube formation capacity of vascular endothelial cells in gestational diabetes mellitus(GDM). Methods Placental exosomes were obtained by using the method ofin vitroculture of placental mass and hypervelocity centrifugation. Transmission electron microscopy(TEM), dynamic light scattering(DLS) and Western blot were used to identify those exosomes; Human umbilical vein endothelial cells(HUVEC) were culturedin vitro. The uptake of exosomes by HUVEC was detected by fluorescence confocal microscopy after double staining; CCK-8 and EdU, scratch and Transwell invasion and tube formation assay were used to detect the effects of different concentrations of exosomes on the proliferation, migration, invasion and angiogenesis of endothelial cells. Results The placental exosomes with diameters ranging from 40 to 130 nm were successfully isolated. The isolated and purified placenta exosomes could be absorbed by endothelial cells and could promote the proliferation, migration, invasion and tube forming ability of endothelial cells in a concentration dependent manner. Conclusion The exosomes from placental tissue of gestational diabetes mellitus can promote the proliferation, migration, invasion and tube forming ability of endothelial cells.

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Objective To explore the effects of interfering with long non-coding RNA CCAT2(lncRNA CCAT2) on mitochondrial function of bladder cancer cells J82 and features of tumor stem cells. Methods The expression of CCAT2 was interfered by constructing lncRNA CCAT2 short hairpin RNA(shRNA),which was verified by RT-PCR.J82 cell lines with the lowest expression level of CCAT2 were selected for the subsequent experiments.The cell proliferation was detected by clone formation assay.The changes in mitochondrial membrane potential(MMP) were detected by flow cytometry.The levels of apoptosis-related proteins were detected by Western blot.The pelletization of tumor cells was observed under microscope.The expression of CD133 in each group was detected by flow cytometry.The levels of OCT4,SOX2 and CD44 were detected by Western blot. Results The expression level of CCAT2 in three kinds of CCAT2-shRNA J82 cell lines was lower than that in shRNA-NC(P<0.05).The colony formation rate,level of c-Myc,tumor pelletization diameter and pelletization rate,number of positive CD133 cells,and levels of OCT4,SOX2 and CD44 in CCAT2-shRNA group were lower than those in shRNA-NC group(P<0.05),while green fluorescence intensity,Bax/Bcl-2,levels of Caspase-3 and Caspase-9 were higher than those in shRNA-NC group(P<0.05). Conclusion Down-regulating the expression level of lncRNA CCAT2 can inhibit the growth of J82 cells,which may be related to the influence of cellular mitochondrial function and tumor stem cell-like features.

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Objective To investigate the effect of miR-33-5 p on the regulation of c-Jun amino terminal kinase 1(JNK1) on the multi-drug resistance of human oral squamous cell carcinoma. Methods CAL-27 cells were divided into CAL-27 group,CAL-27/CBP group,CAL-27/CBP+ scramble inhibitor group,CAL-27/CBP+ miR-33-5 p inhibitor group,carboplatin(CBP) induced drug resistance Cell line,according to the group transfected with scramble inhibitor or miR-33-5 p inhibitor.MTT detected the multi-drug resistance of cells,RT-PCR detected the expression of miR-33-5 p,luciferase report test detected miR-33-5 p With JNK1 targeting,Western blot was used to detect the activation of the JNK1/c-Jun pathway and the expression of MDR1,and flow cytometry was used to detect the results of Rh123 aggregation. Results Compared with CAL-27 group,the multi-drug resistance of CAL-27/CBP increased(P<0.01),the expression level of miR-33-5 p increased(P<0.01),and the activation level of JNK1/c-Jun pathway decreased(P<0.01),the percentage of Rh123 positive cells decreased(P<0.01),the level of MDR1 protein increased(P<0.01),and the luciferase reported experimental results showed that miR-33-5 p targeted JNK1; compared with the CAL-27/CBP group,the multi-drug resistance of cells in the CAL-27/CBP+miR-33-5 p inhibitor group reduced(P<0.05),the expression level of miR-33-5 p reduced(P<0.01),The activation level of JNK1/c-Jun pathway increased(P<0.01),the percentage of Rh123 positive cells increased(P<0.01),and the level of MDR1 protein decreased(P<0.01). Conclusion MiR-33-5 p reduces the multi-drug resistance of CAL-27/CBP drug-resistant cell lines through targeted regulation of JNK1/c-Jun pathway.

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Objective To investigate the regulatory mechanism of zeste gene enhancer human homologue 2(EZH2) on nrf2-are REDOX pathway in drosophila melanogaster in anti-tuberculous drug-induced liver injury(ADLI).Methods 32 kunming mice were randomly divided into four groups: blank control group,model group,experimental group and negative control group.The levels of ALT and AST in serum liver function indexes were detected by microporous plate method.Histopathological changes of liver were observed by HE staining.MRNA and protein levels of EZH2,nuclear factor e2-related factor 2(Nrf2),NADPH quinone oxidoreductase 1(NQO1) and heme oxygenase 1(HO-1) were detected by real-time quantitative fluorescence PCR,ELISA and Western blot,respectively.Results The serum ALT and AST levels of mice in the model group were higher than those in the blank control group(all P<0.05),while those in the experimental group were lower than those in the model group(all P<0.05).Compared with the blank control group,the liver histopathological results of mice in the model group showed obvious injury,while the liver injury of mice in the experimental group was alleviated.Compared with the blank control group,the mRNA and protein levels of EZH2 decreased in the model group( P<0. 05),and further decreased in the experimental group( P<0. 05). Compared with the blank control group,the mRNA and protein levels of Nrf2,NQO1 and HO-1 increased in the model group( P<0. 05),and further increased in the experimental group( P<0. 05). Chip results showed that the level of H3 K27 me3 in the model group was lower than that in the blank control group( P<0. 05),and further decreased in the experimental group( P<0. 05). Conclusion The down-regulation of EZH2 expression in ADLI and the inhibition of EZH2 expression can alleviate the degree of liver injury. The effect of EZH2 on ADLI may be realized through the modification of H3 K27 me3 in the Nrf2 promoter region,and then through the regulation of Nrf2-ARE antioxidant stress pathway.

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Objective Previous study has found that peptide301LARSLKT307can significantly inhibit fetal cardiac developmentin vivo. This study intends to further explore the role and mechanism of301LARSLKT307in inhibiting myocardial differentiation at P19 cell. Methods The structure, conservation, physical and chemical property and precursor proteins of peptide301LARSLKT307were analyzed by PepDraw Tool, Uniprot and ProtParam online. The entry of FITC-labeled peptide301LARSLKT307into P19 cells was observed under a fluorescence microscope. After adding peptide301LARSLKT307, the differentiation of P19 cells was observed by inverted microscope. The expression of troponin cTnT and transcription factor GATA4 were detected by qPCR and Western blot assay to analyze the specific marker gene of myocardium. The expressions of Notch1, Hey1 and Hey2 were detected by qPCR to analyze the effect of peptides on Notch1 pathway in P19 cell. Results Bioinformatics analysis showed that the peptide was located at the 301-307 amino acid site of its precursor protein Talin, which is a small molecule peptide with stable structure, high lipophilicity and high sequence conservation. FITC-labeled peptide301LARSLKT307can enter the P19 cell under the fluorescence microscope. Compared with the control group, the cells in the peptide group were partially necrotic, with disordered growth and morphology. The qPCR and Western blot assay showed that the differentiation related genes GATA4 and cTnT in the peptide group both decreased at different levels. The qPCR showed that the Notch-related genes Notch1, Hey1 and Hey2 in the peptide group decreased at different levels. Conclusion Peptide301LARSLKT307may inhibit the biological function of myocardial differentiation by inhibiting the expression of key factors of Notch1 pathway.

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Objective To explore the role of ERS and protein carbonylation in the development of the rat heart failure cachexia by injected isoprenaline(ISO). Methods Ninety clean male SD rats were randomly divided into the ISO group, the ISO+HYD group, and the control group, with 30 rats in each group.The rats in the ISO and ISO+HYD groups were injected with ISO, rats in the ISO+HYD group were gavaged with HYD, and the control group were injected with saline. Six weeks later, whether the rats were successful in modeling was evaluated by rats′ echocardiograms and changes in body weight. The concentration of BNP, CHOPand GRP78 in serum was detected by ELISA assay kit; UV spectrophotometer detected concentration of the total protein carbonyl in myocardium and skeletal muscle; the relative levels of CHOP mRNA and GRP78 mRNA in cardiac and skeletal muscle of three groups were detected by PCR; the levels of protein carbonyl in myocardium and skeletal muscle of three groups were detected by immunofluorescence. Results (1) The levels of BNP, CHOP and GRP78 were highest in ISO group, followed by the ISO+HYD group and the lowest in the control group(P<0.05).(2) The concentration of protein carbonyl in myocardium and skeletal muscle was highest in ISO group, followed by the ISO+HYD group and the lowest in the control group(P<0.05).(3) The levels of CHOPmRNA and GRP78 mRNA were highest in ISO group, followed by the ISO+HYD group and lowest in the control group(P<0.05).(4) Protein carbonyl expression:compared with the three groups, the ISO group was the highest, followed by the ISO + HYD group, and the control group was the lowest(P<0.05). Conclusion ERS and protein carbonylation are involved in the development of heart failure cachexia, which are related to the level of oxidative stress in the body. The use of HYD to reduce the level of oxidative stress can reduce the expression of ERS and protein carbonylation and prevent the development of heart failure cachexia.

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Objective This study aims to investigate the effects of imperatorin on myocardial injury and oxidative stress in myocardial ischemia-reperfusion rats by inhibiting the activation of extracellular signal-regulated kinase 1/2(ERK1/2)and Nuclear factor-kappaB phosphorylation-65(NF-kappaB p65).Methods SD rats were randomly divided into five groups:healthy Control group(Control group),myocardial ischemia-reperfusion model group(MI/R group),and low,medium and high doses of imperatorin 6.25,12.5 and 25 mg/kg groups.The rat heart rate(HR) and left ventricular shortening fraction score(FS) were detected by small animal ultrasound instrument.Enzyme-linked immunosorbent assay(ELISA) was used to detect creatine kinase-MB(CK-MB)and Cardiac troponin I(cTnI).Myocardial tissue damage was observed by HE staining.Terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labelling(TUNEL) was used to detect the apoptosis of cardiomyocytes.The expression of apoptosis-related proteins Bax,Bcl-2,Caspase-3 and pathway proteins ERK1/2 and NF-κB p65 were detected by Western blot. Superoxide dismutase( SOD) activity and Malondialdehyde( MDA) content were detected by the kit.Results Compared with the control group,HR and FS in MI/R group significantly reduced( P<0. 05); CK-MB and cTnI concentrations increased significantly( P<0. 05); myocardial tissue damage was obvious; apoptotic cells and Bax/Bcl-2 expression ratio significantly increased( P<0. 05); SOD activity significantly decreased( P<0. 05),while MDA content significantly increased( P<0. 05); phosphorylation of ERK1/2 and NF-κB p65 significantly increased( P<0. 05). The above results were reversed in MI/R rats after administration of imperatorin. Compared with the MI/R group,the HR and FS of the medium and high doses imperatorin groups were significantly recovered( P<0. 05); the concentration of CK-MB and cTnI decreased significantly( P<0. 05); myocardial tissue injury improved obviously; apoptotic cells and Bax/Bcl-2 expression ratio decreased significantly( P<0. 05); SOD activity significantly increased( P<0. 05),while MDA content significantly decreased( P<0. 05); phosphorylation of ERK1/2 and NF-κB p65 decreased significantly( P<0. 05). Conclusion Imperatorin can alleviate myocardial injury and oxidative stress in MI/R rats by inhibiting the activation of ERK1/2 and NF-κB p65 pathway proteins.

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Objective To investigate the effect of cancer-associated fibroblasts(CAFs) on the cisplatin resistance of ovarian cancer cells,and to explore the possible mechanisms. Methods CAFs and NFs were isolated and cultured respectively from 10 cases of epithelial ovarian cancer tissue and 10 cases of normal ovarian epithelial tissue.The isolated CAFs were identified by immunofluorescence assay.Indirect co-culture system was established by collecting CAFs or NFs culture supernatants and ovarian cancer SKOV3 cells.Cell proliferation,cisplatin toxicity and cell migration ability were investigated by CCK8 and scratch assay.The content of reactive oxygen species(ROS) in the co-culture system was assessed by DCFH-DA method.The expression of SKOV3 cell resistance-related genes YAP,CTGF and Cyr61 at mRNA and protein levels were detected by real-time quantitative PCR(qPCR) and Western blot. Results Primary ovarian cancer CAFs were successfully separated.The expressions of α-SMA and FAP protein in the separated CAFs were significantly higher than those in the NFs.Co-cultured with CAFs,the proliferation,the half inhibitory concentration(IC50)of cisplatin and migration abilities of SKOV3 cells significantly enhanced.Co-cultured with CAFs,ROS content of SKOV3 cells increased.Co-cultured with CAFs,the expressions of YAP,CTGF and Cyr61 of SKOV3 cells enhanced at mRNA and protein levels. Conclusion CAFs can promote the proliferation and migration of ovarian cancer cells and reduce the sensitivity of ovarian cancer SKOV3 cells to cisplatin.The mechanism is related to ROS promoting autophagy in ovarian cancer cells on the one hand,and it is related to up-regulation of drug resistance genes YAP,CTGF and Cyr61 in ovarian cancer cells on the other hand.

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Objective To investigate the effect of sevoflurane postconditioning on DNA damage and the expression of silent information regulation(SIRT1) of HT22 cell line following oxygen and glucose deprivation/reoxygenation injury. Methods Firstly, HT22 cells in logarithmic phase were randomly divided into normal control group(CON), oxygen and glucose deprivation/reoxygenation 4 h group(OGD/R 4 h), oxygen and glucose deprivation/reoxygenation 4 h+1% sevoflurane postconditioning group(OGD/R 4 h+1%SEVO), oxygen and glucose deprivation/reoxygenation 4 h+2% sevoflurane postconditioning group(OGD/R 4 h+2%SEVO), oxygen and glucose deprivation/reoxygenation 6 h group(OGD/R 6 h), oxygen and glucose deprivation/reoxygenation 6 h+1% sevoflurane postconditioning group(OGD/R 6 h+1%SEVO), oxygen and glucose deprivation/reoxygenation 6 h+2% sevoflurane postconditioning group(OGD/R 6 h+2%SEVO). Secondly, the former four groups in the first part of this study were further investigated the mechanism of sevoflurane postconditiong in oxygen and glucose deprivation/reoxygenation injury. MTT assay was used to detect cell survival rate, PI/Hoechst fluorescence staining and TUNEL assay were used to detect apoptotic and necrosis rate, immunofluorescence staining was used to detect the expression of 8-OHdG and Cleaved-casepase 3, Western blot analysis was used to detect the expression of SIRT1 and apoptotic protein expression. Results In the first part of the experiment, compared with CON group, the cell survival rates in both OGD/R 4 h and OGD/R 6 h groups reduced(P<0.01, P<0.01), and the number of apoptosis and necrotic cells also increased. Compared with the related OGD/R 4 h group, the cell survival rate in the 1% SEVO and 2% SEVO groups both increased( P<0. 01,P<0. 01),and the number of apoptotic cells and necrotic cells also decreased. Moreover,compared with the related OGD/R 6 h group,the cell survival rate in the1% SEVO and 2% SEVO groups increased( P<0. 01,P<0. 01),and the number of apoptotic cells and necrotic cells decreased. The second part of the experiment,compared with CON group,there were more TUNEL positive labeled cells,and increased expressions of Cleaved-casepase3( P<0. 01) and 8-OHdG( P<0. 01),as well as BAX( P<0. 01) in the OGD/R 4 h group,however,SIRT1 and Bcl-2 expression reduced( P<0. 01,P<0. 01).Compared with the OGD/R 4 h group,there were less TUNEL positive labeled cells,and decreased expressions of Cleaved-casepase3( P<0. 01,P<0. 01) and 8-OHdG( P<0. 01,P<0. 01),as well as BAX( P<0. 05,P<0. 01) in the 1% SEVO and 2% SEVO groups,SIRT1 and Bcl-2 expression increased( P<0. 01,P<0. 01; P<0. 05,P<0. 01). Conclusion Sevoflurane postconditioning can alleviate DNA oxidative damage and apoptosis of HT22 hippocampal neurons induced by oxygen and glucose deprivation/reoxygenation,and up-regulate SIRT1 protein expression.

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Objective To investigate the protective effect and mechanism of CP-25 on high insulin-induced podocyte injury. Methods Mouse podocyte were culturedin vitroand divided into blank control group,high-insulin model group,high insulin+CP-25(0.1、1、10 μmol/L) group and Metformin group.The effect of CP-25 on the proliferation of podocyte induced by high insulin was detected by CCK-8 method.The effect of CP-25 on glucose consumption induced by high insulin in podocyte was detected by GOD-POD method.Transwell method was used to detect the effect of CP-25 on the migration of podocyte induced by high insulin.Annexin V-FITC/PI double staining assay was used to detect the effect of CP-25 on high insulin induced apoptosis of podocyte.The effect of CP-25 on the expression of Nephrin protein in podocyte induced by high insulin was detected by Western blot. Results CCK-8 results showed that CP-25 could increase the proliferation capacity of high insulin induced podocyte.The results of GOD-POD showed that CP-25 could improve the glucose uptake ability of high insulin induced podocyte.Transwell experimental results showed that CP-25 could reduce the migration of podocyte induced by high insulin.Annexin V-FITC/PI double staining results showed that CP-25 could inhibit the apoptosis of pdocyte induced by high insulin.Western blot results showed that CP-25 could up-regulate the protein expression of Nephrin in podocyte induced by high insulin. Conclusion CP-25 can improve the injury of podocyte induced by high insulin,and the mechanism may be related to the expression of Nephrin.

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Objective To investigate the role of CCL23 and CCR1 in human epithelial ovarian cancer cells cisplatin resistance. Methods The expression of CCL23 and CCR1 in human epithelial ovarian cancer cisplatin resistant cells C13 K and cisplatin sensitive cells OV2008 was detected by Real-time quantitative PCR(qRT-PCR). CCL23 over expression vector was constructed in OV2008 cells and the positive clones were selected by puromycin. The transfection efficiency was observed by fluorescence microscopy and qRT-PCR. After treated with BX471(CCR1 antagonist), cell proliferation and the half inhibitory concentration of cisplatin(IC50)were assessed by plate clone formation assay and CCK-8 assay respectively. The protein expression of CCL23, CCR1 and YAP(yes-associated protein) was detected by Western blot. Results qRT-PCR showed that the expression of CCL23 and CCR1 in C13 K cells was higher than that in OV2008 cells(P<0.01). Fluorescence microscopy showed that recombinant OV2008 cells were full of green fluorescence. The mRNA level of CCL23 in CCL23 overexpressing cells significantly increased(P<0.001) which proved that the CCL23 gene over expression of OV2008 cells was successfully constructed, but the mRNA level of CCR1 had no significant change(P>0.05). Plate clone formation assay and CCK-8 assay showed that the proliferation ability and cisplatin resistance of OV2008 cell line overexpressing CCL23 increased compared to the empty vector of OV2008 cell line and normal OV2008 cell line(P<0.01). BX471 inhibited the expression of CCL23 and CCR1, meanwhile, downregulated the expression of YAP. Cell proliferation was inhibited and cisplatin sensitivity increased after treated with BX471. Conclusion CCR1/CCL23 axis is involved in EOC cells cisplatin resistance and its mechanism may be related to the regulation of downstream effector YAP by CCL23 and CCR1.

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Objective To investigate the protective effects and mechanism of isoliquiritigenin(ISL) on the brain inflammation with traumatic brain injury(TBI) in rats.Methods TBI model was established with the modified Feeney's method,and adult male SD rats were randomly subjected to either sham operation,model or ISL(15,30,60 mg/kg) group,with 10 rats in each group. Two hours after operation,ISL group was given intraperitoneal injection once,sham operation group and model group were given intraperitoneal injection of equal volume normal saline for 7 days,once a day. 7 days after the TBI,the neurobehavior of rats in each group was evaluated,then the rats were sacrificed to measure the brain water content and inflammatory factors. In addition,the protein and mRNA changes of Toll-like receptor 4( TLR4)-TANK-binding kinase( TBK1)-IκB kinase ε( IKKε) signaling pathway in the brain tissue were also examined. Results ISL improved the neuroethology of TBI rats,reduced the brain water content and expression of inflammatory factors,and inhibited TLR4-TBK1-IKKε signal pathway,with significant difference. Conclusion The protective effects of ISL on the rats with inflammation after TBI may be related to the inhibition of TLR4-TBK1-IKKε signal pathway.

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Objective To explore the mechanism of rosiglitazone(RSG) pretreatment in reducing inflammation and oxidative stress in the procession of acute lung injury(ALI) induced by bacterial lipopolysaccharide(LPS) in mice. Methods 32 SPF CD-1 male mice were randomly divided into Control group, RSG group, LPS 6 h group and RSG+LPS 6 h group. Mice in LPS group and RSG+LPS 6 h group were injected intraperitoneally with a single dose of LPS(2 mg/kg), while mice in RSG+LPS 6 h group and RSG group were given RSG(10 mg/kg) by oral gavage once a day for 4 days before LPS injection. The mice were killed 6 h after LPS injection, and the serum and lung tissue were collected. The pathological changes of lung tissue were evaluated by HE staining and pathological score, the levels of serum inflammation chemokine Keratinocyte-Derived Chemokine(KC) and proinflammatory factor Tumor Necrosis Factor-α(TNF-α) were detected by ELISA method, the protein expressions of NADPH oxidase subunits NOX-2,NOX-4 and nuclear factor-kappa B(NF-κB) signaling pathways(IκBα,p-IκBα,p-p65) in lung tissue were measured with Western blotting method. Results RSG pretreatment significantly attenuated LPS-induced ALI. RSG pretreatment obviously reduced the elevation of LPS-induced KC and TNF-α in lung tissue of mice, and the activation of LPS-induced NF-κB signaling pathway in lung tissue of mice was remarkedly alleviated. RSG pretreatment markedly inhibited the increase of NOX-4 protein in mice lung tissue induced by LPS. Conclusion RSG pretreatment may attenuate LPS-induced ALI in mice by inhibiting pulmonary inflammation and oxidative stress.

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Objective To investigate the effect of silencing SIRT1 gene by RNA interference on the radiosensitivity of human nasopharyngeal carcinoma cell line CNE-1,and to provide a new idea for clinical treatment of human nasopharyngeal carcinoma(PCN). Methods SIRT1 protein in NP69 and CNE-1 cells was detected by Western blot assay.The SIRT1-siRNA interference sequence was transfected into CNE-1 cells.The ability of cell proliferation was evaluated by CCK-8 assay.The cell apoptosis rate was detected by flow cytometry assay.The protein levels of Bcl-2,Bax,and Cleaved-Caspase3 were tested by Western blot assay. Results The results showed that the expression of SIRT1 protein in nasopharyngeal carcinoma tissues and cells was significantly higher than that of para-carcinoma tissues and NP69 cells(P<0.05).The expression levels of SIRT1 gene and SIRT1 protein in the cells of the transfection groups were significantly lower than that in the control group(P<0.05).The results showed after X-ray radiation of 4,6 and 8 Gy on CNE-1 cells,the ability of cell proliferation in the SIRT1 siRNA groups was obviously lower than that in the control group(P<0.01),and the cell apoptosis rate in the SIRT1 silence groups was significantly higher than that in the control group(P<0.01).Meanwhile,compared with the control group,the protein levels of Bax and Cleaved-Caspase3 significantly reduced,Bcl-2 expression significantly increased in SIRT1 siRNA group(P<0.05). Conclusion Silence of SIRT1 gene may enhance the sensitivity of CNE-1 cells to radiotherapy by inhibiting the proliferation and inducing apoptosis of CNE-1 cells,and SIRT1 gene may be a potential target for the treatment of nasopharyngeal carcinoma cancer.

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Objective To study whetherrotavirus(RV) can promote the phosphorylation of signal transduction activator 3(STAT3),induce intestinal epithelial cells to secrete antimicrobial peptide RegⅢ-γ, and regulate intestinal inflammation after infection of BALB/c suckling mice. Methods 7-day-old SPF grade BALB/c suckling mice were administrated with RV by gavage.The experiment was divided into five groups:normal control group,PBS control group,RV infection group,RV + STAT3 inhibitor group and STAT3 inhibitor group.Five suckling mice were killed at 1,2,3,4,5,6,7,8,9 and 10 days after infection,respectively.Enzyme linked immunosorbent assay(ELISA) was used to detect the change of IL-22 content in intestinal homogenate,Western blot was used to detect the expression of STAT3,pSTAT3 and RegⅢ-γ protein at different time points in each group. Results After RV infection,the intestinal tissue showed obvious inflammatory reaction,and the level of IL-22 increased gradually with the infection time which reached the maximum value on the fourth day of infection.The protein levels of pSTAT3 and RegⅢ-γ showed the similar change trend with IL-22,while the protein level of STAT3 showed the opposite trend,and the expression level was the lowest on the fourth day after infection. Conclusion RV can activate IL-22/pSTAT3/RegⅢ-γ signaling pathway and regulate the development of intestinal inflammation.

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Objective To analyze the prognosis of patients with colon cancer risk factors,constructs the nomogram prediction of colon cancer patients 1 year,3 years and 5 years survival rate and to analyze clinical benefit. Methods A total of 53 753 colon cancer patients with pathologically confirmed between 2010 and 2015 were obtained from the Surveillance,Epidemiology,and End Results(SEER) program.All samples were randomly divided into training(n=35 835) and validation(n=17 918) set based on the prognosis status and overall survival time.Univariate and multivariate analysis were performed using Cox proportional hazards regression model to evaluate the prognostic value of 9 clinical parametersincluding age,pathological stage,AJCC 7 th edition T stage,N stage,M stage,positive lymph node ratio(LNR),tissue grade,gender,and chemotherapy.The stepwise regression method was used to identify the potential prognostic risk parameters and a nomogram prognostic model was then established to predict the overall survival(OS).The internal and external calibration curve and decision curve analysis were conducted to evaluate the predictive accuracy of the nomogram. Results Age at diagnosis,T,N,M,LNR,tissue grade,gender and chemotherapy were identified as independent prognostic risk parameters of OS based on the training set in both univariate and multivariate analysis(P<0.01).Four clinical parameters including age,T stage,M stage and LNR were screened by stepwise regression analysis and a nomogram prognostic model was established based on the four factors.The average area under the curve(AUC) of ROC curves for 1-year,3-year and 5-year OS was 0.708(95%CI:0.669～0.753) in the training set and 0.704(95%CI:0.681～0.715) in the validation set.The calibration curve for probability of survival showed excellent consistency between nomogram prediction and actual observation in the entire set,with a bootstrap-corrected concordance index of 0.748(95%CI:0.741～0.755). Conclusion We successfully developed a nomogram incorporating four clinical parametersto predict overall survival of colon cancer patients after surgery resection,which showed better performance than the traditional TNM staging system.The established nomogram might help oncologist make appropriate treatment decisions for colon cancer patients.

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Objective The aim of this study was to investigate the relationship between serum interleukin-6(IL-6) and traditional inflammatory markers in children with kawasaki disease(KD), and to explore the role of IL-6 in predicting clinical classification, intravenous Immunoglobulin(IVIG) reactivity and coronary artery lesion(CAA). Methods 165 Chinese children with KD were enrolled and divided into 6 subgroups, including complete KD, incomplete KD, IVIG-responsive KD, IVIG-nonresponsive KD, coronary artery noninvolvement KD and coronary artery involvement KD. Results (1) Serum IL-6 markedly increased in the acute phase of KD, whereas declined to normal after IVIG therapy; it was positively correlated with C-reactive protein and erythrocyte sedimentation rate.(2) Serum IL-6 was significantly elevated in patients with incomplete KD when compared with their complete counterparts; The area under receiver operating characteristic curve(AUC) value was 0.596 for serum IL-6 in prediction of incomplete KD, and the estimated sensitivity and specificity were 77.80% and 54.40% respectively with a cut-off of IL-6>13.25 pg/ml.(3) Serum IL-6 was significantly elevated in patients with IVIG-nonresponsive KD when compared with their IVIG-responsive counterparts; the AUC value for serum IL-6 was 0.580 in prediction of IVIG-nonresponsive KD, and the estimated sensitivity and specificity were 60.00% and 66.30% respectively with a cut-off of IL-6>26.40 pg/ml.(4) No significant differences in IL-6 were found between KD patients. Conclusion IL-6 is prone to be a candidate biomarker for predicting incomplete and IVIG non-responsive KD rather than CAA.

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Objective To detect the expression of autoantibodies in patients with immune thrombocytopenia(ITP) by flow cytometric immuno-bead array, as well as explore its clinical significance in the diagnosis of ITP patients, and the relationship between the types of platelet specific autoantibodies and the clinical effects of drugs. Methods Flow cytometry immuno-bead array was used to detect the expression of anti platelet membrane glycoprotein Ⅰb(GPⅠb) antibody and antiplatelet membrane glycoprotein Ⅱb/Ⅲa(GPⅡb/Ⅲa) antibody. The results of 71 cases were compared with those of monoclonal antibody capture specific platelet antigen(MAIPA). Patients with ITP were halved into the groups of hormone treatment and recombinant human thrombopoietin( rh TPO) treatment,with49 and 28 cases respectively,and 4 cases from the other groups. Moreover,the patients were divided into effective group and ineffective group based on the clinical effects. Results(1) Results of flow cytometric immuno-bead array: There were comparatively 30 and 29 patients with positive anti GPⅠb antibody and anti GPⅡb/Ⅲa antibody,making the positive rates 42. 25% and 40. 85%. Upon the combined detection of the two antibodies,42 patients were finally positive,with a total positive rate of 59. 15%.(2) Results of MAIPA method: there were 19 and 22 cases of patients with the two antibodies,and the positive rates were 26. 76% and 30. 99% respectively. The combined detection of the two antibodies was 33 cases creating a total positive rate of 46. 48%.(3) The comparison between the above two methods showed no significant difference( P>0. 05).(4) The results of the 49 patients in hormone therapy whose antibody expression was detected by flow cytometry showed that there were 19 and 30 cases with positive anti GPⅠb and with negative anti GPⅠb with the effective rates 42. 11% and 73. 33%. The difference was statistically significant( P<0. 05). Conclusion Compared with MAIPA method,flow cytometric immuno-bead array is more speedy in detecting the platelet-specific autoantibody,but the sensitivity is not different. It is of great significance for the clinical diagnosis of ITP patients. In addition,the reason why the effect of glucocorticoid treatment is not obvious may be related to the positive anti GPIB antibody.

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Objective To assess the clinical effect and complications of non-coplanar nylon templates guided radioactive125I particles implantation, and these templates made by 3 D printer were applied to assist operation in the treatment of elderly lung cancer patients. Methods From January 2016 to December 2019, 80 elderly patients with advanced lung cancer admitted to our department were randomly divided into three groups.Nylon templates guided particle implantation was used in 19 cases as the template group.As traditional group(21 cases), particles were implanted by manual puncture.In these two groups, all patients were treated with chemotherapy after implantation.The third group, only underwent chemotherapy, was chemotherapy group(40 cases).Local control rate, effectiveness, dosimetric parameters, tumor marker levels and complications were compared among three groups. Results The local control rate, efficiency, and dosimetric parameters of the template group were significantly better than those of the traditional group after operation.The levels of carcinoembryonic antigen(CEA) and cytokeratin 19 fragments(CYFRA21-1) among the template group, the traditional group and chemotherapy group were statistically significant at 6 and 12 months after implantation, while the levels of Neuron Specific Enolase in the three groups were not statistically significant.The difference in the incidence of surgical complications between the template group and the traditional group was not statistically significant, and the difference in the incidence of chemotherapy complications among the three groups was not statistically significant. Conclusion The self-made 3 D printed non-coplanar nylon template guided radioactive particle implantation has good local control effect in elderly patients with lung cancer, satisfactory dose distribution, and few surgery-related complications.It is clinically satisfactory and suitable as an adjuvant treatment method for elderly lung cancer patients.

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Objective To investigate the relationship between the level of the human chorionic gonadotropin(hCG) 14 days after frozen embryo transfer and pregnancy outcomes in polycystic ovary syndrome(PCOS) and its related factors. Methods 607 PCOS patients and 1636 controls were analyzed retrospectively in our hospital.The baseline data,hCG level 14 days after frozen embryo transfer and pregnancy outcomes were compared between the two groups.The predictive value of hCG level 14 days after embryo transfer on the live birth of PCOS patients and its related factors were analyzed. Results Compared with the control group,the level of hCG 14 days after frozen embryo transfer in PCOS group decreased,and the live birth rate decreased(P<0.05).The level of hCG 14 days after frozen embryo transfer was an independent predictor of live birth in PCOS patients.The area under the ROC curve(AUC) was 0.903,95%CI=0.878-0.928(P<0.001),the cut-off value was 1 634.25 IU/L with a 74.6% sensitivity and a 88.5% specificity.BMI was negatively correlated with hCG level(P<0.001). Conclusion The level of hCG 14 days after frozen embryo transfer in PCOS patients is low,which is an independent predictor of live birth in PCOS patients and has a high predictive value.hCG level is negatively correlated with BMI.

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The mass-spectrometry-based binding assays(MS binding assays,MSBA) would be established to determine the affinity of small-molecule probes with amyloid-β(Aβ) aggregates in this study.The liquid chromatography-mass spectrometry(LC-MS)-based method and quantitative method were established for the existing positive probe(IMPY) and its deuterated product(IMPY-D6),followed by mass spectrometry-based saturation binding experiments and competitive binding experiments.Finally,the experimental data were analyzed and compared with the results of the radioligand binding assays.The LC-MS method of IMPY and MSBA were established successfully.In the saturation binding experiments,the Kdfor IMPY with Aβ 1-40 and Aβ 1-42 were(3.80±0.39) nmol/L and(18.38±1.11) nmol/L,respectively.In the competitive binding experiments,the Kifor IMPY with Aβ 1-40 and Aβ 1-42 were(18.64±0.76) nmol/L and(25.44±2.38) nmol/L,respectively.These data were consistent with the results of the radioligand binding assays.As a non-radioactive binding experiment method,the results of MSBA were consistent with the radioligand binding assays and could be used for the affinity detection of small-molecule probes to Aβ.This technique can be extended to the determination experiment of the affinity between conventional small-molecule ligands and receptors.

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To explore the effect of preoperative gum chewing on postoperative sore throat(POST) after tracheal intubation using a double-lumen endobronchial tube(DLT). Compared with control group, the incidence of postoperative sore throat and hoarseness at 1, 6 and 24 h after surgery decreased in gum chewing group. The visual analogue scale score for POST also decreased in gum chewing group. Preoperative gum chewing reduced the incidence and severity of postoperative sore throat after tracheal intubation using a DLT.