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Objective To investigate the effects of high glucose-stimulated macrophage-derived exosomes on TGF-β1/Smad3 pathway in kidney tissues of C57 mice. Methods Macrophage-derived exosomes were injected into male SPF-class C57 BL/6 mice via the tail vein. The animals were divided into three groups: high glucose-stimulated macrophage-derived exosomes( HG-Exo),mannitol-stimulated macrophages-derived exosomes( MC-Exo),and normal glucose-stimulated macrophage-derived exosomes group( NG-Exo). The urine and kidney tissues of mice were collected after 8 weeks. The urinary protein excretion rate was detected by the urinary albumin kit. The proliferation of mesangial cells and the degree of matrix expansionwas observed by HE staining and PAS staining. The infiltration of macrophages CD68 and the expression of extracellular matrix,including collagen-Ⅳ( Col-Ⅳ) and fibronectin( FN),in renal tissues were detected by immunohistochemistry. The expression of inflammatory factors TNF-α,IL-1β and extracellular matrix α-Smooth muscle actin( α-SMA),Col-Ⅳ,FN and TGF-β1/Smad3 proteins in kidney tissues were detected by Western blot. Results It showed that the proliferation of mesangium in HG-Exo group was significantly higher than that in NG-Exo group and MC-Exo group by HE and PAS staining,and the expression of CD68,Col-Ⅳ and FN in renal tissue were also higher in the HG-Exo group by immunohistochemistry stain. The expression of inflammatory factors such as TNF-α,pro-IL-1β,IL-1β and extracellular matrix including α-SMA,Col-IV,FN and TGF-β1,Smad3 and p-Smad3 in HG-Exo group were higher than those in NG-Exo group and MC-Exo group by Western blot. Conclusion High glucose-stimulated macrophage-derived exosomes can cause inflammation and extracellular matrix in mouse kidney tissue,and its mechanism may be related toactivation of TGF-β1/Smad3 signaling pathway.

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Objective To investigate the effects of Transforming growth factor-β1(TGF-β1) on rabbit endometrial glandular epithelial cells to establish an epithelial-mesenchymal transition(EMT) model of endometrial epithelial cells. Methods Endometrial epithelial cells of New Zealand white rabbits were isolated and cultured, with different concentrations(0, 10, 60, 110 μg/L) of TGF-β1induced 24 h and 48 h.EMT model was established by screening the optimal concentration and time of induction of TGF-β1.Morphological changes of cells were observed,CCK-8 was used to detect cell proliferation inhibition, and Western-blot and immunohistochemistry were used to detect changes in vimentin(VIM) and E-cadherin(E-cadherin) expression. Results 60 μg/L TGF-β1inducing cell for 24 h significantly inhibited the proliferation of rabbit endometrial epithelial cells, changed cell morphology and decreased cell density to transform cells into mesenchymal morphology. Meanwhile, it down-regulated expression of E-cadherin protein and up-regulated expression of VIM protein. Conclusion The epithelial-mesenchymal transition model of rabbit endometrial epithelial cells can be successfully established with the concentration of 60 μg/L TGF-β1inducing cells for 24 h.

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Objective To investigate the pro-apoptotic effects of hesperidin on human lung adenocarcinoma PC9 cells as well as thevariation of proteins involved in mitochondrial pathway and endoplasmic reticulum stress(ER stress). Methods LDH Cytotoxicity Assay Kit was used to monitor the cytotoxic effect of hesperidin on PC9 cells. Fluorescence-activated cell sorting(FACS) was utilized for determining the apoptosis of PC9 cells pretreated with different dosages of hesperidin. Proteins implicated in mitochondrial apoptosis and ER stress, including Caspase-3, Caspase-9, Apaf-1,Bcl-2, cytochrome C,CHOP, Caspase-4, Bip, p-JNK, p-Perk, IRE1 and ATF6 were measured by western-blotting. Results ① Cytotoxic effect of hesperidin on PC9 cells was conspicuously aggrandized with either the increase of hesperidin concentration or prolongation of incubation time.② Apoptosis, especially early apoptosis among PC9 cells were intensified by the increasing doses of hesperidin. ③ By hesperidin administration, Caspase-3, Caspase-9, Apaf-1 and cytochrome C were upregulated and Bcl-2 downregulated. ④ Hesperidin also induced the expression of ER stress-related proteins or receptors, such as CHOP, Caspase-4, Bip, p-JNK, p-Perk, IRE1 and c-ATF6. Conclusion Hesperidin exhibited cytotoxic and pro-apoptotic efficacies on PC9 cells, which may implicate ER stress.

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Objective To investigate the role of n-acetylcysteine(NAC) in lipopolysaccharide(LPS)-induced apoptosis of vascular endothelial cells, and to explore the possible mechanisms. Methods HUVECs were cultured in vitro and the apoptosis model of human umbilical vein endothelial cells was induced by LPS. Various concentrations of NAC were used to stimulate cells,and cell viability was assessed using the Cell Counting Kit-8( CCK-8) assay.Experiments were separated into four groups: control group,LPS group,NAC group,NAC and LPS group. Apoptosis rate was determined by using flow cytometry; The expression levels of Caspase-3,Bax,and Bcl-2 mRNA were determined by using real-time quantitative polymerase chain reaction( qRT-PCR); the expressions of Bcl-2,Bax,Caspase-3,p-p38 MAPK,and t-p38 MAPK proteins were determined by Western blotting. Results Compared with the control group,the cell viability of HUVECs decreased significantly,the apoptosis rate increased,downregulate the expression of Bcl-2,the expression of Bax,Caspase-3 were up-regulated,and upregulate the expression of phosphorylated p38 MAPK protein in the LPS group. However,these effects were inhibited by pretreatment with NAC.Conclusion NAC inhibits LPS-induced apoptosis of HUVECs through inhibiting the activity of p38 MAPK.

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Objective To explore the effect of β-catenin regulating NF-κB pathway on distraction osteogenesis of premaxillary suture in rats, and to study the effect of β-catenin agonist on NF-κB and related osteogenic factors. Methods 45 SD rats were randomly divided into three groups: control group, maxillary expansion group, SB group(local injection at the time of maxillary expansion), control group and maxillary expansion group, all of which were injected with DMSO once a day. The rats in each group were killed on the 1 st, 4 th and 7 th day after the expansion. The width of the premaxillary suture was measured by Micro-CT, and the histological changes were observed by HE, Masson staining. IHC was used to observe the changes of related factors. Results During the experiment, the width of premaxillary suture in SB group was significantly wider than that in control group. The expression of p65 in SB group was lower than that in maxillary expansion group, the expression of β-catenin was stronger than that in maxillary expansion group, while the IOD value of osteocalatine(OCN), bone morphogenetic protein(BMP-2) in SB group was higher than that in maxillary expansion group(P<0.05). Conclusion β-catenin can down-regulate p65, and inhibit NF-kappa B pathway, and promote BMP-2, OCN expression, and accelerate the formation and calcification of new bone in premaxillary suture.

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Objective To investigate the respective effects of glibenclamide and metformin on antenatal and postpartum glucose metabolism for gestational diabetes mellitus(GDM) mice. Methods Streptozotocin was used to stimulate C57 BL/6 J mice to develop GDM. In addition to GDM control, treatment was respectively given with long-acting insulin, glibenclamide and metformin. The first day of pregnancy was defined as gestational day(GD0), body weight and fasting blood glucose were measured every 2 days from GD0. On GD18, 8 dams in each group were euthanized to collect livers. The remaining 8 dams in each group were housed for natural delivery. After 1 week of post-partum, glucose tolerance test(GTT) and insulin tolerance test(ITT) were measured for all mice. After 5 weeks of post-partum, the GTT and ITT were measured for all mice. The mice were then euthanized to collect livers. The hepatic levels of phosphor-protein kinase B(p-AKT), phosphor-Forkhead box transcription factor O1(p-FoxO1), glucose-6-phosphatase(G6 Pase), phosphoenolpyruvate carboxykinase(PEPCK) were measured respectively. Results Glibenclamide and metformin could decrease fasting blood glucose respectively in GDM mice with antenatal and 1 week postpartum(P<0.05). Compared with the GDM control, p-AKT and p-FoxO1 levels increased(P<0.05) while G6 Pase and PEPCK reduced(P<0.05) respectively in livers from the glibenclamide and metformin intervened groups. However,after 5 weeks of post-partum,the effects of two interventions on the GDM mice disappeared. Conclusions Glibenclamide and metformin may improve the glucose tolerance and insulin resistance for GDM mice with antenatal and 1 week postpartum,but have poor effects on those with 5 weeks postpartum.

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Objective The study was carried out to investigate the respective effects of insulin, glibenclamide and metformin on antenatal and postpartum lipid metabolism for GDM mice. Methods C57 BL/6 J mice were kept under observation for one week to acclimatize the new conditions,then mating. The pregnant mice were grouped as normal control group(N),GDM control group(G),GDM treated with insulin group(GI), GDM treated with glybenclamide group(GG), GDM treated with metformin group(GM).The mild amount of streptozotocin was used to stimulate C57 BL/6 J mice to develop GDM. For animals in the treated groups, treatment was initiated from GD11 to GD18. Results Three interventions respectively in gestational diabetes mellitu(GDM) maternal mice during pregnancy, and decrease total cholesterol(TC), triglyceride(TG), and low density lipoprotein-cholesterol(LDL-C) levels(P<0.05) while the high density lipoprotein-cholesterol(HDL-C) levels were increased significantly(P<0.05). Specifically, p-ACC levels were increased(P<0.05), while sterol regulatory element binding protein 2(SREBP2) were still reduced(P<0.05) respectively in murine livers from all the intervened groups compared with the GDM control. Conclusions Respective interventions of insulin, glibenclamide and metformin may significantly reduce blood lipid levels for maternal GDM mice during pregnancy.

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Objective To investigate the effect of TET1 gene on the proliferation and migration of HeLa cells. Methods The overexpression plasmid of TET1 was constructed by transcription activator-like effectors(TALE)-vp64 system and transfected into cervical cancer HeLa cells. The proliferation of HeLa cells was monitored by thiazolyl blue tetrazolium bromide(MTT), the migration ability of HeLa cells was detected by scratch test, and the invasion ability of HeLa cells was detected by Transwell. Results MTT assay showed that the proliferation ability of TET1 overexpressed cells was significantly lower than that of wild-types(P<0.05).The numbers of HeLa cells passed through the matrigel were(Clone 1, 21±5) and(Clone 2,22±6) in TET1 overexpresed cells, and(38±7) in wild-type HeLa group,the difference between the two group was statistically significant(P<0.05). The migration rates of HeLa cell in TET1 overexpressed(Clone 1, Clone 2) and the wild-type group at 24 h and 48 h were detected by scratch test, the latter was significantly higher than the former(P<0.01).TET1 overexpression could restrain the ability of proliferation, invasion and migration of HeLa cells. Conclusion Overexpression of TET1 gene can inhibit the biological behavior of cervical cancer cells.

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Objective To explore the effects of phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt) signaling pathway on the expression of receptor for activated C kinase 1(RACK1) and ras-related C3 botulinum toxin substrate 1(rac1) in rat pulmonary microvascular endothelial cells(PMVEC) induced by lipopolysaccharide(LPS). Methods Cultured rat PMVEC in vitro were divided into different groups of LPS dose-dependent group, LPS time-dependent group, IGF-1 time-dependent group and LPS+LY294002 group. ① For LPS dose-dependent group, PMVEC were cultured with 0, 1, 5, 10 mg/L LPS for 12 h. ② For LPS time-dependent group, PMVEC were cultured with 10 mg/L LPS for 0, 3, 6, 8, 12, 24 h. ③ For IGF-1 time-dependent group, PMVEC were cultured with IGF-1 for 0, 3, 6, 8, 12, 24 h. ④ For LPS+LY294002 group, PMVEC were cultured with 100 ng/ml LY294002 for 1 h before the treatment of 10 mg/L LPS for an additional 12 h. In addition, blank, LPS and LY294002 groups were set as references. After intervention, the levels of RACK1, rac1 and p-Akt were detected with Western blot. Results ① In LPS dose-dependent group, the relative expression levels of RACK1, rac1 and p-Akt increased in a dose-dependent manner, and there were significant differences among the groups of RACK1(F=120.455,P<0.001), among the groups of rac1(F=165.813,P<0.001)and among the groups of p-Akt(F=309.346,P<0.05), respectively. ② In LPS time-dependent group, the relative expression levels of RACK1 and rac1 increased in a time-dependent manner, and there were significant differences among the groups of RACK1(F=454.034,P<0.001) and among the groups of rac1(F=423.630,P<0.001), respectively. The relative expression level of p-Akt raised at 3 h(0.460±0.089), peaked at 12 h(2.022±0.244), and it was still higher at 24 h(1.264±0.074) than 0 h(0.237±0.063). There were significant differences(F=137.726,P<0.001) among the groups of p-Akt expression. ③In IGF-1 time-dependent group, the relative expression levels of RACK1, rac1 and p-Akt increased in a time-dependent manner, and there were significant differences among the groups of RACK1(F=188.293,P<0.001), rac1(F=115.071,P<0.001) and p-Akt(F=60.175,P<0.001). ④ In LPS+ LY294002 intervention group, the expression of RACK1 was lower than that of the LPS group [(0.732±0.137)vs(1.498±0.167),P<0.001], the expression of rac1 was lower than that of the LPS group [(0.758±0.084)vs(1.384±0.170),P<0.001], the expression of p-Akt was lower than that of the LPS group [(0.492±0.148)vs(1.106±0.219),P<0.001]. Conclusions PI3 K/Akt signaling pathway participates in LPS-induced PMVEC injury by interfering with RACK1 and rac1 expression.

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Objective To construct a lentiviral vector of long non-coding RNA(lncRNA) BC002811 and investigate the effect of stable BC002811 up-regulation on proliferation of gastric cancer HGC-27 cells. Methods BC002811 gene sequence was amplified by PCR, and was ligated with the pLVX-EGFP-IRES-neo vector. After being identified by double enzyme digestion and sequencing analysis, the recombinant vector was co-transfected into HEK293 T cell line with packaging plasmids to produce the lentiviral particles. Lentivirus was used to infect HGC-27 cells, and the cells with stably expressing BC002811 after selection with limiting dilution analysis were amplified and cultured. The expression level of BC002811 was detected by qPCR, and the cell proliferation capacity was determined by MTS. Results The BC002811 recombinant lentivirus plasmid was successfully constructed, which was confirmed by double enzyme digestion and DNA sequencing. The recombinant lentiviral vector was packaged in HEK293 T cells and the titer of concentrated virus was 2.2×1011TU/L. HGC-27 cells infected with the lentivirus, the expression level of BC002811 was enhanced significantly(P<0.05). MTS assay results showed that the proliferation ability of HGC-27 cells in the BC002811 group was increased as compared with the control group(P<0.05). Conclusion The BC002811 recombinant lentiviral vector was successfully constructed, which could stably transfect HGC-27 cells to yield sustained over-expression of BC002811 and promoted cell proliferation.

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Objective To investigate the role of microfilament sketeton in NCI-H1299 cell apoptosis induced by Newcastle Disease Virus( NDV) and the mechanism related. Methods Morphological observation revealed the changes in cell surface structure and ultrastructure after virus infection; CCK-8 assay was employed to evaluate the inhibitory effect of NDV infection on human NCI-H1299 cancer cells. The apoptosis rate of infected NCI-H1299 cells was detected by flow cytometry; The expression levels of RhoA,ROCK2,F-actin and P-MYPT1 proteins,which were closely related to the microfilament sketeton,were evaluated by western blot analysis after NDV infection for 0,24,36,48 h; Scratch assay and cell migration assay were used to observe the effects of virus infection on migration ability. Results Common inverted phase contrast microscope and scanning electron microscope images showed that the cells changed their normal morphology and structure to fusion and dead shapes after NDV F3 treatment. The microvilli on the cell surface became short,swollen or even shedded. CCK-8 assay results showed that NDV inhibited the proliferation of NCI-H1299 cells in a time-and dose-dependent manner( P<0. 05). Flow cytometry results demonstrated that apoptosis rate increased with NDV F3 infection time within 24 h( P<0. 01).Western blot results showed that the expression levels of RhoA、ROCK2、F-actin and p-MYPT1 proteins have decreased with NDV infection time( P<0. 05). Scratch assay and cell migration assay showed the healing rate of NDV-infected group was slower than that of control group and the infected cell'migration ability was inhibited significantly( P<0. 01). Conclution NDV infection inhibits the proliferation and invasion of NCI-H1299 cells via apoptotic mode. The apoptosis mechanism may be related to RhoA/ROCK pathway which is closely involved in the destruction of microfilament sketeton.

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Objective To observe the expression of autophagy-related microRNAs in renal tubular epithelial exosomes under hypoxia. Methods Tubular epithelial cells were cultured in vitro and treated with normoxia and hypoxia respectively. ExoQuick-TC was used to extract the exosomes. The characteristics of the exosomes were quantitatively identified by transmission electron microscopy and protein concentration. RT-PCR was used to detect autophagy-related microRNA30 b, microRNA30 c, microRNA214 and miRNA-183,miRNA-200 a expression in the exosomes of tubular epithelial cells. Results Under hypoxic condition, the secretion of renal tubular cells in vitro did not change significantly, and the secretion of exosome/cell protein content significantly increased after 48 hours of hypoxia than that in normoxia group. With the prolongation of hypoxia time, the expression of autophagy-related exosome gradually increased. The levels of autophagy-related miRNA-30 b, miRNA-30 c, miRNA-214, miRNA-183 and miRNA-200 a in the hypoxia group were higher than those in the normoxic group, and gradually increased with the prolongation of hypoxia. Conclusion Hypoxia can alter the biological function of renal tubular cell secretory exosome, which is manifested by up-regulation of autophagy-related microRNA expression. This may be one of the reasons for the important organ autophagy in patients with chronic kidney disease.

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Objective To analyze the DNA methylation spectrum of mouse islet β cell line(MIN6) using MassARRAY platform, and to explore the changes of methylation model by treating MIN6 cells with DNA demethylation drug 5-aza-2′-deoxycytidine(5-Aza-CdR). Methods The methylation status of miR-375 gene DNA in MIN6 cell line and mouse embryonic fibroblast cell line(NIH3 T3) were detected by MassARRAY platform. The changes of miR-375 gene methylation status were evaluated after treatment with different concentrations of 5-Aza-CdR. The effect of 5-Aza-CdR on the growth and proliferation of MIN6 cells was detected by Thiazolyl blue tetrazolium bromide(MTT). The expression of miR-375 in MIN6 cells and untreated NIH3 T3 cells was detected by qRT-PCR. Results 5-Aza-CdR could inhibit the growth of MIN6 cells. After treated with 5-Aza-CdR for 24,48,72 h, the OD value of 2 μmol/L concentration group was different from that of other groups(P<0.05), and the difference was statistically significant at different time points(P<0.05). After MIN6 cells were treated with different concentrations of 5-Aza-CdR for 24,48,72 h, the DNA of mmu-miR-375 in each group was highly methylated, and the average methylation rate of miR-375 DNA in 50 μmol/L group was lower than that in other groups. The expression of mmu-miR-375 in MIN6 cells treated with 5-Aza-CdR in 50 μmol/L group was significantly higher than that in 0 μmol/L group after 24,48,72 h(P<0.05). The DNA expression of mmu-miR-375 gene in MIN6 cells was significantly lower than that in NIH3 T3 cells(P<0.05). Conclusion These results indicate that promoter hypermethylation of the mmu-miR-375 is a common event in MIN6 cells.

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Objective To observe the effect of amiodarone on L-type calcium channel current in rats single atrial myocytes damaged by H2O2and investigate the effect of amiodarone on electrophysiological characteristics of atrial myocytes. Methods The myocytes were isolated from rats atrial tissues by Langendorff perfusion apparatus,which were randomly divided into three groups: the control group(no treatment),the H2O2group(cells cultured with 100 μmol/L H2O2for 0.5 hour),the amiodarone group(cells cultured with 100 μmol/L H2O2for 0.5 hour and then cultured with amiodarone for 0.5 hour). Whole cell patch clamp technique was used to record ICaL. Results Control group the peak current of ICaLdensity was(-4.99±0.35) pA/pF; H2O2group increased the peak current of ICaLdensity to(-6.97±0.60) pA/pF; Amiodarone group reduced the peak current of ICaLdensity to(-3.16±0.20) pA/pF. Conclusion Amiodarone can reduce ICaLby inhibiting channel activation and recovery after inactivation.

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Objective To investigate the effect of resveratrol(Res) on the formation of renal calcium oxalate stone in rats and its relationship with Keap1-Nrf2/HO-1 signaling pathway. Methods Sixty male SD rats were randomly divided into five groups including control group, model group, Res low dose group(L-Res group), Res high dose group(H-Res group) and Res high dose combined with Nrf2 inhibitor ML385 group(H-Res+ML385), with 12 rats in each group. Except for control group, the other groups were given 1% ethylene glycol+2% ammonium chloride to establish kidney stones model for 4 weeks. Meanwhile, the rats were administered with resveratrol or combined with ML385 for 4 weeks. At the end of the experiment, the 24 h urine volume, urine pH, urine Ca2+concentration, uric acid(Ox) content and the serum of urinary nitrogen(BUN), creatinine(Cr) and Ca2+contents were measured in each group. The kidney tissues in each group were obtained and the content of malondialdehyde(MDA) content, activity of superoxide dismutase(SOD) and levels of reactive oxygen species(ROS) in kidney tissues were determined. The formation of calcium oxalate crystals in rat kidney tissue were observed using Von Kossa staining. The proteins expression of nucleus protein Nrf2 and whole proteins Keap1, HO-1 in kidney tissue of rats were detected by Western blot analysis. Results Resveratrol inhibited the formation of calcium oxalate crystals in the kidney tissue of rats, improved the indexes of urine and blood, reduced the levels of ROS and the content of MDA in kidney tissues, increased the activity of SOD, and up-regulated the proteins expression levels of nucleus protein Nrf2 and whole proteins Keap1, HO-1 in kidney tissues. However, combination treatment with Nrf2 inhibitor ML385 significantly inhibit the effects of resveratrol. Conclusion Resveratrol may be inhibit the oxidative damage of kidney tissue by activating Keap1-Nrf2/HO-1 signaling pathway, thereby inhibiting the formation of calcium oxalate crystals in rat kidney tissue, and has the effect of preventing kidney stones.

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Objective To investigate the effect of miR-25-3 p targeting NOTCH1 gene on invasion, migration and proliferation of esophageal squamous carcinoma cells, and to elucidate the mechanism of miR-25-3 p targeting the regulation of NOTCH1 gene in esophageal squamous cell carcinoma. Methods ECA109 cells were uniformly inoculated into culture dishes at 1 × 106/ml,and each group was given 3 replicate wells. The experiment was divided into two groups. Group 1: miR-25-3 p up-regulated( transfected with miR-25-3 p mimics),miR-25-3 p down-regulated( transfected with miR-25-3 p inhibitor) and NC group; Group 2: NOTCH1 up-regulated( transfected NOTCH1 mimics),NOTCH1 down-regulated( transfected with NOTCH1 inhibitor),and NC group. Real-time quantitative PCR was used to detect the expression levels of miR-25-3 p and NOTCH1 in each group,transwell chamber was used to detect cell invasion ability,CCK-8 was used to detect cell proliferation,bioinformatics analysis and luciferase activity assay were used to determine whether NOTCH1 was Target gene of miR-25-3 p. Results NOTCH1 is the target gene of miR-25-3 p. Results revealed that cell invasion and proliferation increased with the overexpression of miR-25-3 p by miRNA mimics and decreased with the suppression of NOTCH1. Conclusion miR-25-3 p promotes the invasion and proliferation of human ESCC cells by targeting NOTCH1,and miR-25-3 p is expected to become a new target for ESCC treatment.

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Objective To predict and analyze the mechanism of downregulation of HNF4α expression in hepatocellular carcinoma(HCC) by bioinformatics analysis. Methods The promoter sequence of HNF4α gene obtained from NCBI GenBank was analyzed by transcription factor prediction software, including PromotorScan, Patch, P-Match and AliBaba2. Meanwhile, the down-regulated genes in human HCC were screened by Liveratlas. Then the intersected genes were analyzed based on the data extracted from TCGA database. Results The promoter of the corresponding transcript of human HNF4α gene in HCC is about 1 471 bp. After removing duplication, the pool of predicting transcription factor binding sites covers 225 transcription factors by using online software. A total of 2 538 down-regulated genes in HCC were extracted from liveratlas online gene database, and 17 transcription factors were obtained by compared with the transcription factor pool predicted above. The correlation analysis indicated that HLF(r=0.553 4), RREB1(r=0.407 9) and RXRA(r=0.424 7) were positively correlated with the expression of HNF4α. Conclusion Bioinformatics analysis indicates that HLF, RREB1 and RXRA may be involved in the transcriptional regulation of HNF4α expression in liver. The faint expression of one or more of them may be related to the down-regulation of HNF4α expression in the development of HCC.

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Objective To investigate novel antimicrobial agents targeting FtsZ. Methods Identification of potential molecules targeting FtsZ by structural-based virtual screening using Discovery Studio 2017 software in a variety of ways. Determination of antibacterial effect againstS.aureusof small molecule by broth dilution method. Molecular dynamics simulation was used to evaluate the binding stability of small molecule to FtsZ protein. Results Fiftymolecules with potential drug ability and excellent binding ability were obtained by virtual screening. All compounds exhibit certain antimicrobial effects on MSSA and MRSA. Among them, compound 5010-0572 showed best activity against MSSA and MRSA, with MIC value of 1 μg/ml. Molecular dynamics simulation illustrated that compound 5010-0572 with best antimicrobial effects bound to FtsZ better than compound C592-0444 with lowest antimicrobial effects. Conclusion The antimicrobial effects of small molecules targeting FtsZ is closely related to the binding stability with protein. Compound 5010-0572, which has notable antimicrobial activity, may become a leading compound for the development of new antimicrobial agent.

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Objective To investigate the procoagulant properties of red blood cell microparticles(RMPs) and the relationship between this property and acute lung injury in mice. Methods RMPs were isolated, purified and counted,and then the contents of the activated partial prothrombin time(APTT), prothrombin time(PT) and thrombin generation test were detected in the plasma of red cell supernatant under the circumstances before and after the removal of the microparticles and different concentrations of RMPs. In animal experiments,the levels of thrombin-antithrombin complex, plasminogen and plasminogen activator inhibitor-1 in mouse alveolar lavage fluid were measured by ELISA.Histological observation of right lung tissue in the experimental group injected with 3×107RMPs and the control group was conducted. Results The number of RMPs increased gradually with the prolongation of storage time.As the concentration of RMPs added increased, the APTT value of the degranulated plasma decreased slightly, while the PT value did not change significantly.When erythrocyte supernatant and RMPs with a certain range of concentrations were added to the plasma, the lag time and tt-peak could be shortened to some extent, while the peak and ETP were increased significantly with a concentration dependent manner. Compared with the control group, the TATc of BALF was significantly increased in the experimental group injected with 3×107RMPs, and the alveolar tissue inflammation was intensified and the coagulation promoting mechanism was enhanced. Conclusion As the storage time increases, the number of RMPs increases significantly. RMPs can promote thrombin generation and thereby promote systemic coagulation, which may be related to the transfusion-related acute lung injury(TRALI).

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Objective To investigate the effect of homocysteine(Hcy) on the permeability of Caco-2 cells and its mechanism. Methods Caco-2 cells were divided into blank control group,treated with different concentrations of Hcy(10, 20, 50 μmol/L), treated with Hcy + different signaling transduction inhibitors(ERK inhibitor PD98059, MLCK inhibitor ML-7, P38 MAPK inhibitor SB203580, Ca2+/Calmodulin inhibitor KN62, Rho inhibitor Y27632 and PKC inhibitor Staurosporine). The permeability of Caco-2 cells was detected by transepithelial electrical resistance(TEER) and fluorescein isothiocyanate(FITC). The expression levels of MEK, ERK, MLCK, p-ERK, p-MLCK, ZO-1, Occludin, Claudin-1 and Claudin-2 in Caco-2 cells were detected by Western blot. Results Compared with the blank control group, the permeability of Caco-2 cells pretreated with 50 μmol/L Hcy had the faster change. The expression of Claudin-1, Occludin and ZO-1 decreased, while the expression of MEK, ERK, MLCK, p-ERK, p-MLCK and Claudin-2 increased. Compared with Hcy group, pretreatment with ML-7 and PD98059 could reduce the permeability alteration of Caco-2 cells induced by Hcy, which resulted in decreased expression of MEK, ERK, MLCK, p-ERK, p-MLCK, Claudin-2 while increased expression of Claudin-1, Occludin and ZO-1. Conclusion Hcy may regulate the expression of tight junction-related proteins through MEK-ERK-MLCK signaling pathway and affect the permeability of Caco-2 cells.

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Objective To observe the effect of mesencephalic astrocyte-derived neurotrophic factor(MANF) on experimental autoimmune uveitis(EAU) in rats. Methods Lewis rats were immunized with interphotoreceptor retinoid-binding peptide(IRBP) in order to generate EAU model. The experimental group was administered MANF on the 3 rd, 7 th and 11 th day after immunization, and the control group was administered phosphate buffered saline(PBS)as a positive control.The normal group was not treated. Caspi method was used to score the degree of inflammation. The rats were killed during the peak period of inflammation, and the retinal inflammatory reaction and histopathological changes of the three groups were detected by hematoxylin-eosin staining.The expressions of inter-leukin-17( IL-17) and interferon-gamma( INF-γ) were detected by real-time fluorescence quantitative PCR( RTqPCR). Results The inflammation began on the 3 rd day after immunization,and reached the peak on the 14 th day. The pathological and clinical manifestations on the 14 th day after immunization suggested that the ocular inflammation and pathological changes of the experimental group were significantly better than those of the control group. The expression levels of inflammatory factors IL-17 and INF-γ in the experimental group were significantly lower than those of the control group. Conclusion MANF vitreous cavity administration can reduce the inflammation of EAU in rats,and the decrease of the expressions of IL-17 and INF-γ may be a part of the effect,and the specific mechanisms need to be further studied.

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Objective To study the influences of intercellular cell adhesion molecule(ICAM)-1 and interleukin(IL)-17 expressions in myocardial tissues after preservation and transplantation of rabbit heart allograft with three different perfusion methods of HTK solution(simple low temperature immersion, continuous or intermittent 2 h perfusion), and explore the mechanism of HTK infusion types on acute rejection after heart transplantation. Method 18 pairs healthy 2-month-old New Zealand rabbits(mean weight 2.15±0.33 kg, male and female in half) were chosed and divided randomly into three groups: simple low temperature immersion group(simple group,n=5 pairs), continuous group(n=5 pairs) and intermittent 2 h perfusion group(intermittent group,n=8 pairs). Donor hearts were isolated with perfusing HTK solution through aortic root to arrest and protect the heart, and then preserved for 8 h at 4 ℃. According to modified Ono procedure, heart transplantation model was established and donor heart was removed. Then pathological changes with HE staining was observed, quantitative expressions of ICAM-1 and IL-17 were detected by RT-PCR and Western blot, and apoptotic rate was detected by TUNEL. Results HE staining showed that most myocardial tissues in the three groups were intact, accompanied by cell edema, myxoid degeneration or necrosis, inflammatory cell infiltration, adipogenesis and angiogenesis. Semi-quantitative analysis showed that the number of cell edema, inflammatory cells and small vessels in the intermittent group was significantly less than that in the continuous group, which was the most in the simple group(P<0.05). Quantitative detection showed that ICAM-1 and IL-17 mRNAs and protein were significantly lower in the intermittent group than those in the continuous group, which was the highest in the simple group(P<0.05). What's more, the apoptotic rate in the intermittent group was more less than that in the continuous group, and that in the simple group was the most(P<0.05). Conclusion Different perfusion methods of HTK solution have different effects on acute rejection after allograft heart transplantation in rabbits, especially it can significantly reduce ICAM-1 and IL-17 expressions in recipient myocardium and inhibit apoptotic effect with intermittent 2 h perfusion.

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Objective To investigate the expression changes of transient receptor potential melastatin 7(TRPM7) during liver regeneration after partial hepatectomy(PHx) in mice and the effect on proliferation of hepatocytes(L-02). Methods Using 70% PHx liver regeneration model in mice, hematoxylin-eosin(HE) staining was used to observe liver pathology,ELASA was used to detect alanine aminotransferase( ALT) and aspartate aminotransferase( AST) protein levels at different times( 0 h,12 h,24 h,36 h,48 h,72 h,5 d,7 d) after surgery. Western blot analysis was used to detect the expression of TRPM7 and PCNA in liver tissues at different times after operation. L-02 cells proliferation was induced by recombinant human interleukin 6( IL-6),and the expression of TRPM7 was detected by Western blot method. TRPM7 channel was blocked by 2-Aminoethoxydiphenyl borate( 2-APB),L-02 cells viability was detected by MTT assay,and protein expressions of proliferating cell nuclear antigen( PCNA),phosphorylated signal transducers and activators of transcription 3( p-STAT3) were detected by Western blot method. Results After PHx,it was observed that tissue hyperplasia was obvious,the cell volume became larger,the proliferating hepatocyte cord was arranged with different degrees of disorder while inflammatory cell infiltration was accompanied. ALT and AST protein levels increased significantly after PHx and peaked at 12 h,and then gradually decreased to normal levels. The protein level of TRPM7 in mouse liver tissue increased significantly after PHx and lasted for 72 h( P<0. 05),which was correlated with changes in cell proliferation protein PCNA expression levels.IL-6 induced a significant increase in L-02 cell viability and TRPM7 protein levels. IL-6-induced increase of hepatocyte viability was inhibited by 2-APB in a concentration-dependent manner,and PCNA and P-STAT3 levels were also decreased after blocking TRPM7 channel. Conclusion TRPM7 increased expression during liver regeneration and hepatocyte proliferation,and inhibition of TRPM7 channel by 2-APB inhibited hepatocyte proliferation through reducing STAT3 phosphorylation,indicating that TRPM7 plays an important role in hepatocyte proliferation.

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Objective To investigate the association between single nucleotide polymorphisms(SNP) of the TLR5 gene and the risk of non-cardia gastric cancer, and to supply theoretical and experimental basis for early diagnosis and treatment of non-cardia gastric cancer. Methods Using case-control study design, 288 non-cardia gastric cancer patients diagnosed by histopathology were selected as the case group while 281 community physical examinees were selected as the normal control group, and they were all Han population in Baotou. SNP rs34270714, rs5744174, rs851139 and rs17163737 were genotyped using TaqMan method. Haplotype was constructed by Haploview software 4.0. Odds ratios(OR) and 95% confidence intervals(CI) were measured by unconditional Logistic regression analysis to evaluate the correlations of the alleles, genotypes and haplotypes to the susceptibility to non-cardia gastric cancer. Results In Baotou Han population, the SNP rs17163737 of TLR5 was associated with the risk of non-cardia gastric cancer. Compared to GG genotype, the GT genotype was associated with a significantly increased risk of non-cardia gastric cancer(GTvsGG:OR=1.605, 95%CI=1.131～2.280). Other SNPs were not associated with the risk of non-cardia gastric cancer(P>0.05). In addition, there was a strong linkage disequilibrium between rs5744174, rs851139 and rs17163737, which constituted a haplotype region(Block). However, there was no association between any haplotypes and the risk of non-cardia gastric cancer. Conclusion The carriers of TLR5 rs17163737 GT genotype have an increased risk of non-cardia gastric cancer in Baotou Han population.

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Objective To investigate the expressions and clinicopathological relationships of Trophinin associated protein( TROAP) in lung adenocarcinoma. Methods The differential expression of TROAP and its impact on patients prognosis were analyzed by exploring multiple public databases. Immunohistochemical staining was used to detect the histological score( H-Score) of TROAP in 30 patients with lung adenocarcinoma. The differences in expression and clinicopathological features( sex,age,TNM stage,Ki-67 level,smoking history,pleural effusion and Epidermal Growth Factor Receptor( EGFR) mutation status) were analyzed. Results Database analysis suggested that this gene was highly expressed in various solid tumors such as lung cancer. The survival of lung adenocarcinoma patients with high expression of TROAP gene was significantly shortened. Immunohistochemical staining suggested that the expression of this gene in tumor tissues of lung adenocarcinoma patients[H-Score( Histological Score) = 150. 00( 100. 00,180. 00) ]was significantly higher than that in adjacent normal lung tissues [H-Score= 80. 00( 58. 75,125. 00) ]. This difference had statistical significance( P = 2. 69 × 10-5). In addition,the expression of TROAP gene expression was significantly different in disease stages( P = 0. 039),whether there was distant metastasis( P = 0. 031) and different Ki-67 expressions( P = 0. 006). Conclusion TROAP may play an important role in the tumorigenesis,development,invasion and metastasis of lung adenocarcinoma. It could be used as a potential biomarker for pathogenesis,treatment and prognosis.

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Objective Hypoxia-inducible factor 2 A(HIF2 A) and vascular endothelial growth factor A(VEGFA) are important genes in the hypoxia-inducible pathway, the association of rs13419896 in HIF2 A and rs833061 in VEGFA with lung cancer recently becomes the focus. Our study is to demonstrate genetic association rs13419896 and rs833061 with lung cancer in Chinese population. Methods A case-control study was performed between 438 patients with lung cancer and 456 healthy controls. The collected samples were genotyped by Taqman Genotyping method, and the association and clinical characteristics were analyzed. Results The rs13419896 A allele decreased the risk of lung cancer [OR=0.72, 95%CI(0.58～0.88),P=0.001 1], comparing to rs13419896 G allele, and further analysis found that rs13419896-AA significantly decreased risk of lung cancer in the additive model[OR=0.74, 95%CI(0.61～0.90),P=0.002]. After adjustment the effects of smoke, alcohol and age, rs13419896 genotypes still significantly decreased the risk of lung cancer[OR=0.75, 95%CI(0.60～0.93),P=0.009]. However, the rs13419896 genotypes had no significant relationship with pathological types and clinical stages in lung cancer, and there was no association between lung cancer and rs833061 in VEGFA. Conclusion The results show that polymorphism of rs13419896 in HIF2 A is associated with lung cancer in Hainan area of China. It can be used as a potential genetic marker for lung cancer after validation by multi-center and large sample studies.

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Objective To investigate the relationship among hepatic fat content(HFC), serum cystatin C(CysC) and insulin resistance in different glucose tolerance populations. Methods 242 patients were recruited from department of endocrinology, and they were divided into three groups: 141 patients with newly diagnosed T2 DM(NT2 DM), 48 pre-diabetes subjects(PDM) and 53 normal control(NC) with normal glucose tolerance. HFC was measured by Hydrogen proton magnetic resonance spectroscopy(1H-MRS), and the association among HFC, CysC and insulin resistance was analyzed. Results ① The levels of HFC and HOMA-IR orderly increased in NC, PDM and NT2 DM groups(all of P values were less than 0.012 5). Compared with NC group, both of PDM and NT2 DM groups had an elevated CysC level(all of P values were less than 0.001). ② All subjects were divided into four subgroups of Q1, Q2, Q3 and Q4 from low to high according to HFC quartile distribution. There were significant differences of CysC and HOMA-IR among the four subgroups(bothof P values were less than 0.05). Compared with Q1 group, the level of CysC and HOMA-IR were significantly increased in both Q3 and Q4 groups(all of P values were less than 0.008 3). The HOMA-IR in the Q4 group was significantly higher than that in the Q2 group(P<0.008 3). ③Mutilinear regression indicated that CysC was the predictor for HFC whileCysC and HFC were the predictors for the HOMA-IR(both of P values were less than 0.05). Conclusion There are an increase of CysC level in the stage of PDM and HFC(>11.62%), indicating that CysC may be an indicator for the prediction of HFC and HOMA-IR.

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Objective To investigatethe correlation between the entrance morphology of the bicipital groove morphology and biceps tendinitis and to analyze the related factors. Methods A total of 80 cases of people with biceps tendinitis and 80 cases of healthy subjectswere included in this study. The the AW4.6 workstation of PACS system and the Functool software were used to measure the morphological index of cases on axial MRI pictures. Results There was significant difference between opening angle(OA) and the ratio of the depth and width of bicipital groove(R1)(P<0.05) in two groups.However, there was no significant difference(P>0.05) was found on the width of bicipital groove(BGW), depth of bicipital groove(BGD), diameter of biceps tendon(BTD), diameter of humeral head(HHD), medial wall angle(MWA), and the ratio of the depth of bicipital groove and humeral head medial diameter(R2). There was a statistically significant difference in the classification of the entrance between the two groups., while there was no statistical difference between the normal shape of the entrance.In the analysis of related factors,there was a statistical difference in tendon diameter between degree 1 and degree 2 of the biceps tendon.However,there was no statistical difference in the MRI measurements of the intertubercular sulcus according to the classification of the complications of biceps tendon injury and the degree of injury. Conclusion There are a bigger OA and a smaller R1 in people with biceps tendinitis. A shallow groove is a predisposing factor for biceps tendinitis,and the greater the degree of injury of biceps tendon, the smaller the diameter of tendon.

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Objective To investigate the expression of T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains( TIGIT) on peripheral natural killer( NK) cells from patients with rheumatoid arthritis( RA) and its significance and to clarify its relation in the development of RA. Methods The peripheral blood mononuclear cell( PBMC) from 33 patients and 27 healthy controls( HCs) were collected and TIGIT expression in the NK cells were detected by flow cytometry. The correlations of TIGIT expression on NK cells with clinical data were analyzed. Student't test or Mann-Whitney U test was used for comparison between the two groups,and the correlation between the two variables was analyzed by Pearson or Spearman correlation. Results(1) The percentage of NK cells and NK cells counts were significantly decreased in RA patients compared to the HC( P = 0. 005 1; P<0. 000 1);(2) The expression mean fluorescence intensity( MFI) of TIGIT on NK cells was significantly decreased in RA patients compared to the HC( P<0. 000 1);(3) The expression MFI of TIGIT on NK cells in RA patients was found to negatively correlate with CRP( r =-0. 346 3,P = 0. 048 4);(4) MFI of TIGIT expression in NK cells of RA patients is negatively correlated with rheumatoid factor( RF) and positive RF( r =-0. 360 7,P =0. 042 6; r =-0. 400 5,P = 0. 047 3);(5) The expression MFI of TIGIT on NK cells was significantly lower early RA patients than in non-early RA patients( P = 0. 038 5). Conclusion The expression MFI of TIGIT on NK cells is decreased and associated with the inflammation markers,the production of antibodies in RA.

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Objective To investigate the relationship between Toll-like receptor 9(TLR9) gene polymorphisms and Helicobacter pylori(H.pylori) infection. Methods A total of 683 blood samples were collected. The samples were divided intoH.pylorinegative group(n=305) andH.pyloripositive group(n=378) by enzyme-linked immunosorbent assay(ELISA) method. Results TLR9 rs187084, rs352140 and rs164640 were genotyped by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). TLR9 rs187084 TC genotype reduced the risk ofH.pyloriinfection(TCvs. TT:OR=0.520, 95%CI=0.329～0.820). The risk ofH.pyloriinfection increased in rs164640 AG+GG genotype carriers(AG+GGvs. AA:OR=1.569, 95%CI=1.126～2.187). rs352140 was not associated with the risk ofH.pyloriinfection. Conclusion TLR9 rs187084 and rs164640 might play a role inH.pyloriinfection.