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Objective To investigate the effect of mtDNA on monolayer permeability of Caco-2 cells and its mechanism. Methods The Caco-2 cell model was prepared by using Transwell culture plate. The mtDNA was extracted from the liver of C57 BL/6 J mice. Then, different concentrations of mtDNA(0, 1, 5, 10 μg/ml) and FITC-dex-tran were added to the top of the Transwell culture plate to detect the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate at different time,which determined the best mtD NA concentration. Then,the experiment was randomized into four groups: FITC-dextran,mtD NA + FITC-dextran,TLR9 antagonists( ODN TTAGGG) + FITC-dextra,and mtD NA + ODN TTAGGG + FITC-dextran,and the changes of TEER and FITC-dextran content in the bottom chamber of the Transwell culture plate were detected at different time.Meanwhile,Caco-2 cells were pretreated with mtD NA,ODN TTAGGG,mtD NA + ODN TTAGGG,respectively.The expression of TLR9 mR NA was detected by q RT-PCR,and the levels of TNF-lα,IL-6,IL-1β and IL-18 in the supernatant were detected by ELISA. Results Compared with the control group,with the prolongation of the action time and the increase of the concentration of mtD NA,the TEER of the monolayer of Caco-2 cells decreased and the content of FITC-dextran in the bottom chamber increased,and the TEER decreased significantly when the concentration of mtD NA was 10 μg/ml. Compared with 10 μg/ml mtD NA group,the addition of ODN TTAGGG significantly reduced the falling speed of TEER,and reduced the content of FITC-dextran in the bottom chamber,meanwile,the expression of TLR9 mR NA and the levels of inflammatory factors also declined. Conclusion The mtD NA significantly increases the permeability of Caco-2 monolayer,which may be related to the activation of TLR9 signal pathway.

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Objective To prepare liposomes of egg yolk antibodies againstPorphyromonas gingivalis(anti-Pg-IgY liposome), and to study the antibacterial effect of these anti-Pg-IgY liposomes onPorphyromonas gingivalis, a major periodontal pathogen. Methods Anti-Pg-IgY liposomes were preparedby thin film dispersion. Use transmission electron microscope to observe the appearance and characterization of anti-Pg-IgY liposomes.Zeta potential(ζ-potential) and particle size of anti-Pg-IgY liposomes was determined by ultrafiltration centrifugation method. The micro-liquid dilution method was used to study the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) of anti-Pg-IgY liposomes against Porphyromonas gingivalis and draw its kill-time curve. Results The average particle size of the liposomes was(175.43±3.25) nm(n=4), while the polydispersity index was 0.263(n=4), and the zeta potential was(-37.82±0.20) mV(n=4). The encapsulation efficiency was 42.71%(n=4), and the cumulative release rate was 83.4%. The MIC and MBC values of the Pg-Igy liposomes against porphyromonas gingivalis were 4 mg/ml and 8 mg/ml, respectively. When the anti-Pg-IgY liposome reached the MBC value of bacteria, it could completely inhibit the growth and reproduction of bacteria in the culture medium. Conclusion The anti-Pg-IgY liposomes exhibit excellent antibacterial activity, and can kill the bacteria when the minimum germicidal concentration is reached.

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Objective To explore osteogenic properties of strontium doped TiO2nanotube implants. Methods TiO2nanotubes were prepared on the surface of pure titanium by anodic oxidation, strontium was doped into TiO2nanotubes by hydrothermal reaction to construct a new implant coating. The coating's surface morphology,element distribution,crystal phase,hydrophilicity and the release concentration of Sr2+in the coating were detected by scanning emission electron microscopy,X-ray diffraction,atomic force microscopy,surface contact angle measurement instrument and inductively coupled plasma spectrometer. The cytocompatibility and osteogenic activity of MC3T3-E1 on the coating were evaluated by cytological test in vitro,and the osteogenic effect of strontium doped TiO2nanotube implants in vivo was analyzed by Micro-CT. Results Sr was successfully incorporated into TiO2nanotube,mainly in the form of strontium titanate. The surface roughness of the coating had no significant change compared with that of TiO2nanotube,and the hydrophilicity of the coating increased significantly; the strontium doped TiO2nanotube coating promoted the cell adhesion and proliferation,and increased the alkaline phosphatase activity. The strontium doped TiO2nanotube coating implant significantly increased the bone volume fraction around the implant. Conclusion Sr doped TiO2nanotube coating implants show good biocompatibility and osteogenic activity,which can promote bone formation.

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Objective To investigate the killing effect of zinc magnetic ferrite nanomaterials(ZF NPs) on kidney cancer Caki-1 cells and its correlation with ROS and autophagy. Methods ZF NPs were synthesized by aqueous method and characterized by TEM and other methods. The inhibitory effect of ZF NPs on Caki-1 cells was detected by Thiazole Blue(MTT) assay. Intracellular ROS levels were detected by DCFH-DA staining. TEM was used to observe the autophagosomes in Caki-1 cells after ZF NPs treatment. The changes of autophagy effect were detected by Western blot. DCFH-DA staining, MTT and Western blot were used to investigate the regulation mechanism of ROS and autophagy induced by ZF NPs during the killing of CaKi-1 cells. Results ZF NPs with uniform size and shape were synthesized by aqueous method, which exerted paramagnetic properties for MRI. ZF NPs had a dose and time dependent killing effect on Caki-1 cells. In this process, ZF NPs could induce Caki-1 cells to produce dose and time dependent ROS, while NAC could reduce the production of ROS and weaken the killing effect of ZF NPs. ZF NPs could also elicit autophagy in Caki-1 cells. After co-treatment with Wortmannin, the accumulation of LC3-Ⅱ decreased and the subsequent cell viability was higher than that of ZF NPs treated alone, while co-treatment with Trehalose could increase ROS production and autophagic intensity in executing tumoricidal effect of ZF NPs. Among the sensitization process, NAC could reduce the accumulation of LC3-Ⅱ and Wortmannin could decrease ROS generation. Conclusion ZF NPs have the potential to become a MRI contrast agent, and can induce ROS and pro-death autophagy to kill Caki-1 cells.

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Objective To investigate the effects of exosomes derived from Hepa1-6 cellson dendritic maturation and function. Methods Exosomes were extracted from Hepa1-6 cells, and the shape of exosomes was identified by transmission electron microscopy. The expressions of the marker proteins of exosomes, CD63 and TSG101 were determined by Western-blot.Dendritic cellswere impacted with exosomes from Hepa1-6 cells, and the expression of maturation related surface molecules CD40, CD80, CD83, CD86 and MHC-Ⅱ was detected by flow cytometry.Mouse T lymphocytes and exosomes shock DCs from Hepa1-6 cells were co-incubated for 5 d, and the proliferation of T lymphocytes was determined by CFSE staining and flow cytometry. Results The exosomes collected from thesupernatantof Hepa1-6 cells was observed by transmission electron microscopy and exosomesshowed round or circular membranous vesicle-like structure, which was consistent with the typical characteristics of exosomes.Western-blot analysis showed that exosomesderived from Hepa1-6 cells expressed CD63 and TSG101, but did not express Cytochrome C.The exosomes extracted from Hepa1-6 cells were used to shock DCs, and the surface MHC-II molecules, mature marker CD83 and co-stimulating molecules CD40, CD80 and CD86 were up-regulated.Moreover, with the increase of exosomes concentration, the molecular expression of DC surface increased(P<0.05). Exosomes also promoted the proliferation of T lymphocytes(P<0.05). Conclusion Exosomes extracted from Hepa1-6 cells could promote the differentiation of DC2.4 toward mature phenotype and promote the proliferation of T cells.

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Objective To investigate the effect and mechanism of L-cysteine(L-cys) increasing endogenous H2S on hypertension induced by intermittent hypoxia in rats. Methods Male Sprague-Dawley rats were randomly divided into normal oxygen(Control) group, chronic intermittent hypoxia(CIH) group, Control+L-cys group and CIH+L-cys group. The rats in CIH group and CIH +L-cys group were exposed to 15 d intermittent hypoxia, 8 h daily. In L-cys treatment group, the rats were given L-cys(10 mg/kg)each day by the oral administration. Noninvasive measurement of tail artery systolic blood pressure(SBP) was measured by the tail cuff system. The renal sympathetic nerve activity(RSNA) renal sympathetic nerve activity(RSNA) were recordedin vivoon a PowerLab data acquisition system. The plasma concentrations of norepinephrine(NE) and H2S, activity of cystahionine-β-synthase(CBS) and cystahionine-γ-lyase(CSE) were measured by ELISA kits. Results Systolic blood pressure of tail artery significantly increased after CIH exposure, but did not change after normoxia exposure. RSNA and plasma concentrations of NE were also significantly higher in CIH rats than in control rats. The plasma level of H2S in CIH rats were significantly decreased than those in control rats.L-cys increased the level of H2S of CIH rats, and significantly inhibited the increase of blood pressure and excessive activation of sympathetic nerve activity induced by intermittent hypoxia. Conclusion L-cys can improve intermittent hypoxia induced-hypertension in rats by increasing endogenous H2S.

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Objective To screen molecular markers of nasopharyngeal carcinoma(NPC) by establishing a microRNA(miRNA) regulatory network. Methods The miRNA chip GSE70970 and gene chip GSE12452 of NPC were downloaded from Gene Expression Omnibus(GEO) database. The differentially expressed miRNAs(DEMs) and differentially expressed genes(DEGs) were screened by R software and functional enrichment was analyzed. The transcription factors and target genes of DEMs were predicted by FunRich software. The intersection of target genes and DEGs was defined as hub gene, and the miRNA-mRNA regulatory network was established by Cytoscape software. The survival analysis of hub genes was performed by the Kaplan-Meier plotter database. Results A total of 47 DEMs and 797 DEGs were screened. Functional analysis showed that DEMs were found to be associated with signal transduction and transcription regulation, DEGs were mainly involved in cytokine cytokine receptor interaction, cell cycle, small cell lung cancer and other signaling pathways. 23 hub genes were obtained and the miRNA-mRNA regulatory network was established. The survival analysis showed that the expression level of 4 hub genes were associated with prognosis of NPC patients, including multiple inositol-polyphosphate phosphatase 1(MINPP1),sentan(SNTN), zinc and ring finger 3(ZNRF3) and tubulin beta 2 A(TUBB2 A). The corresponding miRNAs were hsa-miR-199 b-5 p,hsa-miR-520 e,hsa-miR-107,hsa-miR-421. Conclusion By establishing miR NA-mRNA regulatory network and survival analysis,molecular markers of NPC are screened to provide potential targets for diagnosis and treatment of NPC.

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Objective To research the effects of different concentrations of galangin( Ga) on the proliferation and migration of the A549 and PC9 cells of human lung adenocarcinoma and their possible mechanisms. Methods A549 and PC9 cells were cultured in vitro and were divided into control group( DMSO solvent control group,0 Ga control group) and experimental groups, which contained different concentrations( 12. 5,25,50 and 100μmol/L),respectively. CCK-8 test and cell scratch test were used to detect the effects of the proliferation and migration of lung adenocarcinoma cells in each group. The changes of lactic acid( LD) production,lactate dehydrogenase( LDH) activity and ATP production were detected by biochemical tests. The expression of E-cadherin( Ecad) and N-cadherin( N-cad) in cells was detected by ELISA kits. Differences between groups in each index were compared by statistical analysis. Results CCK-8 results showed that Ga had an inhibitory effect on the proliferation of lung adenocarcinoma cells. Cell scratch results showed that Ga had an inhibitory effect on the migration of lung adenocarcinoma cells. Biochemical detection results showed that Ga had an inhibitory effect on LD production,LDH activity and ATP production. ELISA results showed that E-cad protein expression in the supernatant in the experimental groups was higher than that in the control group,while N-cad protein expression was lower than that in the control group. Conclusion Ga can inhibit the proliferation and migration of A549 and PC9 cells of lung adenocarcinoma in vitro,possibly by inhibiting aerobic glycolysis and epithelial-mesenchymal transition.

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Objective To explore the relationship between methylation of ras-association domain family 1 A(RASSF1 A) gene and iodine nutrition status. Methods 40 SD rats were randomly divided into low iodine group, suitable iodine group, 5-fold high iodine group, 10-fold high iodine group and 20-fold high iodine group. Urine iodine level was determined by ICP-MS method, thyroid hormone level in serum was determined by chemiluminescence immunoassay method, and methylation rate of RASSF1 A gene was determined by BSP method. Results After feeding SD rats with different iodine concentrations for 3 months:(1) the serum FT3 value of rats in each group decreased with the increase of iodine concentration(P<0.05), but FT4 and TSH values had no significant difference among the groups;(2) The methylation rates of CpG island 1 and CpG island 2 in the promoter region of RASSF1 A gene were not significantly different among the groups. Conclusion There is no statistical difference in methylation rate of RASSF1 A gene among groups under iodine induction for 3 months. Iodine may have no effect on methylation of RASSF1 A gene or the body is in compensatory stage.

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Objective To investigate the effect of secreted protein, acidic and rich in cysteine(Sparc) on the proliferation and apoptosis of human keloid fibroblasts(HKF) and normal fibroblasts(NFs), as well as the expression of collagen type Ⅰ and collagen type Ⅲ in the extracellular matrix. Methods HKF and NFs were isolated and cultured. The two types of cells were both divided into experimental group and control group in which the experimental group was incubated with Sparc while the control group was incubated with the same amount of DMEM. CCK-8 experiment was used to test the influence of Sparc on each cell proliferation; flow cytometry was used to test the influence of Sparc on each cell apoptosis; Western blot and RT-PCR were used to detect the expression of collagen type Ⅰ and collagen type Ⅲ on each group. Results SPARC could significantly promote the proliferation and inhibit the apoptosis of cells in each group, and SPARC could obviously promote the expression of collagen type I and Collagen type Ⅲ in each group. Conclusion SPARC is an important protein to promote the formation of keloids, which can be targeted to regulate the formation of keloids.

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Objective To explore the protective effect and mechanism of human umbilical cord derived mesenchymal stem cells(hUC-MSCs) on neurons by transplanting hUC-MSCs and treating hypoxic ischemic encephalopathy(HIE) in neonatal rats induced by hypoxia and ischemia. Methods About 10 cm umbilical cord of normal full-term healthy newborns born was collected under sterile conditions, and hUC-MSCs were cultured by tissue block adherent method, and then infected with recombinant adenovirus carrying green fluorescent protein. 18 healthy 7-day-old SD rats were randomly divided into normal control( sham operation) group,model group and h UC-MSCs treatment group,6 in each group. After 24 h of modeling,hUC-MSCs were injected into the left ventricle of the treatment group. After 48 h of h UC-MSCs transplantation,h UC-MSCs were detected in the brain of HIE rats and TTC staining was used to observe the infarcted brain tissue. The mR NA levels of Beclin-2 and Caspase-3 in hippocampus were detected by RT-qPCR. Western blot was used to detect the expression of Beclin-2 and Caspase-3. Results 48 h after hUC-MSCs transplantation in HIE group,there was no infarct volume in normal control group,and the infarct volume ratio was( 41. 67 ± 4. 17) %; in hUC-MSCs treatment group,the infarct volume ratio was( 16. 65 ± 3. 43) %,which was significantly lower than that in hypoxic-ischemic brain injury group. 48 h after HIE modeling,the expression of endogenous beclin-2 mR NA and caspase-3 mR NA,beclin-2 protein and caspase-3 protein were significantly up-regulated. 48 h after hUC-MSCs transplantation,the expression of beclin-2 mR NA,caspase-3 mR NA,beclin-2 protein and caspase-3 protein decreased. Conclusion h UC-MSCs can effectively reduce the infarct volume of rats with hypoxic-ischemic injury,which has a protective effect on the injured rats; at the same time,hUC-MSCs can significantly reduce the apoptosis of rat brain cells,and significantly down regulate the levels of beclin-2 mR NA,caspase-3 mR NA,beclin-2 protein and caspase-3 protein in hippocampus of rats with hypoxic-ischemic brain injury.

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Objective To explore the expression and clinical significance of CXC cell chemokine ligand-5(CXCL5) and matrix metalloproteinase-9(MMP-9) in intrauterine adhesion(IUA) endometrial. Methods Endometrial tissue was obtained from 48 patients who underwent IUA. The transcervical resection of adhesions(TCRA) was performed, and the surgical effect was evaluated by hysteroscopy one month after TCRA. Endometrial tissue was obtained again during hysteroscopy. 48 women who underwent hysteroscopy to exclude endometrial disease during the same period served as a control group. Immunohistochemistry was performed to determine the relative expression of CXCL5 and MMP-9 in endometrial tissue. Western blot was used to detect the relative expression of protein, and RT-PCR was used to detect the relative expression of mRNA. Results The uterine cavity of 48 patients with IUA recovered well after TCRA surgery, and 1 patient had severe adhesion. TCRA surgery was performed again. The results of immunohistochemistry showed that CXCL5 and MMP-9 were relatively low in the IUA group and increased in the IUA postoperative group, but the expression levels of the two groups were lower than those of the control group, and the difference among the three groups was statistically significant(P<0. 01). Western blot and RT-PCR results showed that the expression levels of CXCL5, MMP-9 protein and mRNA in the endometrium of the IUA group were lower than those of the IUA postoperative group, and both groups were lower than the control group. The difference between the three groups was statistically significant(P<0.05); the expression of CXCL5 and MMP-9 were correlated and showed consistent changes. Conclusion CXCL5 and MMP-9 are low expression in the endometrium of IUA, and increase in the endometrium of IUA patients after transcervical resection of adhesions. CXCL5 affects the level of MMP-9, and CXCL5 and MMP-9 may be involved in the occurrence of the IUA.

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Objective To analyze the breeding and genotype of Agtr2-/-mice by constructing Angiotensin Ⅱ type 2 receptor(Agtr2-/-) mice with CRISPR/Cas9 technique. Methods Agtr2-/-F0 generation mice were obtained by using CRISPR/Cas9 gene targeted knockout technology. The genotypes of mouse tail were identified by PCR. Agtr2-/-mice of F1 generation were procured from F0 generation Agtr2-/-mice mating with the same litter wild-type mice after sexual maturity. Western blot was used to check on the dependability of PCR identification results from protein levels. Results The Agtr2-/-mice were successfully constructed by CRISPR/Cas9 technology. The obtained mice were bred and genotypes were identified. Agtr2+/+, Agtr2+/-, and Agtr2-/-stable Agtr2 gene mice were obtained. Western blot results confirmed that Agtr2 protein was almost not expressed in the heart, spleen, thymus, liver and kidney of Agtr2-/-mice. Conclusion The Agtr2-/-mouse model was successfully constructed through CRISPR/Cas9 technology, and the homozygous Agtr2-/-mice were obtained through reliable identification methods and appropriate breeding methods.

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Objective To explore the mechanism by which the inhibitor WZB117 affects the stemness and glycolysis via the downregulation of Yes-related proteins in patient-derived gastric cancer cells. Methods Patient-derived gastric cancer cells were obtained via the trypsin digestion of clinical gastric cancer tissues and incubation. The cells were divided into normal group,low glucose control group and WZB117 treatment group. Afterwards,qRTPCR was used to detect the expression levels of stemness-related genes( Nanog,oct-4 and sox-2),invasion-related genes matrix metalloproteinase-2( MMP-2) and matrix metalloproteinase-9( MMP-9),and apoptosis-related genes( Bcl-2,bax and Cyt-C). Western blot was applied to determine the expression levels of YAP,metabolism-related proteins hexokinase 2( HK2) and pyruvate kinase M2 subtype( PKM2) and activated cysteine-containing aspartate proteolytic enzyme 3( Cleaved Caspase-3). Thiazolyl blue( MTT),plate clone,scratch test,Transwell and kit were performed to measure the cell proliferation,clone formation,migration,invasion,and content of adenosine triphosphate( ATP) and lactic acid,respectively. Results After WZB117 cultured cells,the YAP expression level was significantly lower than that of the normal group and the low glucose control group( P<0. 05),and that of stemness-related genes( Nanog,oct-4 and sox-2) and cell proliferation,clone formation,invasion,and migration were also lower( P<0. 05). The levels of Cleaved Caspase-3 and apoptosis in group treated by WZB117 were higher than those in control groups( P<0. 05),and the expression level of Bcl-2 decreased with the increase of WZB117 concentration( P<0. 05),and the expression levels of Bax and CYT-C increased with the increase of WZB117 concentration( P<0. 05). The content of ATP and lactic acid in supernatant and the protein expression levels of HK2 and PKM2 were lower than those in control groups. Conclusion WZB117 can down regulate the expression of Yap,HK2 and PKM2 in gastric cancer cells,inhibit glycolysis and reduce ATP level.

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Objective To investigate the effect of inhibiting proprotein convertase subtilisin/kexin type 9(PCSK9) on the calcification of human aortic endothelial cells(HAEC) induced by dexamethasone combined with L-ascorbic acid and β-glycerophosphate. Methods Different concentrations(0, 10, 30, 50 mmol/L) β glycerophosphate + 50 μg/ml L-ascorbic acid + 100 nmol/L dexamethasone were used to stimulate HAEC, and the optimal concentration was selected to induce cellular calcification. The mRNA and protein expression of PCSK9 in HAEC was verified. Small interfering RNA(siRNA) was used to knock down the expression of PCSK9 in HAEC, and cell calcification was induced by the same conditions, and the expression changes of PCSK9 and major calcification indexes in HAEC were detected after PCSK9 knockdown. Alizarin red S staining was used to observe the calcification deposition in the cells. Results (1) When the β glycerophosphate concentration was 30 mmol/L, the mRNA and protein expression levels of the main calcification indicators bone morphogenetic protein(BMP)2, BMP4 and Runt-related transcription factor 2(RUNX2) in HAEC reached the peak, and the effect of inducing calcification was the best;(2) The mRNA and protein expression levels of PCSK9 significantly increased after HAEC calcification was induced;(3) Knockdown of PCSK9 expression in cells could significantly inhibit HAEC calcification inducedin vitro, the expression of calcification-related genes and proteins significantly reduced, and the intracellular calcium salt deposition significantly reduced. Conclusion The expression of PCSK9 is related to the calcification level of HAEC. Knockdown of PCSK9 can inhibit the calcification of HAEC induced by dexamethasone combined with ascorbic acid and β-glycerophosphate.

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Objective To study the effect of circular RNA PIP5 K1 A(circPIP5 K1 A) on the growth and metastasis of oral squamous cell carcinoma(OSCC) cells and its possible mechanism. Methods The expression of circPIP5 K1 A in OSCC tissues and cells was detected by RT-qPCR. After transfecting si-circPIP5 K1 A into OSCC cells, cell viability was measured by CCK-8 assay, colony formation ability was detected by colony formation assay,cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI staining,cell migration and invasion were detected by Transwell assay,and the expression levels of proteins related to Wnt/β-catenin pathway were detected by Western blot. After treating OSCC cells transfected with si-circPIP5 K1 A with Wnt/β-catenin signaling agonist LiC l,cell viability,colony formation,apoptosis,migration and invasion were determined. Results CircPIP5 K1 A was up-regulated in OSCC tissues and cells( P<0. 05). After silencing of circPIP5 K1 A,the cell viability,colony formation,migration and invasion in OSCC cells decreased( all P<0. 05),cell apoptosis significantly increased( P<0. 05),and the expression levels of Wnt1 and β-catenin protein decreased( P<0. 05). LiC l inhibited the inhibitory effect of silencing circPIP5 K1 A on the growth and metastasis of OSCC cells( P<0. 05). Conclusion Silencing of circPIP5 K1 A can inhibit the growth and metastasis of OSCC cells by inhibiting the Wnt/β-catenin signaling pathway.

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Objective To explore the effect of sodium aescinate(SA) on hepatic fibrosis induced by bile duct ligation. Methods 54 male SD rats were randomly divided into 3 groups.Rats in the control group(Sham) were only given equivalent normal saline(NS) injection each day after laparotomy.Rats in obstructive jaundice group(OJ group) were only given equivalent NS injection each day after operation. Rats in SA group were given SA injection at the dose of 3.6 mg/kg each day after operation.There were 18 rats in each group.The expression of alanine aminotransferase(ALT),aspartate aminotransferase(AST), total bilirubin(TBIL) was detected by ELISA.The changes of morphological structure and the collagen fiber of rats' liver were revealed by HE and Masson staining. The expression of collagen Ⅰ and collagen Ⅲ was detected by immunofluorescence. The expression of P-4 EBP1、eIF-4 E was detected by immunohistochemical and Western blot analysis. Results In OJ group, the structures of liver were disordered, the expression level of ALT,AST and TBIL in SA group significantly increased, the expression level of collagen Ⅰ and collagen Ⅲ significantly increased, and the expression of eIF-4 E and P-4 EBP1 in hepatocytes decreased.Compared with OJ group, the expression levels of ALT,AST and TBIL in SA group significantly decreased, the expression levels of collagen Ⅰ and collagen Ⅲ significantly decreased, and the expression of eIF-4 E and P-4 EBP1 in hepatocytes increased. Conclusion SA can reduce liver fibrosis by promoting the phosphorylation of 4 EBP1 and activating the expression of eIF-4 E to protect liver cells.

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Objective To investigate the effects of high-salt environment on store-operated calcium entry(SOCE) and contractile function in rat coronary artery smooth muscle. Methods After culturing the isolated rat coronary arteries in different concentrations of high-salt culture media(with 137.65～167.65 mmol/L NaCl respectively) for 24 h, the distribution and expression of Orai1, STIM1, IP3R and ETAin coronary artery smooth muscle were detected by immunohistochemistry assays, U46619, ET-1 and SOCE-induced coronary vasoconstriction were examined by microvascular tension measurement system, and the SOCE mediated Ca2+influx in RCASMCs was detected by calcium imaging technology. Results High-salt environment significantly enhanced ET-1 and SOCE-induced coronary vasoconstriction(P<0.01) and SOCE mediated Ca2+influx in the RCASMCs(P<0.01), and increased the expression of Orai1, STIM1 and IP3R in coronary artery smooth muscle(P<0.05). Conclusion The extracellular high-salt environment can enhance the contractile function of rat coronary artery induced by agonist and SOCE, and the mechanism may be associated with the up regulation of key proteins related to the IP3R-SOCE pathway and the enhancement of Ca2+influx mediated by SOCE in the rat coronary artery smooth muscle.

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Objective To clone and express the 453 bp fragment of P116 ge-ne FP2 ofMycoplasma pneumoniae, and to investigate the adhesion of P116 protein to A549 cells. Methods The immunogenicity and titer of purified P-116 protein fragment were detected by Western blot and ELISA in serum and antibody ofMycoplasma pneumoniaeinfected. Immunofluorescence was used to evaluate the adhesion and adhesion inhibition ofMycoplasma pneumoniaeto A549 cells by P116-FP2 polyclonal antibody.Mycoplasma pneumoniaeadhesion, adhesion inhibition and surface exposure were evaluated using the anti-P116 polycl-onal antibody and A549 cells adhesion assay. Results The results showed that P116-FP2 had immunoreactivity, and the ELISA method established with this protein had high sensitivity and specificity. The P116-FP2 antibody was prepared to block the adhesion ofMycoplasma pneumoniaeto A549 cells. With the increase of titer of polyclonal antibody, the adhesion rate ofMycoplasma pneumoniaeto A549 cells decreased significantly. Conclusion The P116-FP2 protein is proven to be immunoreactive preliminarily. The P116-FP2 polyclonal antibody can inhibit the adhesion ofMycoplasma pneumoniaeto A549 cells.

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Objective To investigate the effect of long non-coding RNA(LncRNA) H19 on the apoptosis of rat pancreatic acinar cells AR42 J induced by caerulein. Methods An AP modelin vitrowas established by stimulating with caerulein. The mRNA and protein expression levels of Caspase 3 and Caspase 9 were detected by qRT-PCR and Western blot after H19 silencing and overexpression. The apoptosis rate of AR42 J cells in each group was detected by Annexin V-FITC/PI assay. Results After H19 silencing, the mRNA and protein levels of Caspase 3 and Caspase 9 significantly decreased, and the apoptosis rate was also relatively reduced(P<0.05). After H19 overexpression, the mRNA and protein levels of Caspase 3 and Caspase 9 significantly increased, and the apoptosis rate also significantly increased(P<0.05). Conclusion The silencing of LncRNA H19 can significantly reduce the apoptosis of cerulean-induced pancreatic acinar cells, suggesting that it may be a potential effective target for the treatment of acute pancreatitis.

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Objective To investigate the expression of dehydroepiandrosterone(DHEA) and melatonin in peripheral blood of patients with acquired premature ejaculation(APE) and their clinical significance. Methods The peripheral blood from 27 APE patients and 30 healthy controls were collected. The expression of DHEA gene and melatonin receptor 1 gene were detected by qPCR. The secretion levels of DHEA and melatonin in serum were analyzed by Elisa. Student'ttest 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 The gene expression of DHEA and melatonin receptor 1 gene in peripheral blood of patients with APE was lower than that of the control group(P<0.01,P<0.01). The serum concentrations of DHEA and melatonin secretion in the premature ejaculation group were lower than those in the control group(P<0.05,P<0.001). Changes in serum melatonin concentration in APE patients were correlated with Chinese Index of Sexual Function for Premature Ejaculation-5(CIPE-5) score, which was statistically significant(r=0.773 8,P<0.05). Conclusion The expression of DHEA and melatonin significantly decrease in APE patients. Melatonin is associated with disease severity.

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Objective To investigate the correlation between hypertension and the risk and pathological changes of renal clear cell carcinoma. Methods The clinical data of 201 patients undergoing renal clear cell carcinoma surgery and renal pathology were retrospectively analyzed in this study. According to the ratio of age and sex, 1 005 normal people who underwent physical examination in the physical examination center of Anqing Municipal Hospital were used as the control group. The differences in metabolic syndrome, smoking were compared between the two groups. By using Logistics multi-factor regression analysis, the correlation between hypertension and renal cancer was analyzed, and the renal cancer group was divided into the hypertensive group(group A) and the non-hypertensive group(group B), and the differences of relevant pathological data were analyzed. Results There was no significant difference in total cholesterol, triglyceride, high-density lipoprotein(HDL), low-density lipoprotein(LDL) and uric acid between the two groups. Hypertension, smoking, diabetes and obesity prevalence in two groupswere compared(χ2=109.318, 4.949, 4.142, 15.589;P<0.05), and the differences were statistically significant. Logisitic regression analysis suggested that hypertension was positively correlated with the risk of renal cancer. There were statistical differences between the two groups in the incidence of arterial plaque in the abdominal aorta of the two groups, the incindence of clinical stage, Furman nuclear grade ≤ grade 2 detection rate, Ki67 index ≤5% detection rate, microvascular density(P<0.05). There was statistically meaningless in microvascular invasion. Conclusion The incidence of hypertension in patients with renal clear cell carcinoma is higher than that in the normal population. Hypertension may be an associated factor involved in the development and progression of renal clear cell carcinoma. Early reasonable intervention and prevention of hypertension are recommended in the high-risk population of renal carcinoma.

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Objective To explore the changes of cognitive function before and after endovascular treatment of middle cerebral artery stenosis and its possible mechanism. Methods Twenty-two patients with middle cerebral artery stenosis were grouped into the treatment group and control group according to whether they received endovascular treatment or not. All subjects were evaluated at 3 d before operation, 3 d after operation and 3 months after operation.The changes of cognitive function between the two groups were compared. The cognitive function and emotion of patients were evaluated by Mini Mental State scale(MMSE), Montreal Cognitive Assessment scale(MoCA), Cambridge Geriatric Cognitive examination scale(CAMCOG-C), language fluency test, Wechsler visual replication,digit span test and neuropsychological scales,such as Hamilton Anxiety Scale( HAMA) and Hamilton Depression Scale( HAMD). Vascular stenosis,cerebral perfusion and collateral circulation were evaluated by CT angiography( CTA),CT perfusion imaging( CTP) and digital subtraction angiography( DSA). Results In the treatment group,the delayed memory in MoCA and the C1 of Wechsler visual replication were significantly improved during the 3-month follow-up period,and the score during the follow-up period was higher than that in the control group. While there was no significant difference in the abstract comparison of MoCA subitems between the treatment group and the control group,the abstract score in the treatment group was significantly higher than that in the control group during the follow-up period. The digital span sequence score of treatment group were significantly improved at 3 days after operation and 3-month follow up compared to that before operation. The collateral circulation in the treatment group measured by Multiphase( mC TA) and DSA was significantly improved after operation. In addition,the cerebral perfusion in the treatment group was significantly increased after operation,and the average transit time( MTT) was significantly decreased( P<0. 05). Conclusion Endovascular treatment of middle cerebral artery stenosis may improve the cognitive function of patients by improving collateral circulation and increasing blood perfusion.

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Objective To explore the value of golgi protein 73(GP73) and heat shock protein 90α(HSP90α) in the diagnosis of primary hepatic cancer(PHC) with low levels of alpha-fetoprotein(AFP). Methods ELISA was used to analyze the serum levels of GP73 and HSP90α in 50 cases of PHC with low levels of AFP, 30 cases of liver cirrhosis, 30 cases of hepatitis, 40 cases of healthy controls. Serum levels of GP73 and HSP90α among groups were compared. Results The serum levels and positive detection rates of GP73 and HSP90α were significantly higher in PHC group with low levels of AFP than those in the group of liver cirrhosis, hepatitis and healthy controls, and the difference was statistically significant(P<0.01). Serum levels of GP73 and HSP90α in PHC patients with low levels of AFP with lymph node metastasis and portal vein invasion were significantly higher than those in PHC patients without lymph node metastasis and portal vein invasion, and the difference was statistically significant(P<0.05). Sensitivity, specificity and accuracy rates in the diagnosis of PHC with low levels of AFP by GP73 alone were 78.0%, 77.0%, 77.3% respectively, while thoseviaHSP90α alone were 84.0%, 95.0% and 91.3% correspondingly. In the combined detection, the highest sensitivity of GP73 or HSP90α positive diagnosis was 94.0% and the highest specificity of GP73 and HSP90α positive diagnosis was 96.0%. Conclusion Both Serum levels of GP73 and HSP90α can be used as effective tumor biomarkers in the diagnosis of PHC with low levels of AFP and their combination significantly improves diagnostic accuracy with a higher sensitivity.

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Objective To analyze the expressionof brain-expressed X-linked protein1(BEX1) and brain-expressed X-linked protein 4(BEX4) in oral squamous cell carcinoma(OSCC)and adjacent tissues, and to explore its relationship with the occurrence and development of OSCC. Methods The expression of BEX1 and BEX4 proteins in OSCC tissues and non-tumor tissues was analyzed by immunohistochemistry method. Kaplan-Meier survival analysis in TCGA was performed to analyze the correlation between BEX1 and BEX4 expression in OSCC and the overall survival. The location of BEX1 and BEX4 in signal transduction and its upstream and downstream genes related to it were analyzed by the String-DB database. Results Among 55 cases of OSCC, 25 cases were positive for BEX1 protein(45.45%), 19 cases were positive for BEX4 protein(34.55%), 24 cases were positive for BEX1 in non-tumor tissues adjacent to cancer(80.00%), 27 cases were positive for BEX4 in non-tumor tissues adjacent to cancer(90.00%). The differences were statistically significant compared with adjacent tissues(P<0.01). The expression of BEX1 protein and BEX4 protein was related with the stage of oral squamous cell carcinoma, lymph node metastasis and differentiation degree, and the difference was statistically significant(P<0.001); but neither of them was related to gender, age and tumor site. The correlation analysis showed that the correlation intensity of BEX1 protein and BEX4 protein expression was 0.489(P<0.001). Kaplan-Meier survival analysis showed that OSCC patients with low expression of BEX1 had a poor prognosis. Conclusion BEX1 and BEX4 may serve as biomarkers for predicting metastasis of OSCC.

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Objective To investigate the characteristics and correlative factors of sialorrhea in patients with primary Parkinson's disease in order to provide individualized management procedures. Methods 59 patients with idiopathic Parkinsonʼs disease were collected. Data of demographic characteristics, clinical features and history of medications were collected. Patients were assessed with a comprehensive serie of scales which include the third part of Unified Parkinson's Disease Rating Scale(UPDRS Ⅲ), modified Hoehn&Yahr Scale(H&Y), levodopa equivalent dose(LED), the freezing of gait questionnaire(FOG-Q), Dynamic Parkinson gait scale(DYPAGS), 39-item Parkinson's Disease Questionnaire(PDQ-39), the Schwab&England Activities of Daily Living Scale(S&E) and the Scales for Outcomes in Parkinson's Disease-Autonomic questionnaire(SCOPA-AUT). The orofacial motor function and swallowing function of patients were evaluated by Radboud Oral Motor Inventory for Parkinson's disease test(ROMP), the water swallowing test and the Nordic Orofacial Test-Screening(NOT-S). Saliva secreted in five minutes from the participants was collected and weighed to assess salivary secretion volume. Results Among 59 patients with Parkinson's disease, 38 patients had sialorrhea, and the incidence of sialorrhea was 64.4%. Severe sialorrhea accounted for 21.1%, while non-severe sialorrhea accounted for 78.9%. The age, scores of Hoehn-Yahr stage, levodopa equivalent dose(LED), SCOPA-AUT scores, PDQ-39 scores, saliva secretion, ROMP-language scores, ROMP-swallowing scores, scores of NOT-S, UPDRS Ⅲ and DYPAGS scores, UPDRS Ⅲ-19 scores in sialorrhea group exceeded those in non-sialorrhea group, while in non-sialorrhea group, S&E scores were much lower. LED and NOT-S scores were independent risk factors of sialorrhea(OR=1.004, 95%CI1.001～1.008,P<0.05;OR=2.379,95%CI1.289～4.392,P<0.05). In addition, sialorrhea severity was positively correlated to age, LED, UPDRS Ⅲ scores, ROMP-language scores, ROMP-swallowing scores, DYPAGS scores, scores of NOT-S, FOG-Q scores, SCOPA-AUT scores, UPDRS Ⅲ-19 scores(r=0.330, 0.327, 0.414, 0.454, 0.416, 0.390, 0.399, 0.477,0.435,0.353,P<0.05).The scores of NOT-S was an independent risk factor of sialorrhea severity(OR= 6.635, 95%CI1.335～32.990,P<0.05). Conclusion Sialorrhea is correlated with many factors, such as age, motor dysfunction severity, equivalent dose of levodopa, severity of autonomic nerve dysfunction, saliva volume, severity of speech and swallowing disturbance, camptocormia, orofacial dysfunction severity. Sialorrhea is closely related to the impaired neuromuscular function of mouth, face, pharynx and larynx. Orofacial motor dysfunction and equivalent dose of levodopa are independent risk factors of sialorrhea.

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Objective To explore a new serum marker of carotid artery plaque instability——pregnancy-associated protein-A(PAPP-A) and to evaluate its prognosis in patients with ischemic stroke. Methods 84 patients with ischemic stroke were selected and divided into stable plaque group and unstable plaque group according to carotid ultrasound examination. Then the serum levels of PAPP-A were compared between the two groups. Besides, the National Insititutes of Health stroke scale(NIHSS) scores and activity of daily living scale(ADL) scores of the two groups were assessed respectively at three timepoint: before treatment, the 4 th week and 12 th month after treatment during 1 year's following up. Simultaneously, the changes of the scores were compared, to explore their roles in evaluating the prognosis of patients with ischemic stroke. Results The serum levels of PAPP-A in the unstable plaque group were significantly higher than those in the stable plaque group(P<0.01).The area under the curve(AUC) was 0.811 when assessing the predictive value of PAPP-A towards unstable plaque. The NIHSS scores in the unstable plaque group was higher than that in the stable plaque group, while there was no statistical difference in ADL scoresbetween the two groups. Conclusion The serum level of PAPP-A is related to the instability of carotid artery plaque.Patients with ischemic stroke with unstable plaques are more likely tohave poor prognosis.Therefore, rigorous clinical follow-up is recommended.

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Objective To investigate the effects of protective ventilation on the production of interleukin(IL)-1β,IL-18 and endogenous melatonin and prognosis of patients with esophageal cancer. Methods Ninety esophageal cancer patients were randomized to receive “conventional”ventilation( Vt = 10 ml/kg) or lung protective ventilation [Vt = 5 ml/kg along with 5 cm H2O positive end-expiratory pressure( PEEP) ]. IL-1β,IL-18 and melatonin in bronchoalveolar lavage fluid( BALF) and serum were measured before and at the end of the operation. Respiratory variables during two-lung ventilation,one-lung ventilation for 30 min and at the end of the operation were recorded. Results of blood gas analysis on the first day after surgery and outcomes were evaluated. Results Compared with “conventional”ventilation group,lung protective ventilation decreased peak airway pressure( Ppeak),plateau airway pressure( Pplat) and driving pressure( ΔP). Lung protective ventilation increased the partial pressure of oxygen on the first day after surgery and lowered the blood lactic acid values( P<0. 001). In addition,lung protective ventilation reduced the incidence of postoperative pulmonary complications( P = 0. 045) and the rate of major postoperative complications( P = 0. 028). Moreover,lung protective ventilation not only suppressed alveolar and serum IL-1β and IL-18 secretion,but also restored the level of endogenous melatonin( P<0. 001).Conclusion Lung protective ventilation suppresses IL-1β and IL-18 secretion,restores the level of endogenous melatonin and reduces the incidence of postoperative lung and total complications.

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Objective To explore the clinical value of computed tomography colonography(CTC) based colonic imaging features evaluating incomplete colonoscopy. Methods 71 patients who underwent both CTC and colonoscopy were retrospectively collected and divided into complete group(n=30) and incomplete group(n=41) according to whether the colonoscopy reached the ileocecal region. The clinical factors and imaging features related to incomplete colonoscopy were analyzed, and the value of imaging features in predicting incomplete colonoscopy was evaluated. Results Compared with the complete group, the incomplete group had a higher age(P<0.001), higher sigmoid parietal height(P<0.001),longer colon length(P<0.001), longer abdominal circumference in supine position(P=0.005) and more tortuosity(P=0.003). The area under the ROC curve of sigmoid apex height, colon length, supine abdominal circumference, tortuosity and combined with these featuress were 0.741, 0.795, 0.685, 0.704, 0.850, respectively. Conclusion Age, high sigmoid length and abdominal circumferences in supine position are associated with incomplete colonoscopy, and the combination of multiple imaging features can better predict whether the colonoscopy can be completed.

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Objective To analyze the factors of the elderly over 60 years old to vaccinate COVID-19 vaccine by understanding the intention and affecting factors of Corona Virus Disease 2019(COVID-19) vaccine inoculation for the elderly over 60 years old in Hefei and investigating the vaccination status of the elderly. Methods The elderly over 60 years old in Hefei city were selected to investigate their willingness to receive COVID-19 vaccine by using online questionnaire.Single factor and multifactor analysis were conducted to learn about the affecting factors. The elderly over 60 years old in Hefei were followed up to understand the actual vaccination rate and the reasons for non vaccination. Results 76.8% of the elderly over 60 years old in Hefei city were willing to receive the vaccine. Age, gender, education level, chronic underlying diseases and influenza vaccination were not affecting factors for the elderly to receive the new vaccine. The possibility of being infected with COVID-19 after the vaccination, the side effects and effectiveness of the vaccine, the necessity of getting vaccinated in China which is doing quite well in the prevention of the disease and the new cases in the city were the main affecting factors for the elderly to consider getting vaccinated.The result of follow-up survey showed that 208 elderly over 60 years old in Hefei had been vaccinated with COVID-19 vaccine, and the actual vaccination rate was 46.2%. Plan to be vaccinated after a period of time, think that the vaccination of people over 60 years old has not been opened; elderly over 60 years old who have basic diseases will not be allowed to be vaccinated temporarily and inconvenient to queue up for a long time when the weather is hot were top four reasons for the elderly over 60 years old who have not been vaccinated. Conclusion The promotion of safety, effectiveness and knowledge of COVID-19 vaccination, strict grasp of the vaccination taboo, increase resources for vaccination and speed up vaccine production, are very important to improve the willingness and the actual vaccination rate of the elderly over 60 years old to receive the vaccination.

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Collagen antibody-induced arthritis(CAIA) is a novel mouse model of arthritis. Compared with collagen-induced arthritis(CIA) model, CAIA does not require more than 7 weeks to develop arthritis phenotype, and has a better induction effect for CIA model insensitive strains. However, the high dose inducers reported in previous literatures, on the one hand, caused serious adverse reactions and even death in mice, and on the other hand, resulted in unnecessary waste of 5-clone collagen antibodies cocktail. Reducing the dosage of 5-clone antibody cocktail and lipopolysaccharide(LPS) effectively induced the arthritis model of C57 BL/6 mice while avoiding the death of the model mice and saving the cocktail of 5-clone antibody, thus providing an improved method for the future research on the pathogenesis of RA.

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In order to study the inhibition effect of MTAN(a mixture of Nisin, acid, and detergent)and MTAN+(ethylene diamine tetraacetic acid, EDTA)onS.sanguinis,F.nucleatumandP.gingivalispaired mixed bacteria solutions and three-in-one mixed bacteria solution, MTAN and MTAN+EDTA with different Nisin concentrations were used to treatS.sanguinis,F.nucleatumandP.gingivalispaired mixed bacteria solutions and three-in-one mixed bacteria solution respectively, and the OD method was adopted to measure MIC and MBC. The results showed that MTAN had inhibitory effect on all mixed bacteria solutions. With the increase of concentration, the inhibitory effect was enhanced. When Nisin concentration was ≥250 μg/ml, bacteria could be killed. MTAN+EDTA with different concentrations of Nisin inhibited all mixed bacterial solutions, and when the concentration of Nisin≥2.5 μg/ml, bacteria could be killed. Therefore after MTAN acts on all the mixed bacteria solutions, it has obvious bacteriostatic effect, and this effect is obviously improved with the addition of EDTA.

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To investigate the fracture resistance and fracture mode of the tooth after IPS e-max Press occlusal veneer repair. The occlusal surfaces of 7 mandibular premolars were ground evenly for 1. 5 mm,simulating the morphology of occlusal surface of severe wear tooth,and the occlusal surface was prepared with IPS e-max Press. After the adhesive was cemented,static loading test was performed on each specimen until cracked,and the fracture load value and fracture mode were recorded. The fracture load of the specimens was much higher than the normal masticatory force,which could meet the clinical repair needs. The fracture mode was mostly limited to the fragmentation of the restoration or the damage of the tooth in a small range,which was conducive to the secondary repair. IPS e-max Press occlusal veneer is a minimally invasive and ideal method for the restoration of severe tooth wear in premolar.

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To explore the breeding and genotype analysis of Lanosterol synthase(LSS) gene knockout mice. The introduced heterozygous knockout mice were reared and bred, the tails of progeny mice around 2 weeks old were cut, their genomic DNA was extracted, the target gene fragments were amplified by PCR reaction and identified by agarose gel electrophoresis Genotype of mice; Western blot analysis was used to analyze and compare LSS protein expression in livers and skin tissues of heterozygous and wild-type mice. A batch of LSS knockout mice were successfully bred, and their progeny mice were identified by PCR with two genotypes: wild type(LSS+/+, Wt) and heterozygous(LSS+/-), which did not appear Homozygous knockout mice; Western blot results showed that compared with wild-type mice, LSS gene knockout heterozygous mice expressed less LSS protein in liver and skin tissues; the viability of heterozygous knockout mice was weaker than that of wild-type mice. Successfully obtaining LSS gene knockout mice, PCR identification of mouse genotypes has the advantages of fast, simple and stable, which lays the foundation for further study of the biological function of LSS.

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To explore the clinical efficacy of the double-window approach for the treatment of intra-articular calcaneal fractures, and to compare it with the extended lateral approach, to evaluate the application value of the double-window approach.The clinical data of 110 patients(123 feet) with intra-articular calcaneal fractures were retrospectively analyzed.Among them, 58 patients(66 feet) were operated via the double-window approach and 52 patients(57 feet) were operated via the extended lateral approach.The imaging indicators of the two groups at the last follow-up were improved compared with preoperatively, and the difference was statistically significant(P<0.05).There was no significant difference in the imaging indicators and the AOFAS scores between the two groups before and after operation(P>0.05).The incidence of wound complications(3.0%) and the rate of subtalar joint stiffness(9.0%) in the double-window approach group were lower than those in the extended lateral approach group(21.0% and 40.4%),and the difference was statistically significant(P<0.05).The double-window approach is fully exposed.It can achieve the reduction results and efficacy equivalent to that of the extended lateral approach, and significantly reduce the incidence of wound complications and subtalar joint stiffness.