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Objective To study the biological function ofLamtor2 gene and its mechanism in the process ofLamtor2 infection, the model ofLamtor2 macrophage conditioned knockout mice infected with KP was established. Methods Lamtor2 flox /+mice were introduced for self-crossing, andLyz2-Cre mice were hybridized withLamtor2 flox /+to obtain offspring mice withLamtor2 flox / flox Lyz2-Cre-/-andLamtor2 flox /+Lyz2-Cre+/-. The two genotypes were hybridized to obtain control mice(Lamtor2 flox / flox Lyz2-Cre-/-) andlamtor2 conditional knockout mice(Lamtor2 flox / flox Lyz2-Cre+/-). The toe genomic DNA of mice was extracted and amplified by polymerase chain reaction(PCR). The genotypes of offspring mice were identified by agar-gel electrophoresis and alveolar macrophages were extracted. TheLamtor2 gene knockout effect was verified by RT-qPCR and Western blot. Mice were infected with Kp and their survival conditions were observed. The expression levels of TNF-α, IL-1β and Cxcl1 after Kp infection with alveolar macrophages were detected by qRT-PCRin vitro, and the comparison between the two groups was performed byttest. Western blot was used to verify the expression level of iNOS. Results Lamtor2 conditional gene knockout mouse model was successfully established withviableand fertile offspring. Compared with control mice, experimental mice had lower survival rate after Kp infection. The levels of TNF-α(t=17.54, 0.44±0.050,P=0.003 2), IL-1β(t=15.37, 0.35±0.043,P=0.004 2), and Cxcl1(t=37.82,0.23±0.043,P=0.000 7) fromLamtor2 flox / flox Lyz2-Cre+/-mouse alveolar macrophages reduced, and the iNOS expression decreased. Conclusion Lamtor2 knockout mice are constructed, and the survival rate of mice and iNOS expression of alveolar macrophges decreases after infected with Kp.

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Objective To explore the construction and identification of miR-100 gene knockout mouse model. Methods The introduced miR-100flox/floxmice were mated with EIIa-Cre mice to produce F1 heterozygous mice. The male and female mice with miR-100flox/-genotype were further mated to obtain F2 generation mice. When the mice were 1 week old, the tail tissueswere cut to extract DNA,the target gene fragment was amplified by PCR,and the genotype was determined by agarose gel electrophoresis. Results Among them, there were miR-100 knockout homozygous mice. If the obtained knockout homozygous mice were mated, more knockout homozygous mice could be obtained. By cutting the mouse ears to extract the RNA,the mouse RNA could be extracted without affecting the life of the mouse. Therefore, the ears of miR-100 knockout homozygous mice, miR-100flox/floxmice, and miR-100 knockout heterozygous mice were cut to extract RNA,and then qPCR was performed to verify the expression of miR-100. The breeding and identification of miR-100 gene knockout mice were successful, and stable genetic knockout mice were obtained. Three genotypes were found in F2 generation mice mated with F1 heterozygous mice: miR-100flox/flox,miR-100flox/-,and miR-100-/-. The genotypes of the offspring mice were successfully identified by PCR. qPCR results showed that the expression of miR-100 in the ears of the whole-body knockout homozygous mice was almost zero. Conclusion In this study, a mouse model of whole-body deletion of miR-100 gene is successfully cloned, providing a basis for subsequent experiments.

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Objective To study the effect of miR-181 on the migration and invasion of gallbladder cancer cells by targeting tissue inhibitor of metalloproteinase-3(TIMP3). Methods Gallbladder cancer GBC-SD cells were cultured and transfected with miR-181 mimic, NC mimic, TIMP3 siRNA, NC siRNA, recombinant plasmid expressing TIMP3, blank plasmid. The expression of miR-181 was detected by fluorescence quantitative PCR, the expression of TIMP3 was detected by Western blot, the cell migration and invasion activity were detected by Transwell, and miR-18 target TIMP3 was verified by bioinformatics analysis and luciferase reporter gene experiment. Tumorbearing mouse model was established by subcutaneous injection of GBC-SD cells transfected with miR-181 mimic or NC mimic, then the quality of the transplanted tumor and the expression of miR-181, TIMP3 were detected. Results Cell experiments showed that miR-181 mimic could promote cell migration and invasion, inhibit the expression of TIMP3 and luciferase activity of double luciferase reporter gene; TIMP3 siRNA could promote cell migration and invasion; after transfection of recombinant plasmid expressing TIMP3, the effect of miR-181 mimic on cell migration and invasion was weakened.Animal experiments showed that miR-181 mimic could reduce the weight of transplanted tumor and increase the expression of TIMP3. Conclusion miR-181 promotes the migration and invasion of gallbladder cancer cells, and targeting inhibition of TIMP3 is one of the molecular mechanisms.

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Objective To investigate the role of ubiquitin carboxyl-terminal hydrolase L1(UCHL1) in the occurrence and development of myocardial fibrosis in a mouse model. Methods Male mice(n=6 per group) were continuously perfused with angiotensin II(Ang II) for 14 days to establish a mouse myocardial fibrosis model. UCHL1 specific inhibitor LDN57444 was injected intraperitoneally into these mice. Blood pressure was measured at the tail. The change in cardiac structure was evaluated by M-mode echocardiography. The severity of myocardial fibrosis was assessed after sirius red staining. The primary mouse myocardial fibroblasts were randomly divided into 3 groups: control group, P group(PDGF-DD) and PL group(PDGF-DD + LDN57444). The mRNA and protein expressions were detected by qRT-PCR and Western blot. Results Compared with the control group, the mean systolic pressure increased significantly and the cardiac structure and function changed in the animals treated with Ang II. Treatment with UCHL1 specific inhibitor LDN57444 could significantly inhibit the above Ang II-induced effects, and the severity of myocardial fibrosis decreased markedly.The mRNA expression of type I collagen in the PL group was markedly lower than that in the P group, the mRNA expression of UCH-L1 in the P and PL groups was significantly higher than that in the control. The protein expression levels of PDGFRβ/pPDGFRβ, UCH L1, pRb/Rb, and Col Ⅰ in the P group was significantly higher than those in the control group, the protein expression levels of PDGFRβ/pPDGFRβ, ColⅠ and pRb/Rb in the PL group was markedly lower than those in the P group. Chromatin immunoprecipitation assay confirmed NF-κB regulated the transcription of downstream UCH-L1. Conclusion UCH-L1 is involved in the regulation of mouse cardiac fibroblasts proliferation. In this process, the PI3 K/AKT/NF-κB pathway regulates the expression of UCH-L1 and then regulates downstream Rb protein expression, which subsequently regulates the cell cycle to affect the proliferation of cardiac fibroblasts. Inhibiting UCH-L1 can reduce myocardial fibrosis in mice.

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Objective To explore the effect mechanism of miR-483-5 p onpalmitic acid-injured podocytes. Methods Podocytes were treated with different concentrationsof palmitic acid, miR-483-5 p inhibitor, miR-483-5 p mimic and KLF2 plasmid, respectively. The binding relationship between miR-483-5 p and KLF2 was analyzed by starbase and dual-luciferase reporter assay. The cell viability was indicated by CCK-8 assay, the levelsof miR-483-5 p and KLF2 were indicated by qRT-PCR, the cell apoptosis was indicated by flow cytometry assay, and the levels ofmalonaldehyde(MDA), superoxide dismutase(SOD)were indicated by colorimetry assay, the protein levels of C caspase3, Nephrin and Podocin wereindicated by Western blot. Results Palmitic acid inhibitedviability(P<0.05) and promoted miR-483-5 p expression(P<0.05) onpodocytesin a concentration-dependent manner; miR-483-5 p inhibitor could partially reverse the inhibitory effect of palmitic acid(0.5 mmol/L) on cell viability(P<0.05), and reverse the stimulative effect on apoptosis(P<0.05) and oxidative stress(P<0.05).The miR-483-5 p was directly targeted to KLF2, and miR-483-5 p mimic further enhanced the inhibitory effect of palmitic acid on cell viability(P<0.05), Nephrin and Podocin expression levels(P<0.05), and enhanced the stimulative effect on oxidative stress(P<0.05) and C caspase3 expression level(P<0.05), which was partially reversed by KLF2 overexpression. Conclusion miR-483-5 p can regulate palmitic acid-induced podocytes injury through regulating KLF2 expression, which is helpful for the research of prognosis in patients with diabetic nephropathy.

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Objective To investigate the effects ofArmillariellatabescens polysaccharides(ATPS) on cell activity, occludin and ZO-1 in IEC-6 intestinal epithelial cells induced by lipopolysaccharide(LPS), and to reveal the protective mechanism ofArmillariellatabescens polysaccharides on intestinal mucosal barrier injury. Methods IEC-6 intestinal epithelial cell injury model induced by LPS was established. The injured cells were randomly divided into control group, LPS group, LPS+ATPS group and LPS+matrine group. The cell viability was observed by microscope and MTT; the cell apoptosis was detected by immunofluorescence and flow cytometry; the expression of ZO-1 and occludin was detected by Western blot. Meanwhile, Western blot was used to detect the expression of p-PERK,p-eIF2α,GRP78,CHOP and Arrb1 proteins. Results With the increase of ATPS concentration, the cell activity in the LPS+ATPS group increased, and the apoptosis rate decreased by immunofluorescence and flow cytometry compared with control and LPS group. The expression levels of occludin and ZO-1 protein were significantly higher in LPS+ATPS group than those in LPS group. The levels of p-PERK, p-eIF2α, GRP78, chop and arrb1 protein in LPS+ATPS group decreased compared with LPS group. Conclusion Armillariellapolysaccharide can protect intestinal epithelial cells from LPS mediated inflammatory injury by inhibiting the damage of occludin and ZO-1 proteins. The mechanism may be related to endoplasmic reticulum stress protein which mediates apoptosis and Arrb1(an important molecule) regulating intestinal mucosal injury.

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Objective To observe the differential expression of ferroportin(FPN) in non-small cell lung cancer(NSCLC) and to explore the mechanism of FPN effect on NSCLC cell migration and proliferation. Methods Immunohistochemistry(IHC) was used to detect the expression level of FPN in 60 cases of lung cancer and 20 cases of normal lung tissue. Western blot was used to detect the expression level of FPN in cell lines.FPN overexpression system(PCDH-FPN-F) and knockdown system(shFPN) were constructed respectively, and verified by Western blot and qRT-PCR.The effect of FPN on lung cancer cell migration was detected by scratch test.Growth curve method was used to detect the effect of FPN on proliferation of lung cancer cells. Results Compared with normal lung tissue, the expression of FPN in lung cancer tissues significantly decreased(P=0.000). Compared with normal lung cell lines, the expression of FPN in lung cancer cell lines significantly decreased(P<0.000 1).Compared with the control group, the expression of FPN in the overexpressed group significantly increased(Western blot: P<0. 01,qRT-PCR: P<0. 000 1). Compared with the control group,FPN expression significantly decreased in the knockdown group( Western blot: P<0. 001,qRT-PCR: P<0. 001). After overexpression of FPN,the wound healing distance between cell scratches was significantly lower than that of the control group at 18 h and 36 h. The number of cells in 2 d and 4 d was significantly lower than that in the control group. When FPN was knocked down,the wound healing distance between cell scratches at 18 h and 36 h was significantly higher than that of the control group. The number of cells in 2 d and 4 d was significantly higher than that in the control group. Conclusion After low expression of FPN,the migration and proliferation of lung cancer cells were enhanced,which may be the reason for the differential expression of FPN in lung cancer.

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Objective To investigate the effects of miR-30 a-5 p on the proliferation, migration, cycle and apoptosis of laryngeal squamous cell carcinoma cells by targeting the regulation of MTA1. Methods The target gene MTA1 of miR-30 a-5 p was predictedby biological information technology and verified by RT-qPCR and dual-luciferase reporting experiment. miR-30 a-5 p mimics and NC were transfected into LSCC cell line Hep2 at logarithmic phase. Cell proliferation was detected by CCK-8 assay. Transwell assay was used to detect cell migration. Flow cytometry was used to detect cell cycle and apoptosis. Results In the real-time quantitative PCR results, the post-transfection expression levels of experimental group and control group were(12.62±0.97) and(1.04±0.32), respectively, with statistically significant difference(P<0.05). In the results of the double luciferase report experiment, the experiment levels of miR-30 a-5 p mimics+ pGL4.10+pGL 4.73 were(0.41±0.06) and(0.91±0.01), respectively, when compared with the wild-type and mutant-type, and the differencewas statistically significant(P<0.05).In the results of CCK-8 experiment, the absorbance value of the experimental group was lower than of the control group at 24,48,72 h, and the difference was statistically significant(P<0.05). In the Transwell cell migration experiment, the number of the experimental group and the control group was(207.67±7.23) and(369.00±41.22), respectively, with statistically significant differences(P<0.05). The results of flow cytometry detection of cell cycle showed that the G1,S and G2 phases of the experimental group were smaller than those of the control group, with cell cycle arrest. The results of cell apoptosis showed that the apoptosis rates of the experimental group and the control group were(22.61±5.70)% and(4.52±2.12)% respectively, with statistically significant differences(P<0.05). Conclusion The miR-30 a-5 p/MTA1 axis may have a certain inhibitory effect on the pathogenesis of laryngeal squamous cell carcinoma.

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Objective To rescueSRSF9 gene in human glioma U87SRSF9 knockout monoclonal cell line using CRISPR/Cas9 system and to research its function. Methods SRSF9-WT andSRSF9-sgMu overexpression vector which was synonymous mutated 5 bases on site ofSRSF9-WT were conducted and packaged into lentivirus. U87SRSF9 knockout monoclonal cell lines were infected with lentivirus to constructSRSF9 gene rescue cell lines. Western blot and immunofluorescence assays were used to detect the localization of rescued protein, proliferation experiment and tumor stem cell sphere formation assay were used to detect the rescue functions of rescued gene. Results SRSF9-WT andSRSF9-sgMu overexpression vector were successfully constructed and packaged into lentivirus. Western blot and immunofluorescence assays confirmed that the rescue was successful and therescuedSRSF9 protein was predominantly expressed in nucleus. Theresultsofproliferation and tumor stem cell sphere formation assays showed that the rescuedSRSF9 could also rescue its function(P<0.05). Conclusion SRSF9-sgMu can successfully rescue the expression ofSRSR9 and its function of proliferation and tumor stem cell sphere formation in human glioma U87SRSF9 knockout monoclonal cell line.

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Objective To observe its effect on the proliferation, invasion and apoptosis of human glioma cells by the overexpression recombinant plasmid targeting to long non-coding maternally expressed gene 3(LncRNA MEG3) gene transfects the human glioma cells in vitro.Methods The expression of LncRNA MEG3 mRNA in normal brain tissue cells(N) and glioma cell line U251 cells was detected by qRT-PCR. Eukaryotic overexpression vector ofpCI-MEG3 was constructed to transfect the human glioma cell line U251 cells as pCI-MEG3 overexpression plasmid group(pCI-MEG3 group). Those transfected with empty pCI plasmid were divided into empty vector pCI group(pCI group) and those without any transfection were divided into Control group. The expression of LncRNA MEG3 gene was analyzed by qRT-PCR. The expression of MDM2 and P53 of LncRNA MEG3-related protein was confirmed by Western blot. The proliferation ability of glioma cells was analyzed by techniques of MTT and cell cloning. Transwell was used to analyze the invasion ability of glioma cells,and flow cytometry was used to analyze the apoptosis rate of glioma cells. Results Compared with normal brain tissue cells,the expression level of LncRNA MEG3 mRNA decreased in glioma cells. The results of vitro experiments showed that both the mRNA of LncRNA MEG3 and protein expression levels of p53 increased,and the expression levels of MDM2 protein were down-regulated( P<0. 05) in p CI-MEG3 group when compared with the control group and the p CI group( P<0. 05). Cell proliferation and cell invasion were inhibited( P<0. 01). The apoptosis rate increased( P<0. 01). Conclusion The expression of LncRNA MEG3 in human glioma cells is lower than that in the normal human brain tissue cells. Overexpression of LncRNA MEG3 in human glioma cells can increase the expression of LncRNA MEG3 thereby inhibiting the proliferation and invasion of human glioma cells and increasing the apoptosis rate.

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Objective To investigate the effect of quercetin(Que) on brain injury in rats with subarachnoid hemorrhage(SAH) and its mechanism. Methods Internal carotid artery puncture was used to prepare rat models of subarachnoid hemorrhage, and the rats were randomly divided into sham group, SAH group, SAH + Que 50 mg/kg group and SAH+Que 100 mg/kg group with 15 rats in each group. After 30 minutes of successful modeling, quercetin was injected intraperitoneally, and Garcia nerve function scores were performed in each group after 23 hours of intervention. The water content of brain tissue was measured by wet and dry weight method. The blood-brain barrier permeability was measured by Evans blue staining. The proportion of TUNEL-positive and microtubule-associated protein light chain 3(LC3)-positive cells were detected by immunofluorescence staining, and Cleaved caspase-9, Bax, Bcl-2, Nrf2, HO-1 and NQO1 protein levels were detected by Western blot. Results Compared with the sham group, the Garcia neurological score was higher in the SAH group, the brain tissue water content increased. Evans blue content increased, the proportion of TUNEL/NeuN double positive cells increased, the proportion of LC3/NeuN double positive cells increased, cleaved caspase-9 and bax protein expressions were up-regulated, Bcl-2, Nrf2, HO-1 and NQO1 protein expressions were down-regulated. Conclusion Quercetin can reduce brain neurons damage in rats with subarachnoid hemorrhage and improve neural function, and the mechanism may be related to the inhibition of autophagy.

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Objective To isolate, culture and identify rat femoral artery endothelial cellsin vitro, and to provide anin vitrocell model for pathological and physiological studies. Methods The rat femoral artery blood vessels were taken aseptically, and rat femoral artery endothelial cells were isolated by collagenase digestion; the purity of endothelial cells was detected by flow cytometry; CD31 positive cells were sorted out by flow cytometry, and endothelial cells were identified by laser confocal; the proliferation ability of endothelial cells was detected by CCK-8 method and high-content cell imaging method, and their migration and tube function were detected by Transwell method and Matrigel gel method. Results The primary endothelial cells of the femoral artery vascular obtained by collagenase digestion and separation had covered more than 2/3 of the area of the culture flask at 14-16 d, and the cells were typical paving stones.The purity was 38.34%, and the sorted cells were identified by CD31 fluorescence staining. The cells were all CD31 positive; PGE2can significantly upregulate the proliferation, migration, and tube formation of CD31 positive endothelial cells. Conclusion In this study, a simple method is used to successfully isolate the primary endothelial cells of femoral arteries, providing anin vitrocell model for studying the function of primary endothelial cells derived from relatively small blood vessels.

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Objective To investigate the effect of cathelicidin LL-37 on the apoptosis of gastric cancer AGS cells and its molecular mechanism. Methods Gastric cancer AGS cells were treated with different concentration of LL-37 for 24 h, MTT was used to detect theactivity of cell proliferation, Annexin V-FITC/PI double staining flow cytometry was used to detect cell apoptosis, qRT-PCR method was used to detect the mRNA expression of p53, immunofluorescence method was used to detect the protein localization of p53, Western blot was used to detect the protein expression of p53 in cell nucleus and apoptosis-related proteins such as Cleaned-caspase-3, PUMA, Bcl-2 and Bax in cells; interference with p53 gene expression verified whether LL-37 induced apoptosis of gastric cancer AGS cells by activating p53 signaling. Results LL-37 dose-dependently inhibited the proliferation of gastric cancer AGS cells, elevatedthe rate of apoptosis, up-regulated the mRNA and protein expression level of p53 and promoted the nuclear translocation of p53 protein, while increased the apoptosis proteinsexpression of Cleaved-caspase-3, PUMA and Bax, decreased the anti-apoptotic proteinexpression of Bcl-2.Interference with p53 gene expression inhibited LL-37-induced apoptosis of AGS cells. Conclusion LL-37 can induce apoptosis of gastric cancer AGS cells, and its mechanism may be related to the activation of p53 expression.

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Objective To study the mechanism of sevoflurane general anesthesia and exploratory laparotomy on cognitive dysfunction in aged rats. Methods Thirty 16-month-old SD rats were randomly divided into 3 groups: control group, anesthesia group, anesthesiaoperation group(10 cases in each group); control group did not do any treatment; the anesthesia group was induced by inhalation of 4% sevoflurane, and the anesthesiaoperation group was induced by inhalation of 4% sevoflurane. At the same time, exploratory laparotomy was performed. Morris water maze experiment was performed before and after the operation of all rats to evaluate their learning and memory function, and then the mice were taken blood and decapitated to remove the brain; immunohistochemical staining method was used to detect the number of astrocytes in hippocampal CA1 area, ELISA method was used to detect serum GFAP concentration. Results The results of the Morris water maze experiment showed that both the anesthesia group and the operation group could cause cognitive dysfunction in elderly rats, and the cognitive dysfunction of the elderly rats in the anesthesiaoperation group was more severe, with statistically significant difference(P<0.05); immunohistochemical staining showed that star-shaped glue plasma cells of the anesthesia group and the operation group decreased, and the anesthesiaoperation group decreased more, with statistically significant difference(P<0.05); ELISA results showed that the serum GFAP concentration of the anesthesia group and the operation group increased, and the anesthesiaoperation group increased more, with statistically significant difference(P<0.05). Conclusion Sevoflurane general anesthesia and abdominal exploration can cause cognitive dysfunction in aged rats, and the cognitive dysfunction in aged rats is related to the decrease in the number of astrocytes.

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Objective To investigate the role of nuclear factor of activated T cells 5(NFAT5) in inflammol/lation cascade in sepsis induced kidney injury. Methods Cultured mouse kidney medullar collecting duct cells 3 were stimulated by lipopolysaccharide(LPS)(10 ng/ml) for 12 hours with or without siRNA transfected. qRT-PCR was used to detect the transcription level of cytokine tumor necrosis factor α(TNFα) and chemokinemonocyte chemotactic protein-1(MCP-1) respectively, ELISA was applied to measure the protein of secreted in the culture medium. Chromatin immol/lunoprecipitation(ChIP) was used to detect whether NFAT5 bind the promotor of TNFα and MCP-1 respectively. Results After LPS stimulation for 12 hours,compared with the control group,the secretion of protein and transcription level of mRNA of TNFα and MCP-1 were upregulated( P<0. 05),however,they did not increase after transfection of NFAT5 siRNA. Western blot was difficult to measure the expression of NFAT5 isolated from the nuclear after LPS stimulation for 12 hours,however,NFAT5 was detected binding to the promotor of TNFαand MCP-1 respectively by ChIP. Conclusion NFAT5 plays a role in sepsis induced acute kidney injury by mediating inflammolation,specifically speaking,by controlling the transcription of cytokine TNFα and chemokine MCP-1 in LPS stimulated mouse cell line iM CD3.

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Objective To investigate the construction of silver-carrying coating on titanium implants and its antibacterial effect againstPorphyromonas gingivalis(Pg), and to provide a new method for antibacterial modification of titanium implants. Methods Titanium dioxide nanotube structure was prepared by anodicoxidation on the surface of pure titanium, the silver-loaded chitosan and heparin were adsorbed alternately on the surface of the nanotube by layer by layer self-assembly method, and the silver-loaded multilayer film coating was constructed. The samples were characterized and detected by scanning electron microscopy, contact angle measuring instrument, X-ray photoelectron spectroscopy and Ultraviolet spectrophotometer. The samples were cocultured with Pg to observe its antibacterial effect on bacteria. Results Characterization test confirmed that the silver-loaded multilayer film was deposited successfully on the surface of the titanium sheet and its hydrophilicity was improved. The bacteriological test showed that the antibacterial property of titanium implants was significantly improved, and the antibacterial effect was enhanced with the increase of the concentration of silver. Conclusion The silver coating on the surface of titanium implants can effectively inhibit the growth of Pg and the formation of biofilm, which is expected to improve the antibacterial properties of titanium implants.

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Objective To investigate the effect of captopril on lipid metabolism in diabetic mellitus(DM) rats and its relationship with diabetic retinopathy(DR). Methods Thirty male SD rats aged 8 weeks were randomly divided into normal control group(n=10) and model group(n=20). The model rats were induced by high-fat diet(HFD) combined with streptozotocin(STZ) to establish the model of type 2 diabetes mellitus rats. The model rats were randomly divided into captopril group(DM+CAP) and normal saline group(DM+NS) with 10 rats in each group. Rats in DM + CAP group were gavaged with captopril 25 mg/(kg·d), and DM+NS group were gavaged with equal volume normal saline. Body weight and fasting blood glucose were measured weekly. After 8 weeks of intervention, the rats were euthanized, the whole blood was collected, serum was separated and retinal tissue was collected. The concentrations of total cholesterol(TC) and triglyceride(TG) in serum were detected; the structure of retina was observed, including the structure changes of inner and outer nuclear layers, and the number of new blood vessels; the levels of cholesterol regulatory element binding protein 1(SREBP1) and cholesterol regulatory element binding protein 2(SREBP2) were detected. Results Captopril could not reduce fasting blood glucose in rats. Compared with DM+NS group, the concentration of TC and TG in serum of DM+CAP group decreased(P<0.01); the retinal tissue structure improved: the inner and outer nuclear layer cells were tightly connected, the number of new blood vessels was less; the expression levels of SREBP1 and SREBP2 decreased(P<0.01). Conclusion Captopril can reduce the blood lipid related indexes of diabetic rats, improve the retinal tissue morphology, and reduce the key enzymes of lipid synthesis in the retina of diabetic rats. The mechanism may be related to its inhibition of lipid synthesis.

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Objective To explore the effects of intraperitoneal injection of tetramethylpiperidine(Tempol) on the interleukin(IL)-22 and IL-2 in the liver injury of trichloroethylene(TCE) sensitized mice and the influence of malondialdehyde(MDA) levels.Methods Forty 6～8 weeks Balb/c female mice were randomly sampled and divided into blank group, solvent group, TCE group and TCE+Tempol group to establish a TCE percutaneous sensitization model. The animals in TCE+Tempol group were injected intraperitoneally with Tempol solution(100 mg/kg)before the last challenge. 24 hours after the last challenge, the animals were divided into sensitization positive group or sensitization negative group according to the skin sensitization result score. The pathology of liver sections was observed by HE staining method, serum was separated for the detection of aspartate aminotransferase(AST) and alanine aminotransferase(ALT), and MDA content in liver was detected by TBA method, IHC and Western blot method to detect the expression level of IL-22 and IL-2 in liver tissue.Results The sensitization rates of TCE group and TCE + Tempol group were 0. 40 and 0. 33,respectively. Pathological examination found that the liver cells of the blank group,solvent group and non-sensitized group had no obvious pathological changes. There was a large number of vacuolar degeneration in the liver cells of the TCE sensitization positive group,with cell edema and cytoplasm in a loose state,while the TCE + Tempol sensitization positive group showed hepatocyte edema in some areas; Compared with the blank group,the MDA activity,IL-22 and IL-2 content in the liver tissue of mice in solvent group and sensitization negative group were not statistically significant. The relative expression levels of MDA,IL-22 and IL-2 in the liver tissue of mice in the sensitization positive group were higher than those in the solvent group and the sensitization negative group( P<0. 05),and the TCE sensitization positive group was higher than the TCE at the same point TCE + Tempol sensitization positive group( P<0. 05). Conclusion Tempol may reduce the levels of IL-22 and IL-2 to protect the immune liver injury caused by TCE.

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Objective To observe the expression of TNFR2 in the liver of mice and to explore the relationship between TNFR2 and the immune liver injury induced by trichloroethylene in mice. Methods Twenty-one BALB/c mice from 6 to 8 weeks of age were randomly divided into blank control group(n=5), solvent control group(n=5) and TCE treatment group(n=11). A TCE-sensitized mouse model was established according to the pre-sensitization method of the research group. Twenty-four hours after the last challenge, the skin sensitization response of the mice was scored, and the mice in the TCE group were divided into the TCE-sensitized group and the TCE non-sensitized group. The mice were sacrificed 72 hours after the last challenge, and the blood was collected from the venous plexus of the eye to measure liver function indexes such as alanine aminotransferase(ALT) and aspartate aminotransferase(AST). The liver of the mice was taken out and made into paraffin sections. HE staining was used to observe the pathological features of the liver. Immunohistochemistry method and Western blot method were used to determine the expression of TNF-α and TNFR2 in mouse liver.Real-time fluorescence quantitative PCR method was used to detect the level of TNFR2 gene in mouse liver. Results The sensitization rate of the TCE group was 45.45%(5/11); the serum ALT and AST levels of the TCE-sensitized group were significantly higher than those of the blank control group, solvent control group and TCE unsensitized group, and the difference was statistically significant(P<0.05). HE staining results showed that the blank control group, the solvent control group and the TCE unsensitized group had normal liver cell morphology and even staining. The TCE-sensitized group showed cell swelling and vacuolar degeneration. Immunohistochemical experiments showed that the TCE-sensitized group had a higher expression of TNF-α, a large number of TNFR2 positive expressions could be observed in the TCE-sensitized group compared with other groups, and the difference in scores was statistically significant(P<0.05). Real-time fluorescence PCR results showed that the expression of TNFR2 gene in the TCE-sensitized group was also significantly higher than that in the other groups(P<0.05). Western blot method was used to determine the expression level of TNFR2 protein in the liver of mice, and it was found that the protein expression level of TCE-sensitized group was higher than that of the other three groups(P<0.05). Conclusion The expression of TNFR2 in the liver of TCE-sensitized mice is significantly higher than that of other groups, suggesting that the expression of TNFR2 may be related to the immune liver injury in TCE-sensitized mice.

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Objective To elucidate the risk factors for lower extremity amputation patients of diabetic foot ulcer(DFU) disease. Methods The clinical data of 287 patients with diabetic foot ulcer based on the University of Texas(UT) wound classification system were analyzed retrospectively.Demographic characteristics, laboratory data, disease history were considered as independent variables to predict the therapeutic outcome(lower extremity amputation and non-amputation). Multivariate logistic regression was used to analyze the possible factors affecting amputation. Results The amputation rate among diabetic foot patients was 35.2%. A total of 46 variables were included in the analysis. In univariate analysis, 22 variables in the amputation group were significantly different from those in the non-amputation group(P<0.05). Binary logistic regression analysis showed that grade 2 of the UT wound classificationsystem(OR:10.090, 95%CI: 2.696-37.760,P=0.001) and grade 3(OR:35.327, 95%CI:7.648-163.678,P<0.001), ulcer history(OR:6.320, 95%CI:2.278-17.554,P<0.001), toe(OR:24.880, 95%CI:7.565-82.24,P<0.001), increased glycosylated albumin(OR:1.156,95%CI:1.040-1.284,P=0.007), elevated white blood cell count(OR:1.137, 95%CI:1.003-1.288,P=0.045) and the risk of malnutrition(OR:3.460, 95%CI: 1.348-8.878,P=0.010) were associated with increased risks from diabetic foot ulcer to amputation, while hemoglobin(OR:0.966, 95%CI: 0.941-0.991,P=0.008) was a protective factor for amputation. Conclusion For DFU patients, early treatment, control of infectionaggravation, improvement of nutritional status, and stability of short-term blood glucose are very important for the prevention of amputation.

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Objective To explore the expression and its efficacy in predicting neoadjuvant chemoradiation therapy sensitivityof human antigen R(HuR) in rectal cancer. Methods Fifty-six paired middle-low rectal cancer samples and clinicopathologic characteristics were analyzed. HuR expression in middle-low rectal cancer tissuesand exploratory biopsies was detected by quantitative real-time PCR and immunochemistry. The capability of HuR to influence DNA DSBs inthe presence of chemo-radiation was determined by using comet assay. Results HuR was mostly up-regulated in middle-low rectal cancer tissues and over expression HuR was significantly associated with invasive clinicopathologic characteristics such as advanced TNM stage, positive lymph node invasion and vascular infiltration. The Kaplan-Meier plot of overall survival from TCGA database demonstrated that high HuR expression patient subgroup had a shorter median survival period compared with the low HuR expression subgroup. Moreover, patients with high HuR expression were of inferior therapeutic sensitivity to chemo-radiation therapy. Conclusion HuR is over expressed in rectal cancer and possessed the capabilities to resist chemo-radiation therapy. In addition, inhibition of HuR can improve chemo-radiationtherapeutic response.

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Objective To explore the application of low depth whole genome sequencing technique combined with karyotype analysis in prenatal diagnosis of high risk pregnant women. Methods Amniocentesis was performed in 165 high-risk pregnant women. The G-banding karyotype of fetal amniotic fluid samples was analyzed while the genomic DNA of fetal amniotic fluid cells was detected by Low-pass whole genome sequencing technology. The sequencing reads were precisely mapped using the human genome(GRCh37, UCSC release hg19) as a reference. Then, the pathogenicity of CNVs was interrogated in ISCA, Decipher, ClinVar and other databases. The differences between G-banded chromosome karyotype analysis and Low-pass whole genome sequencing results were compared. Results Among 165 amniotic fluid samples, 44 cases of trisomy 21, 10 cases of trisomy 18, 1 cases of trisomy 13 were detected by Low-pass whole genome sequencing. The result was consistent with that of G-banding karyotype analysis. In addition, there were 2 cases of sex chromosome abnormality detected by Low-pass whole genome sequencing,1 case more than that detected by G-banding chromosome karyotype analysis,and 10 cases of other types of chromosome abnormality,4 cases more than that detected by G-banding chromosome karyotype analysis. The total detection rate of chromosomal abnormalities by Low-pass whole genome sequencing was 53. 33%,which was 3% higher than that by chromosome G-banding karyotype analysis. Conclusion Low-pass whole genome sequencing has a good application value in prenatal diagnosis of pregnant women with high risk in non-invasive prenatal testing. The combined application of Low-pass whole genome sequencing and G-banding chromosome karyotype analysis can effectively improve the detection rate of chromosome abnormalities in pregnant women with high risk in non-invasive prenatal testing,reduce birth defects and improve population quality.

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Objective To investigate the relationship between neutrophils extracellular net(NET) and follicular helper T cells(Tfh) in patients with anti-neutrophil cytoplasmic myeloperoxidase antibody-associated vasculitis(MPO-AAV) and its significance in the pathogenesis. Methods 35 patients with untreated MPO-AAV(patient group) and 35 healthy volunteersas control(control group) were enrolled in this study.Flow cytometry(FCM) was employed to detect percentage of Tfh in CD4+T cells(Tfh%), percentage of Tfh expressing ICOS in CD4+T cells(ICOS+Tfh%) and mean intensity(MFI) of Tfh expressing ICOS in their peripheral blood, while enzyme linked immunosorbent assay(ELISA) was used to measure the level of MPO-ANCA,NET and IL-23 intheirblood. Their demographic documents were collected and the disease activity of all patients were recorded by Birmingham Vasculitis Activity Score-version 3(BVAS-V3). Results Compared with those in control group, the detected Tfh%,ICOS+Tfh%, MFI, NET and IL-23 increased[(25.7±3.9)%vs(20.7±5.3)%,P<0.001;(1.9±1.0)%vs(0.8±0.4)%,P<0.001;(59.5±10.3)vs(48.7±6.4)(MFI),P<0.001;(0.6±0.2)vs(0.2±0.1)(absorbance),P<0.001; and(0.753 5±0.201 9)vs(0.237 5±0.087 0)(pg/L),P<0.001, respectively].By bivariate correlation analysis, the MPO-ANCA had positive correlation with the Tfh% and the MFI of ICOS+Tfh(r=0.737,P<0.001;r=0.628,P<0.001, respectively), whereas, multivariate linear regression analysis indicated the MPO-ANCA only was associated with Tfh%(P<0.001).Statistical analysis also showed IL-23 was positively correlated with Tfh%, the MFI of ICOS+Tfh, and the NET(r=0.475,P=0.004;r=0.359,P=0.034; andr=0.806,P<0.001, respectively). Although the Tfh% was positively correlated with serum NET and IL-23(r=0.714,P<0.001 andr=0.480,P=0.004, respectively) by bivariate correlation analysis, multivariate linear regression analysis demonstrated that the NET levelwasthe only independent influence factor on Tfh%(P<0.001).By bivariate correlation analysis, the renal lesions had positive correlation with the NET(r=0.390,P=0.021). Conclusion To participate in the pathogenesis of MPO-AAV, NET may increase Tfh% by activating myeloid dendritic cells to assist B cells to produce specific autoantibody MPO-ANCA.

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Objective To investigate the relationship between DNA damage repair gene XPG rs873601 polymorphism and the efficacy and prognosis of chemotherapy for lung cancer platinum drugs, so as to provide a scientific theoretical basis for accurate treatment of lung cancer patients. Methods 120 patients with lung cancer who received platinum-based chemotherapy were confirmed by pathology. After informed consent was obtained, we extracted peripheral blood DNA from 2-3 ml of peripheral venous bloodand detected the polymorphism of XPG rs873601 locus by mass spectrometry. WHO′s solid tumor efficacy evaluation criteria were used to determine the efficacy of chemotherapy for patients, and the survival of patients was obtained by telephone follow-up and other methods. Results Among 120 patients with lung cancer, the frequency distribution of the three genotypes of the XPSrs873601 was 53(44.2%), 61(50.8%), and 6(5%), which complied with the Hardy-Weinberg law of genetic balance(χ2=4.81).The total effective rate of chemotherapy was 32.5%. Patients with the XPG gene rs873601 AA genotype had the highest effective rate of chemotherapy, reaching 43.4%, while the GG genotype had the lowest, only 16.7%. The difference in efficacy between different genotypes was statistically significant(χ2=7.747,P=0.046). The total one-year survival rate of 120 lung cancer patients was 73.2%, the five-years survival rate was 20.8%, and the XPG gene rs873601 AA genotype had the lowest survival rate, the one-year survival rate was 60.4%, the five-years survival rate was 18.3%, and there was no statistically significant difference in survival rates between SNP(χ2=3.300,P=0.348). Conclusion XPG rs873601 polymorphism is associated with the efficacy of platinum-based chemotherapy for lung cancer, and it can be used as one of the markers for predicting the effectiveness of chemotherapy for lung cancer.

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Objective To study the characteristics and possible influencing factors of stigma in adult patients with epilepsy, and to explore the correlation between stigma and ecological executive function in adult patients with epilepsy.Methods Through the questionnaire survey, 83 patients with primary or possibly symptomatic adult epilepsy and 58 healthy adults with matching gender, age, marriage and education level were used to use the Chinese version of Kilifi stigma scale for epilepsy,the behavior rating inventory of executive function-adult version( BRIEFA) evaluation. Results(1) In the epilepsy group,the proportion of stigma obtained was as high as 97. 6%,and there were differences in the stigma obtained in different places of residence,family income,control status,and medication types.(2) Multivariate stepwise linear regression analysis suggested that the number of years of education in patients with epilepsy( t =-2. 383,P = 0. 019),per capita household income( t =-4. 812,P<0. 01) were negatively related to stigma score; the long-term residence of patients with epilepsy( t = 2. 329,P<0. 05),epilepsy was controlled or not( t = 2. 206,P = 0. 03),the type of medication( t = 6. 496,P<0. 01) were positively related to stigma score.(3) The scores of global executive composite( GEC),behavioral regulation index( BRI) and mmetacognition index( MI) of BRIEF-A and various factors( inhibition,conversion,emotional control,self-monitoring,task initiation,working memory,planning,organization,task monitoring) scores were significantly higher than those of health in the control group,and the difference was statistically significant( P<0. 05).(4) Spearman correlation analysis suggested that stigma was significantly positively correlated with the scores of BRIEF-A( including GEC,BRI,MI and all sub factors)( P<0. 01). Conclusion Adult patients with epilepsy have obvious stigma,and the severity of stigma is related to years of education,per capita family income,long-term residence,epilepsy control or not,and type of medication; adult patients with epilepsy have ecological executive function impairment; adult epilepsy stigma of patients is closely related to impairment of executive function.

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Objective To probe into the influencing factors of subendocardial viability ratio(SEVR) based on pulse wave analysis. Methods Height, weight and blood pressure were measured in 592 physical examination subjects. Fasting venous blood was collected to analyze blood glucose, blood lipids and other biochemical indicators. Pulse wave pressure waveforms were collected with cardiac function instruments of the Chinese academy of sciences, and SEVR values were calculated based on diastolic and systolic area. Results Multivariate linear regression analysis showed that SEVR values were significantly different between males and females(P<0.05), heart rate was negatively correlated with SEVR values(P<0.05), and creatinine was positively correlated with SEVR values(P<0.05). Conclusion Gender, heart rate and serum creatinine may be the main influencing factors of SEVR value. According to this study, SEVR is a non-invasive evaluation indicator of myocardial perfusion with few interferences, which has certain clinical application value.

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Objective To investigate the characteristics of gait disorders in patients with cerebral small vessel disease(CSVD) and the correlation between the number and location of cerebral microbleeds and gait disorders. Methods 102 patients with cerebral small vessel disease were enrolled and divided into CSVD with cerebral microbleeds group(n=29) and CSVD without cerebral microbleeds group(n=73). In addition, age and sex-matched patients who without CSVD on cranial MRI were selected as the control group(n=30). The quantitative gait parameters of all subjects were measured, and the differences of gait parameters between cerebrovascular disease group and normal control group were compared. The gait parameters included step length, gait speed, stride frequency, and the percentage of bipedal support phase. The correlation between the number and location of cerebral microbleeds and gait parameters such as step length, gait speed, gait frequency and the percentage of bipedal support phase were statistically analyzed. Results Compared with the normal group, CSVD with cerebral microbleeds group was significantly shorter and slower(P<0.05) while CSVD without cerebral microbleeds group was shorter and faster(P<0.05). There was no significant difference between CSVD with cerebral microbleeds and CSVD without cerebral microbleeds. The number of cerebral microbleeds was negatively correlated with step length(r=-0.295,P=0.001) and positively correlated with foot support(r=0.225,P=0.011), the more the number of cerebral microbleeds, the shorter the step length and the longer the support phase of feet. The cerebral microbleeds in frontal lobe, temporal lobe and deep part were associated with shorter step length and slower walking speed(P<0.05). Conclusion As an independent factor, cerebral microbleeds are associated with gait disorders in patients with cerebrovascular diseases. Frontal lobe, temporal lobe and deep cerebral microbleeds are correlated with gait disorders.

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To study the effect of candesartan targeting TRAIL-DR5-mediated AMPK signaling pathway on autophagy protection of cervical cancer cells. Human cervical cancer HeLa cells were used as the study objects. The levels of TRAIL-DR5, AMPK, ATG4 A, LC3 Ⅰ, LC3 Ⅱ, Bax and Bcl-2 in HeLa cells were measured. Compared with 0 μmol/L candesartan, the apoptosis rate of HeLa cells and levels of Bax and TRAIL-DR5 increased after treatment with 15.0, 30.0 and 60.0 μmol/L candesartan(P<0.05), the levels of Bcl-2, ATG4 A, LC3Ⅱ/LC3Ⅰ and p-AMPK decreased(P<0.05), in a concentration dependent manner. Candesartan can inhibit HeLa cells proliferation and promote apoptosis, which may be related to TRAIL-DR5-mediated AMPK signaling pathway reducing the autophagy protection of HeLa cells.

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203 thyroid micro-nodules pathologically confirmed were retrospectively analysed, including 148 cases of thyroid microcarcinoma and 55 cases of micronodular goiter.The software(AK) was used to perform radiomics analysis on CT plain and enhancement scan images of thyroid micro-nodules. Six models were used for evaluation: Forest model, SVM model, KNN model, Tree model, Bayes model and Logistic model. 70% of the cases were randomly selected as the training group, and the testing group was the remaining 30%. The accuracy, specificity, sensitivity and AUC of different models for the differential diagnosis of two types of micro-thyroid nodules were obtained.The diagnostic efficiency of plain scan and enhanced scan were analyzed and Forest model was the best. The diagnostic accuracy, specificity, sensitivity and AUC of the plain scan were 99.3%, 97.9%,100% and 0.999,respectivelyfor training group while 87.9%, 81%, 91.1% and 0.884 for the testing group. For the enhancement scan, those of the training group were all 100% and the test group were 82.8%, 93.3%, 71.4% and 0.905. For CT radiomics, Forest model is of certain value in the differential diagnosis of benign and malignant thyroid micro-nodule. Plain scan and enhanced CT have high sensitivity and specificity, respectively.

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80 patients who needed medium-thickness skin grafts were selected and randomly divided into 4 groups A, B, C, and D. The donor sites were implanted with advanced platelet rich fibrin(A-PRF) membrane(group A), platelet-rich fibrin(PRF) membrane(B group) and recombinant bovine basic fibroblast growth factor gel(C group) respectively. Group D was the blank control group. All groups were covered with petrolatum gauze and compression bandage. The wound healing time, postoperative pain and scar hyperplasia of the four groups were compared. All three biological materials could promote wound healing,and A-PRF had the best clinical effect. APRF and PRF could reduce postoperative pain and scar hyperplasia,and the clinical effect of A-PRF was better than that of PRF. A-PRF could speed up the healing of medium-thickness skin donor site wounds,reduce scar hyperplasia,and improve post-healing morphology. A-PRF can provide new ideas and methods for clinicians to choose biomaterials to promote the healing and regeneration of soft tissues in the medium-thickness skin donor area.

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Clinical and pathological data of 127 patients with basal cell carcinoma(BCC)and dermoscopic data of 42 patients were analyzed retrospectively. The main clinical types of BCC were nodular/ulcerative type, pigmented type and superficial type, accounting for 54.3%,37.8% and 7.9% respectively. Among the pathological types, the proportion of nodular/cystic type was the highest. The main dermoscopic features were blue-gray oval nest, dendritic vessels, maple leaf structure and spoke like structure. Bright red white unstructured area and short capillary dilation were mainly seen in superficial type. The misdiagnosis rates of pigmented type and superficial type were higher. Dermoscopy can improve the accuracy of clinical diagnosis of BCC.