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Objective To investigate the effect of apigenin on the proliferation, migration, invasion, apoptosis and autophagy of human lung squamous carcinoma NCI-H520 cells. Methods Human lung squamous carcinoma NCI-H520 cells were culturedin vitro, and the CCK-8 method was used to detect the effects of different concentrations of apigenin(2.5, 5, 10, 20 μmol/L) or cisplatin(2.5, 5, 10, 20 μmol/L) on cell viability. Carboxyfluorescein diacetate, succinimidyl ester(CFDA SE) was used to detect the effect of apigenin or cisplatin on cell division. Scratch test and Transwell test were used to detect the effect of apigenin on cell migration and invasion. Annexin V/PI double staining method and Hoechst 33258 nuclear staining method were used to detect the effect of apigenin on cell apoptosis. Transmission electron microscopy was used to observe the generation of autophagic vesicles in cells. Acridine orange(AO) staining was used to observe the changes of acidic organelles in cells. Western blot was used to detect microtubule-associated protein 1 light chain 3 B-Ⅱ(LC3 B-Ⅱ) and p62 protein expression. Results Compared with the control group, CCK-8 assay and CFDA SE showed that apigenin or cisplatin reduced proliferation of NCI-H520 cells(P<0.05). Scratch test and Transwell test showed that apigenin reduced the migration and invasion levels of cells(P<0.01). Annexin V/PI double staining showed that apigenin increased apoptosis rate(P<0.05). Hoechst 33258 nuclear staining showed that apigenin promoted nuclear condensation and hyperchromatism of cells. AO staining fluorescence value enhanced, the autophagy bimolecular structure appeared under transmission electron microscopy, and the results of LC3 B-Ⅱ and p62 protein expression levels in Western blot showed that apigenin increased the autophagy level of cell NCI-H520(P<0.05). Chloroquine(CQ) failed to increase the protein levels of LC3 B-Ⅱ and p62 in apigenin treated cells. Conclusion Apigenin at different concentrations can inhibit the proliferation, migration and invasion of human lung squamous cell carcinoma NCI-H520, and its inhibition of cell growth and metastasis may be related to the induction of apoptosis and autophagy, and the increase of autophagy may be caused by blocking autophagosome degradation.

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Objective To investigate the distribution characteristics and role of GRK5-Moesin pathway in glioma. Methods Glioma cell lines with GRK5 up-regulated/down-regulated were prepared by lentivirus transfection, which were identified by Western blot and qRT-PCR. Effect of GRK5 changes on Moesin expression was detected by Western blot. Immunofluorescence analyzed subcellular localization and distribution characteristics of GRK5 and Moesin in glioma tissues. Proliferation, migration, invasion and apoptosis of U87 cells were detected by CCK-8 assay, Cell scratch assay, Transwell assay and flow cytometry, respectively. Results GRK5, Moesin and CD44 were co-located in glioblastoma cell membrane. GRK5-Moesin were enriched in glioma niches and glioma blood vessels, but the distribution of GRK5 and Moesin in each blood vessel was different. Targeting GRK5-Moesin could affect the biological activity of U87 glioma cells. Conclusion GRK5-Moesin can promote the malignant progression of glioma and is closely related to glioma stem cells.

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Objective To explore the effects and mechanisms of NADPH oxidase 4(NOX4) and nucleotide-binding oligomeric structural domain protein-like receptor protein 3(NLRP3) inflammasome on aging-associated renal injury in mice. Methods The 6, 16, 20, and 24-month-old mice were used in this study. The levels of serum creatinine(SCr) and blood urea nitrogen(BUN) were detected by the kit. Frozen sections of kidney tissue were used to detect the levels of β-Galactosidase(β-Gal) and reactive oxygen species(ROS). The pathological changes of the kidney were observed by H&E, PAS, and Masson staining. The expressions of collagen Ⅳ and NLRP3 were detected by immunohistochemistry. Western blot was used to detect the expression of related proteins in the NOX4-NLRP3 signaling pathway in kidney tissues. Results The results showed that, compared with 6-month-old mice, the levels of BUN and SCr in serum, β-Gal activity, and ROS level in the renal cortex increased, the glomerular and tubular injury was mild, and there was no obvious renal fibrosis change in 16-month-old mice. However, in 20 and 24-month-old mice, these indexes increased, the damage of glomeruli and renal tubules increased, and renal fibrosis appeared. In addition, expression of NOX4 and NLRP3 inflammasome-associated proteins mediating ROS production was upregulated in the kidneys of 20-and 24-month-old mice. Conclusion The NOX4-NLRP3 signaling pathway may activate and promote renal aging and renal fibrosis during aging.

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Objective This study aimed to explore the role of SGK-1 in the occurrence of temporomandibular joint(TMJ) osteoarthritis(TMJ-OA). Methods Sixteen rats were randomly divided into Control group(Control group) and TMJ-OA group(TMJ-OA group), and TMJ-OA group was injected with sodium iodoacetate(MIA) intra articular cavity while Control group was injected with 0.9% sodium chloride solution. Pain behavior was assessed by measuring the head withdrawal threshold(HWT) with a von-Frey apparatus. Hematoxylin-eosin(HE) and Safranin O-fast green stains were used to observe the histological structure changes of the condyle of TMJ-OA rats. Real-time PCR was performed to exam the expression levels of mRNA of SGK-1, MMP-13, IL-1β, COX-2 in mandibular condylar cartilage(MCC). HE stain was used to observe the histological structure changes of the trigeminal ganglion(TG) of TMJ-OA rats. Real-time PCR was performed to exam the expression levels of mRNA of SGK-1, COX-2 in TG. Results MIA injection induced typical OA-like lesions in the TMJ within 28 days. Administration of MIA led to the significant decrease in HWT, disordered of the condyle cartilage and subchondral bone structure, demyelination aggravated of nerve fibers in TMJ rats. Compared with rats in Control groups, the expression levels of SGK-1 in MCC and TG of rats in TMJ-OA group were upregulated. Conclusion In the pathological process of TMJ-OA, SGK-1 may plays an important role not only in cartilage structural damage but also in pain transmission.

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Objective To explore the high expression level of serum heat shock protein 90α(HSP90α) and gene HSP90 AA1 in cancer tissue, and to discover the prognosis of lung cancer. Methods A total of 109 cases of lung cancer were collected as the experimental group; 38 lung inflammation groups as the reference group; and 30 healthy controls. The serum HSP90α levels between the three groups were compared; the correlation between HSP90α and clinical parameters were analyzed. The TCGA data were used to analyze the correlation between the expression level of HSP90 AA1 and various pathological features, as well as its influence on the prognosis of lung cancers. Results The serum HSP90α concentrations in the experimental group were higher than those of the reference group and the control group(P<0.05). The ROC curve area of HSP90α in the diagnosis of lung cancer was 0.898(P<0.05); the expression of HSP90α in the lung squamous cell carcinoma group(LUSC) and the small cell lung cancer group(SCLC) were higher than those in lung adenocarcinoma group(LUAD)(P<0.05); the level of HSP90α decreased when condition alleviated, while increased significantly when disease progressed(P<0.05); Further TCGA database showed that the expression of HSP90 AA1 in cancer tissues was higher than that of adjacent cancer tissues(P<0.05),meanwhile, remarkably increased in LUSC compared with in LUAD(P<0.05). The expression of HSP90 AA1 has no significant correlation with the age, gender, clinical stage, and tumor residue of lung cancer patients. Survival analysis and Cox regression analysis showed that high expression of HSP90 AA1 reduced the overall survival(OS) of lung cancer patients; HSP90 AA1 was an independent prognostic factor for lung cancer patients. Conclusion Serum HSP90α and HSPAA1 in cancer tissues are elevated in patients with lung cancer, which should be used as an auxiliary diagnosis method for lung cancer. Meanwhile they should be used for therapeutic effect observation and survival status prediction.

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Objective To compare the efficiency of different methods for extracting rhesus monkey lung fibroblasts and their effects on functions, so as to provide a method for obtaining primary lung fibroblasts that are closer to human fibroblasts. Methods Two extraction methods for rhesus monkey lung fibroblasts were used, direct tissue block adhesion method and collagenase combined digestion with tissue block adhesion method. The cell morphology was observed with the inverted microscope, the purity of isolated rhesus monkey lung fibroblasts was identified by immunofluorescence, cell viability was detected by CCK-8, the expression of α-SMA was detected by flow cytometry and the effect of long-term in vitro culture on cell apoptosis was detected by apoptosis kit. Western blot was used to detect the expression of α-SMA protein. Results The combined digestion with collagenase and tissue block adhesion method could see small and bright cells crawling out in 24 hours, and cells could be seen crawling out in a large area after 48 hours. The cells were in a long spindle shape, after 4 days to 5 days, a single layer of cells could be formed. Identified by immunofluorescence, all cells expressed α-SMA. Tissue adhesion method showed small and bright cells crawling out after 72 hours. After 4 days to 5 days, the cells crawled out in a small area and showed a long spindle shape. After a week, the cells crawled out in a large area and formed a single layer of cells and the cells are all expressed α-SMA by immunofluorescence. The experimental results showed that the cell viability of the cells crawled out by the collagenase digestion method was significantly higher than that of the tissue adhesion method. After TGF-β1 stimulates the cells, the cells extracted by collagenase digestion method proliferated faster and expressed α-SMA more obviously. Conclusion Both methods can isolate rhesus monkey lung fibroblasts in vitro, but the collagenase digestion method extracts cells in a shorter time and in better condition. The expression of related proteins is more stable after stimulation by stimulants, which is an effective method to obtain rhesus monkey lung fibroblasts, and it is also an effective method to obtain primary lung fibroblasts that are closer to human.

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Objective To explore the inflammatory changes and the changes of β-amyloid in the brain of rats with experimental periodontitis induced by ligation. Methods Eighteen Sprague Dawley rats were randomly divided into 3 groups(n=6): the negative control group, chronic periodontitis group and chronic periodontitis treated with intraperitoneal injection of TAK-242 group. The experimental periodontitis model was established by ligation of the necks of bilateral maxillary first molar and inoculation ofPorphyromonas gingivalis(P.gingivalis). At the end of the second month after the successful modeling, the samples were collected from the rats. The damage of the alveolar bone was analyzed by Micro-CT. The mRNA expression levels of interleukin(IL)-6, IL-1β and tumor necrosis factor-α(TNF-α) in gingival tissue and hippocampal tissue, the mRNA expression level of Toll like receptors-4(TLR4), leukocyte differentiation antigen 14(CD14) and NF-κB in hippocampal tissue of rats were detected by qPCR. The protein expression levels of IL-6, IL-1β, TNF-α, myloid-beta protein-40(Aβ40) and Aβ42in hippocampal tissue of rats were evaluated by ELISA. Results Experimental periodontitis model of rats could be successfully established by ligation of the neck of the rat's bilateral maxillary first molars and inoculation with porphyromonas gingivalis. The results of qPCR and ELISA showed that experimental periodontitis up-regulated the expression levels of inflammatory factors(IL-6, IL-1β and TNF-α) in hippocampus of rats and the result of ELISA showed that the level of Aβ42in hippocampus of experimental periodontitis rats increased. But the pretreatment with TAK-242 intraperitoneal injection could reduce the up-regulated the expression of inflammatory factors and Aβ42by down-regulating the TLR4/NF-κB signaling pathway. Conclusion Experimental periodontitis in rats induced by ligation combined with inoculation of porphyromonas gingivalis can result in inflammation in the brain and promote the accumulation of Aβ42in the brain, and it is reasonable to speculate that inflammation may play an important role in the correlation between periodontitis and systemic diseases such as Alzheimer's disease.

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Objective To investigate the locations of sodium retention and the mechanism of its occurrence in the rat model of adriamycin nephrotic syndrome. Methods The nephrotic syndrome model was established by tail vein injections of adriamycin twice two weeks(4 mg/kg in first week and 2 mg/kg in second week). Urine and blood biochemical parameters were detected; the contents of water and sodium in rat skin were detected respectively by desiccation and atomic absorption spectrometry; the content of glycosaminoglycan was analyzed by ELISA; the expression of tonicity-responsive enhancer binding protein(TonEBP), vascular endothelial growth factor C(VEGFC), vascular endothelial growth factor receptor 3(VEGFR3) and lymphatic vessel hyaluronan receptor-1(LYVE-1) in the renal cortical was detected by Western blot; the lymphatic vessels distribution in the renal cortex was measured by immunofluorescence. Results the model rats had increased urinary protein excretion, and abnormal renal function and lipid, which suggested the model was successfully established. The excretion of Na+in urine decreased, the content of skin sodium and glycosaminoglycan in skin obviously increased, the expression of TonEBP, VEGFC, VEGFR3, LYVE-1 in renal cortical markedly increased, and density of lymphatic vessels in renal cortical notably increased(P<0.05 orP<0.01). Conclusion Sodium retained in nephrotic syndrome rats may be stored in the skin, which is related to the TonEBP/VEGFC/VEGFR3 pathway.

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Objective To investigate the effect and mechanism of α-lipoic acid(ALA) on the formation of hypertrophic scar(HTS) in rabbit ears. Methods Eighteen New Zealand white rabbits were randomly divided into normal group, model group and 4%ALA group. After 28 days of treatment, the changes of scar related indexes were evaluated by hematoxylin-eosin(HE) staining, Masson pine(Masson) staining and immunohistochemical staining, the changes of oxidative stress related indexes were evaluated, and the expression of antioxidant pathway related proteins was detected by Western blotting. Results The color, texture and volume of scar in 4%ALA group were lighter and softer than those in model group. The results of HE, Masson staining and immunohistochemical staining showed that the number of fibrous cells and the degree of fibrosis in the 4%ALA group were less than those in the model group. In the 4%ALA group, the content of malondialdehyde(MDA) was lower than that of the model group(P<0.001), the content of hydroxyproline(Hyp) was lower than that of the model group(P<0.01), the level of glutathione(GSH) was higher than that of the model group(P<0.01), the activity of T-SOD was higher than that of the model group(P<0.001). Western blotting showed that the content of NRF2 and the expression level of downstream antioxidant proteins in the 4%ALA group were higher than those in the model group. Conclusion ALA up-regulates NRF2 signal pathway to reduce the level of oxidative stress in HTS and inhibit scar proliferation in rabbit ears.

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Objective This research aimed to explore the application of the combination of region growth and U-Net in the retinal vessel segmentation system, retinal vessels were automatically segmented through the developed system. This research provides doctors with accurate information on changes in the retinal blood vessel structure, which will improve the efficiency of diagnosis and treatment. Methods Combined with U-Net network and region growth, the pre-processed retinal blood vessels were automatically segmented, and the algorithms were integrated into the retinal blood vessel segmentation system through the design of controls. Results The average values of blood vessel segmentation performance indexes— accuracy, sensitivity, and specificity were 0.977 7, 0.768 4, and 0.982 1, respectively, and regional iterative growth could improve the segmentation effect of fine retinal blood vessels. Conclusion The system has the characteristics of simple interface and convenient operation.It realizes automatic retinal blood vessel segmentation with high precision and visualization, provides an effective application platform for doctors to observe the changes of retinal vascular structure, and also provides a thinking direction for doctors to judge the nature of lesions.

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Objective To investigate the expression of uteroglobin-related protein 1(UGRP1) in thyroid cells induced by interleukin-1β(IL-1β) and its correlation with Fas/FasL mediated apoptosis. Methods Control group, IL-1β group and IL-1β+anti-FasL antibody group were established, rat thyroid cells(FRTL-5 cells) were culturedin vitro. The expression levels of UGRP1 and Fas mRNA of each group were detected by real-time PCR and the apoptosis rate of each group was detected by flow cytometry. Results Compared with control group, the mRNA expression levels of UGRP1 and Fas in IL-1β group and IL-1β+anti-FasL antibody group increased, and the difference was statistically significant(P<0.05). Compared with control group, the early apoptosis rate of thyroid cells in IL-1β group increased(7.49%±1.91%vs28.46%±3.17%), and the difference was statistically significant(P<0.001). Compared with IL-1β group, the early apoptosis rate of thyroid cells in IL-1β+anti-FasL antibody group decreased(28.46%±3.17%vs19.20%±1.75%), and the difference was statistically significant(P<0.05). Compared with IL-1β group, the expression levels of UGRP1 mRNA(2.22±0.31vs2.66±0.28) and Fas mRNA(2.75±0.18vs3.03±0.16) in IL-1β+anti-FasL antibody group were not significantly different(P>0.05). Conclusion IL-1β induces the high expression of UGRP1 and Fas and apoptosis of thyroid cells, the high expression of UGRP1 is not associated with Fas/FasL mediated apoptosis.

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Objective To explore the role of miR-30b in the trigeminal ganglion( TG) of trigeminal neuralgia( TN) model rats. Methods An infraorbital nerve-chronic constriction injury( ION-CCI) model of TN was produced in the rat. Von Frey hair was used to determine the time-course of changes in mechanical pain threshold.qRT-PCR and Western blot were used to detect the expression of miR-30b and activating transcription factor 3( ATF3) in the TG. In addition,miR-30b agomir and agomir NC were injected into ION-CCI rats by the infraorbital foramen injection. The mechanical pain threshold and the expression changes of ATF3 were determined after intervention. Results Compared with the sham-operated group,the ION-CCI group had a decreased mechanical pain threshold 2 days after surgery,reached the lowest observed level by day 12,and this threshold reduction lasted more than 28 days( P<0. 05). Compared with the sham-operated group,the ION-CCI group had a lower miR-30b expression and a higher ATF3 expression in postoperative TG( P<0. 05). After the overexpression of miR-30b in ION-CCI group,the mechanical pain threshold increased,and the expression of ATF3 decreased in TG( P<0. 05). Conclusion These results suggest that miR-30b may participate in the regulation of TN. miR-30b may be a potential therapeutic target for TN.

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Objective To investigate the effect of vitamin D on cognitive function in mice with chronic kidney disease(CKD) induced by adenine. Methods Fifty-six male C57 BL/6 J mice were randomly divided into five groups: control(CON), model(CKD), and low, medium and high vitamin D(LVD, MVD and HVD) supplement. The renal failure was induced in mice by chronic dietary adenine intake. Mice in the vitamin D-treated groups were injected with calcitriol(20, 100, 500 ng/kg) three times a week for six weeks. Then, blood samples were collected from the orbit, and serum creatinine, urea nitrogen and the activity of superoxide dismutase(SOD) were detected. Pathological changes were observed using HE and Masson staining. The cognitive function was assessed using the Morris water maze. Results Compared with CON group, serum creatinine and urea nitrogen increased in CKD group(P<0.05). Kidney damage such as glomerular atrophy, renal interstitial fibrosis could be observed in CKD group. In the probe trial testing, compared to CON group, the escape latency of mice in CKD group was longer(P<0.05). And the escape latency of MVD and HVD group was shorter than that in CKD group(P<0.05). In the hidden-platform trial, time and travel distance spent in the goal quadrant in CKD group was lower than that in CON group(P<0.05), and the time and distance in MVD and HVD group was higher than that in CKD group(P<0.05). The activity of SOD decreased in CKD group compared to CON group(P<0.05), and increased in LVD and MVD group compared to CKD group(P<0.05). Conclusion The cognitive function of CKD mice descends. Vitamin D can improve cognition in CKD mice through its antioxidant function.

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Objective To investigate the value of whole-body dynamic18F-flurodeoxygluse positron emission tomography with computed tomography(18F-FDG PET/CT) Patlak imaging for evaluating the radiosensitization early efficacy in C6 rat glioma. Methods Twenty-four C6 rat glioma models were established and randomly divided into control group, Oleanolic acid(OA) group, radiotherapy group and OA+radiotherapy group, with 6 rats in each group. 75 min whole-body dynamic18F-FDG PET/CT Patlak imaging was performed before and 48 h after treatment. The left ventricular time-activity curve(TAC) of tumor-bearing rats was used as input function for Patlak analysis to obtain Ki images. The last frame of the dynamic PET scan was selected for static analysis, and the changes of maximum standardized uptake value(SUVmax), Ki values and tumor volume were observed, and the survival time of tumor-bearing rats was recorded. Then, the tumor samples were dissected for Ki-67 and glucose transporter-1(GLUT-1) immunohistochemical staining. Results Before treatment, there were no significant differences in SUVmaxand Ki values among the 4 groups(F=0.909, 0.858,P>0.05). 48 h after treatment, SUVmaxand Ki values of control group and OA group increased significantly(t=-10.697,-17.516,P<0.01;t=-19.321,-9.887,P<0.01), SUVmaxand Ki values of radiotherapy group and OA+radiotherapy group decreased(t=5.629, 6.916,P<0.01;t=7.241, 10.490,P<0.01), there was no significant difference in SUVmaxbetween radiotherapy group and OA+radiotherapy group 48 h after treatment(t=1.233,P=0.246), while the Ki values in OA+radiotherapy group was significantly lower than that in radiotherapy group, the difference was statistically significant(t=3.051,P<0.05). The immunohistochemistry staining results suggested that the expression of Ki-67 and GLUT-1 in OA+radiotherapy group was lower than that in the other 3 groups(F=63.887, 40.720,P<0.05). In addition, Ki values showed positive correlations with the expression of Ki-67 and GLUT-1(r=0.931, 0.936,P<0.01). Kaplan-Meier analysis showed that the survival time of tumor-bearing rat in OA+radiotherapy group was longer than that in the other three groups, and the difference was statistically significant(χ2=19.817,P<0.01). Conclusion OA has radiosensitization effect on C6 rat glioma. Whole-body dynamic18F-FDG PET/CT Patlak multi-parameter imaging can evaluate the radiosensitization early efficacy in C6 rat glioma, which is more sensitive than PET/CT static imaging.

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Objective To observe the histomorphological features of the hypothalamic-pituitary-adrenal axis in macaques to provide a reference for simulating the physiological functions and pathological responses of the human hypothalamic-pituitary-adrenal axis. Methods After euthanasia of macaques, hypothalamus, pituitary and adrenal tissues were removed intact, fixed by PFA, and paraffin sections and frozen sections were prepared; the basic structure and cellular distribution were observed by HE staining; the secreted hormones and receptors were detected by immunohistochemistry; the effects of staining in frozen and paraffin sections were compared, and the cellular composition of some hypothalamus tissues was identified. Results The hypothalamic region was hollow and funnel-shaped, the pituitary gland resembles a pea, and the right and left adrenal glands were located between the liver and kidneys; HE staining showed that the hypothalamic region was mainly composed of neurons and microglia, the pituitary gland was divided into neuro-pituitary and adeno-pituitary, and the adrenal gland was composed of cortex and medulla; immunohistochemical results showed that the hypothalamus secretes CRH and expresses GR, the pituitary gland secretes ACTH and expresses CRHR1 and GR, and the adrenal gland expresses ACTHR; immunofluorescence of frozen sections better showed that the hypothalamus contains neurons and microglia. Conclusion In this study, sections of hypothalamus, pituitary and adrenal gland tissues from macaques were successfully produced, and the relevant anatomical and morphological features were observed and examined, which provided a reference method for simulating the physiological and pathological responses of the human hypothalamic-pituitary-adrenal axis.

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Objective To investigate the relationship between the ubiquitin ligase E3 cirRNA(circHERC4/hsa＿circ＿0007113) and the senescence in Human embryonic lung fibroblasts(IMR-90)cells. Methods The whole gene sequence of circHERC4 was obtained from circBase database and cloned into cirRNA expression vector pLC5-ciR. The recombinant pLC5-ciR(+) hsa＿circ＿0007113 was constructed in vitro. The recombinant plasmid was identified by PCR, enzyme digestion and sequencing. The recombinant plasmid was transfected into HEK293 T and IMR90 cells with liposome 2000 transfection reagent. The expression of circHERC4 in normal cells, empty vector group and recombinant vector group was detected by RT-PCR, and the senescence of cells was detected by SA-β-Gal staining. Cell proliferation was detected by CCK-8 method. Results The overexpression vector of circHERC4 was successfully constructed by correct sequencing, and circHERC4 could be efficiently transfected in HEK293 T cells. Compared with the control group, the positive rate of SA-β-Gal staining in the recombinant vector group decreased(P<0.05), and the proliferation rate of cells increased(P<0.05). Overexpression of circHERC4 could improve the proliferation and inhibit the senescence in IMR90 cells. Conclusion It suggests that circHERC4 has potential function of anti-senescence, which lays a foundation for further study on the function of circHERC4.

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Objective To investigate the effect and mechanism of liposome curcumol( LC) on cisplatin resistance of human ovarian cancer cells. Methods Ovarian cancer cisplatin resistant cell line SKOV3/DDP was used. Cells were divided into five groups: blank control group,negative control group( containing 12. 5 μg/ml liposome),cisplatin group( 4 μg/ml),LC group( 20 μg/ml),and combined group( cisplatin( 4 μg/ml) + LC( 20 μg/ml).Cell proliferation was detected by CCK8 assay,the content of cisplatin in cells was detected by high performance liquid chromatography-tandem mass spectrometry( HPLC-MS),and apoptosis was detected by flow cytometry. The level of P-glycoprotein( P-gp) mRNA was detected by qRT-PCR and the expression of P-gp protein was detected by immunohistochemistry and Western blot. Results The cell proliferation ability of the combination group was the lowest while the apoptosis rate was the highest( P<0. 05) among the groups. HPLC-MS showed that the content of cisplatin in combined group was higher than that in cisplatin group( P<0. 05). P-gp mRNA and protein in the combined group were down-regulated( P<0. 05). Conclusion LC can reduce cisplatin resistance of human ovarian cancer cell line SKOV3/DDP by inhibiting the expression of P-gp.

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Objective To investigate the protective effect and mechanisms of silent information regulator 1(SIRT1) agonist resveratrol on monocrotaline-induced Hepatic sinusoidal obstruction syndrome(HSOS) in rats. Methods Thirty-two male Sprauge-Dawley(SD) rats were randomly divided into three group, with 8 rats in the control group and 12 rats in each of the monocrotaline group and resveratrol group. The monocrotaline group and resveratrol group were given monocrotaline(160 mg/kg) by single gavage. The resveratrol group was intraperitoneally injected with resveratrol solution [30 mg/(kg·d)] one day before intragastric administration. The experiment was terminated 2 days after the monocrotaline administration. Serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBiL), glutathione(GSH) and malondialdehyde(MDA) in liver homogenate were detected. The pathological change of liver tissue was observed. The protein expression levels of SIRT1, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF) in liver were detected by Westernblot. Results Compared with the control group, the serum ALT, AST and TBiL in monocrotaline group increased(allP<0.01), GSH in liver homogenate decreased(P<0.01), MDA increased(P<0.01). Disordered arrangement, degeneration and necrosis of liver cells, congestion and dilation of hepatic sinuses and damage of central vein endothelium were observed. SIRT1 decreased(P<0.01), HIF-1α and VEGF protein expression increased(allP<0.01). After resveratrol treatment, serum ALT, AST and TBiL obviously decreased(P<0.05;P<0.05;P<0.01). GSH in liver homogenate increased(P<0.01), MDA decreased(P<0.01). And resveratrol also could inhibit the liver pathological damage caused by monocrotaline. SIRT1 protein expression increased(P<0.01), HIF-1α and VEGF protein expression also decreased(allP<0.01). Conclusion Resveratrol can improve Hepatic sinusoidal obstruction syndrome caused by monocrotaline in rats, and the mechanism is related to its activation of SIRT1, inhibition of HIF-1α/VEGF signaling pathway and antioxidanion.

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Objective To investigate the mechanism of cathepsin L(CTSL)-mediated kidney injury in trichloroethene(TCE)-sensitized mice. Methods 41 BALB/C mice were randomly divided into blank group(n=5), solvent group(n=5), TCE treatment group(n=15) and TCE+CTSLi treatment group(n=16). TCE percutaneous sensitization mouse model was established, and the mice were evaluated as positive group and negative group according to skin sensitization score. The renal pathology of mice was observed by electron microscopy and HE staining, the renal function level of mice was assessed by serum urea nitrogen(BUN). The expression of CTSL was detected by immunofluorescence, the apoptosis of renal cells was assessed by TUNEL staining, and the activation of renal protein kinase C(PKC) signal molecule was detected by Western blot. Results The sensitization rates of TCE treatment group and TCE+CTSLi treatment group were 53.3%(8/15) and 50.0%(8/16), respectively, and there was no statistical difference in sensitization rates(P>0.05). Pathological results showed that TCE sensitized-mice showed edema and vacuolar degeneration of renal tubular cells, thickening of glomerular basement membrane, fusion of podocytes and mitochondria vacuolar degeneration. The results of renal function showed that the serum BUN level of TCE sensitized mice was higher than that of other groups. Immunofluorescence results showed that the expression level of CTSL in the kidney of TCE-sensitized positive mice increased(P<0.05,F=82.438), and the apoptosis level of renal structure cells was also higher than that of other groups(P<0.05). Western blot showed that the phosphorylation of PKC protein in the kidney of TCE-sensitized mice increased, while the expression of PKC protein in TCE+CTSLi sensitized mice was down-regulated after CTSLi pretreatment(P<0.05,F=35.686), the level of renal cell apoptosis decreased, and renal damage was improved. Conclusion CTSL might aggravate renal damage via activation of PKC signaling in TCE-sensitized mouse.

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Objective To investigate the mechanical property,biocompatibility,and osteogenic differentiation ability of chitosan-β-TCP( CS-β-TCP) scaffold,meanwhile,to study the possibility of the composite as a scaffold to repair bone defect. Methods Briefly,the CS-β-TCP composite scaffold was fabricated utilizing bidirectional lyophilization technique. Then,the scaffold micro-structure was observed by scanning electron microscopy( SEM),X-ray diffraction( XRD) and energy disperse spectroscopy( EDS) were employed to analyze the ingredients and elements distribution of scaffold,respectively. Additionally,the compression strength of the scaffold was tested by mechanical universal testing machine. The biocompatibility of the scaffold and the cell viability research were characterized via CCK-8 assay and Live/Dead staining,respectively,and the cell adhesion was studied by DPAI/Phalloidine fluorescence staining. qRT-PCR was employed to investigate the expression level of osteogenic-related gene such as BMP2,RUNX2 and COL1. ALP staining was carried out to measure the osteogenic differentiation effect of BMSCs. Results The CS-β-TCP scaffold was comprised of bulk parallel,aligned and thin lamellas with many porous structures. β-TCP particles were evenly distributed over CS framework layers and the CS-β-TCP scaffold possess excellent elastic property and biocompatibility,moreover,the cell seeded on scaffold revealed high cell viability and continuous proliferation. qRT-PCR and ALP staining results demonstrated that the CS-β-TCP scaffold could induce osteogenic differentiation of BMSC. Conclusion To sum up,the CS-β-TCP scaffold expressed desired mechanical and biological properties,and could induce BMSC differentiate into osteoblast,the composite scaffold provided a promising strategy for bone defect regeneration.

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Objective To investigate the role of miR-15 a-5 p in DOX resistance of lung cancer cells, and to elucidate the relationship between miR-15 a-5 p and DOX resistance. Methods miR-15 a-5 p inhibitor and miR-15 a-5 p mimics were used to transfect A549 and A549/DOX resistant cells(A549/D). MTT assay was used to detect cell viability, flow cytometry was used to detect cell apoptosis, Western blot was used to detect the expression of EMT related protein and P53 protein, and QRT PCR was used to detect the expression of miR-15 a-5 p. The potential target genes of miR-15 a-5 p were analyzed by bioinformatics prediction, dual luciferase reporter. A549/D cells were used to establish the xenograft tumor model in nude mice, and the effect of miR-15 a-5 p overexpression on DOX in vivo was analyzed. Results MTT analysis showed that the knockdown of miR-15 a-5 p increased the cell viability of A549 cells(IC50value: 8.86±0.32 μmol/L), and the overexpression of miR-15 a-5 p decreased the cell viability of A549/D cells(IC50value: 1.92±0.11 μmol/L). Blocking miR-15 a-5 p in A549 cells reduced apoptosis(P<0.001), and increasing the expression of miR-15 a-5 p in A549/D cells promoted apoptosis(P<0.001). The role of DOX in regulating the EMT was reversed by the transfection of miR-451 a inhibitor through Western blot. Bioinformatics prediction showed that there was a specific binding site between P53 and miR-15 a-5 p. miR-15 a-5 p inhibition reduced the expression of P53 protein in A549 cells(P<0.001), and miR-15 a-5 p over-expression increased the expression of P53 protein in A549/D cells(P<0.01). In vivo experiments showed that combination of agomir-miR-15 a-5 p and DOX could reduce the tumor volume and the expression level of N-cadherin, and enhance the expression levels of P53 and E-cadherin. Conclusion miR-15 a-5 p over-expression may inhibit EMT by targeting P53 and enhance the sensitivity of lung cancer cells to DOX therapy.

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Objective To detect the difference in the incidence of small intestinal bacterial overgrowth(SIBO) in patients with liver cirrhosis and chronic hepatitis fibrosis(CHF) with the lactulose hydrogen breath test(LHBT), and to explore the relationship between SIBO and inflammatory factors and oxidative stress related indicators. Methods LHBT was performed on 38 CHF patients, 60 cirrhosis patients and 31 healthy controls to evaluate the incidence of SIBO. The patients were further divided into SIBO-positive and negative group. Then we compared related clinical symptoms and laboratory tests between the two groups and detected lipopolysaccharide(LPS), interleukin(IL)-6, tumor necrosis factor(TNF)-α, IL-10 level, and several kinds of oxidative stress indicators such as diamine oxidase(DAO), superoxide dismutase(SOD), glutathione(GSH), catalase(CAT). Statistical analysis was conducted to analyze the correlation between the concentration of LPS, IL-6, TNF-α, DAO, SOD, GSH, CAT and LHBT summation value. Results (1) The positive rate of SIBO in CHF group, cirrhosis group and control group was 36.84%, 60.00% and 9.68%, respectively. The difference was statistically significant(P<0.01).(2) The differences in CTP classification and ascites were statistically significant between the two subgroups with and without SIBO(P<0.05).(3) The levels of LPS, IL-6, CAT, DAO and SOD in SIBO-positive group were higher than those in negative group(P<0.05). However, the concentrations of IL-10, TNF-α and GSH were similar between the two groups.(4) The LHBT summation value was positively correlated with the concentrations of LPS, IL-6, DAO, SOD and CAT in serum(P<0.05), but had no significant correlation with TNF-α, IL-10 and GSH. Conclusion Compared with healthy controls, CHF and cirrhosis patients are more likely to develop SIBO. It is also related to the increase of inflammatory factors and oxidative stress indexes in peripheral blood. SIBO may aggravate the inflammatory response of CHF and cirrhosis patients through intestinal flora dysregulation and oxidative stress, thus aggravating the disease change.

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Objective To explore the predictive value of CHADS2and CHA2DS2-VASc scores for late recurrence after catheter ablation( CA) of atrial fibrillation. Methods 92 patients with atrial fibrillation of CA who hospitalized in the Second Affiliated Hospital of Anhui Medical University for the first time from July 2018 to December2020 were collected. Results From the 3rd postoperative month to the 6th postoperative month,atrial fibrillation outpatients should follow-up each month. 92 patients were collected,of which 3 were missing during follow-up,89were followed up,and late postoperative recurrence ratio was 24%. Compared with patients without recurrence,recurrent patients were significantly higher in age,smoking( 30%),diabetes mellitus( 38%),heart failure( 29%),platelet count,left atrial diameter( LA),CHADS2,and CHA2DS2-VASc scores; furthermore Logistic regression analyzed variance factors that were not included in the score: CHADS2( OR = 13. 473,95% CI: 3. 804-47. 713),LA( OR = 1. 193,95% CI: 1. 049-1. 356)( P<0. 05); CHA2DS2-VASc( OR = 5. 363,95% CI: 2. 822-10. 193),LA( OR = 1. 245,95% CI: 1. 087-1. 426)( P<0. 05). ROC curves predicted the values of two sets of scores for late recurrence after CA,AUC( CHADS2) = 0. 8578( 95% CI: 0. 7618-0. 9539),AUC( CHA2DS2-VASc) = 0. 8848( 95% CI: 0. 7985-0. 9711). Conclusion LA,CHADS2and CHA2DS2-VASc scores are independent risk factors for late recurrence after CA of atrial fibrillation,and CHA2DS2-VASc scores have better predictive value.

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Objective This study is designed to compare the changes of intestinal microflora and cytokine levels in patients with schizophrenia in different periods, analyze the correlation between intestinal microflora and disease symptoms, and explore the influence of intestinal microflora changes on the symptoms of patients with schizophrenia. Methods 40 schizophrenic patients in different periods were included in the study, with their demographic data of age, body mass index(BMI), sex and course of disease collected. For each subject, serum was first collected for the levels of cytokines to be determined by an Meso Scale Discovery. The severity of schizophrenia was then assessed using negative and positive symptom scales. Finally macrofactor sequencing of intestinal flora was performed using MetaGeneMark. Results Serum levels of interleukin-6(IL-6), interleukin-10(IL-10), interleukin-17(IL-17), interleukin-23(IL-23), and tumor necrosis factor-α(TNF-α) in the acute stage were higher than those in remission stage, and the results were statistically significant(P<0.05). Negative and positive symptom Scale(PANSS) positive factor scores were negatively correlated with TNF-α and IL-17(R=-0.312,P<0.05;R=-0.399,P<0.05); IL-23 was positively correlated with the negative factor score of the scale(R=-0.344,P<0.05). IL-6 was positively correlated with scale cognitive factor score(R=-0.339,P<0.05). IL-23 was positively correlated with the total score of the scale(R=-0.370,P<0.05). The microbial diversity detected in stool samples of patients with acute schizophrenia was lower than that of patients with remission, and certain difference was detected in the intestinal flora species composition between patients in the acute stage and in the remission stage. Conclusion The level of serum cytokines in the acute stage of schizophrenia is higher than that in the remission stage, and some cytokines levels are correlated with clinical symptoms.

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Objective The main purpose of this study is to explore the relationship between hepafin-binding epidermal growth factor-like growth factor(HB-EGF) and urokinase-type plasminogen activator receptor(uPAR) with the pathogenesis of preeclampsia. Methods Firstly, the differentially expressed proteins were screened by antibody chip technology, a large number of literatures were searched, and uPAR and HB-EGF were selected for verification. Serum samples from 30 normal pregnant women and 50 preeclampsia pregnant women were collected, and the expressions of HB-EGF and uPAR in serum were determined by ELISA. Placental tissues of 10 preeclampsia pregnant women and 6 normal pregnant women were selected for further validation by qRT-PCR and immunohistochemistry. Results The expression of u PAR in serum in preeclampsia pregnant women was higher than that in normal pregnant women( P<0. 05),and the average level of HB-EGF in serum was lower than that of normal pregnant women( P<0. 05). The results of qRT-PCR showed that the expression level of u PAR mRNA in placenta was low or no,and the expression level of HB-EGF mRNA in preeclampsia pregnant was lower than that in normal pregnant women( P<0. 05). Immunohistochemistry showed that the expression of u PAR and HB-EGF in the placenta of preeclampsia pregnant was lower than that of normal pregnant women. Conclusion The abnormal expression of HBEGF and u PAR is related to preeclampsia. This study proves that HB-EGF and u PAR are related to the pathogenesis of preeclampsia,and the difference in the expression of HB-EGF and u PAR in blood may play a guiding role in the early diagnosis of biomarkers.

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Objective To investigate the correlation between clinicopathologic features like human epidermal growth factor receptor type 2(HER2) status and lymph node metastasis, meanwhile, to establish a prediction model for the risk of lymph node metastasis in early gastric cancer. Methods Multiple data of 157 patients who underwent early gastric cancer radical gastrectomy were involved and analyzed. Further, the risk prediction model was established for lymph node metastasis of early gastric cancer. Results Among these 157 patients, 31 cases were reported lymph node metastasis, with a rate of 19.7%. Analysis showed that female patients, HER2 IHC(3+), infiltrating into submucosa, poor degree of differentiation and vascular invasion accounted for a large proportion in lymph node metastasis(P<0.05). Analysis also showed that HER2 IHC(3+), infiltrating into submucosa, poor degree of differentiation and vascular invasion were independent risk factors for lymph node metastasis in early gastric cancer(P<0.05). According to the above analysis results, the prediction models for lymph node metastasis risk of early gastric cancer were established respectively included or excluded HER2 status, and the area under the ROC curve(AUC) was 0.800 and 0.759, respectively. Meanwhile, differentiated tumor accounted for a large proportion in HER2 IHC(3+)(P<0.05). Conclusion HER2 IHC(3+), infiltrating into submucosa, poor degree of differentiation and vascular invasion were independent risk factors of lymph node metastasis in early gastric cancer. The new established model including HER2 status has good sensitivity and specificity, which may provide a reference for predicting the risk of lymph node metastasis in early gastric cancer.

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Objective To explore the level of secreted phosphoprotein 24(SPP24) and its association with cardiac valve calcification(CVC), cardiovascular events(CVE) in patients with hemodialysis patients. Methods Eighty-eight maintenance hemodialysis patients were enrolled in the study. All patients were assessed for cardiac valve calcification by echocardiography. According to the results of echocardiography, patients were divided into two groups: cardiac valve calcification group and the group without cardiac valve calcification. The levels of SPP24 were measured by enzyme-linked immunosorbent assay. Serum SPP24 levels were compared between CVC group and the group without cardiac valve calcification. Binary logistic regression was used to evaluate the association between SPP24 and cardiac valve calcification. The factors for calcification of the mitral and aortic valves were explored. The cases were followed up and cardiovascular events were recorded. COX regression model was used to analyze the factors for cardiovascular events. Results There were 47 patients in CVC group, the levels of SPP24 in CVC group were lower than those in the group without cardiac valve calcification(P=0.040). Twenty-two patients had calcification of the mitral and aortic valves. Logistic regression analysis showed that older age(OR=1.055), hyperphosphatemia(OR=8.234) were risk factors for CVC, higher SPP24(OR=0.997) was a protective factor. Older age(OR=1.086) and hyperphosphatemia(OR=7.393) were risk factors for calcification of the mitral and aortic valves, higher SPP24(OR=0.964) level was a protective factor. Patients were followed up, the follow-up intervals were from 2 to 14 months, and it was found that CVC(HR=4.156) increased the risk of cardiovascular events in hemodialysis patients, high SPP24 level(HR=0.976) reduced the risk of cardiovascular events. Calcification of the mitral and aortic valves increased the risk for cardiovascular events(HR=3.071). Conclusion The incidence of cardiac valve calcification in maintenance hemodialysis patients is as high as 53.41%. Older age and hyperphosphatemia are risk factors for CVC, while high SPP24 level is a protective factor for CVC. CVC is an independent risk factor for cardiovascular events, and high SPP24 level is a protective factor for cardiovascular events.

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Objective To quantity the degree of acromial droop tilt and explore its correlation and diagnostic value for subacromial impingement syndrome. Methods The degree of quantification of shoulder tilt and droop(including Angle α、β、γ、Δ、ε、ζ) was analyzed by preoperative MRI of 40 patients undergoing arthroscopic surgery(experimental group) and 40 healthy shoulder MRI(control group).The correlation and difference of each quantitative index in each group and group were compared to discuss their diagnostic value for rotator cuff injury and subacromial impingement syndrome. Results There was no significant difference between the patients group and the control group in the acromion droop and tilt correlation the independent sample T test of angle α(P>0.05), respectively, and the difference was statistically significant(P<0.05). The region below the ROC curve of β,γ,Δ,ε,ζ were 0.677, 0.864, 0.707, 0.848, 0.886, respectively, and the region below the ROC curve of β and ζ combined was 0.906, indicating excellent diagnostic performance. Conclusion Acromial droop and tilt correlation angle have certain value in the diagnosis of subacromial impaction syndrome. The quantitative indexes γ,ε,ζ have significant significance in the diagnosis of SAIS, and the combined diagnosis performance of β and ζ is better.

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Objective To compare the short-term efficacy and long-term survival rate of radical radiotherapy combined with concurrent chemotherapy and sequential chemotherapy in esophageal cancer patients, and to investigate the dominant population of esophageal cancer patients who were suitable for concurrent chemoradiotherapy. Methods Two hundred and eighty patients with esophageal cancer treated with radical radiotherapy from July 2015 to June 2020 were recruited, including 140 patients with concurrent chemotherapy and 140 patients with sequential chemotherapy. The short-term efficacy and long-term survival rate between two groups were compared. Kaplan-Meier survival analysis, Logistic regression analysis and Cox regression analysis were used to analyze the risk factors for short-term efficacy, long-term survival rate and the dominant population suitable for concurrent chemoradiotherapy. Results There was no significant difference about the short-term efficacy between the two groups(P>0.05), but in patients with TNM<Ⅳ, the complete remission(CR) rate in concurrent chemotherapy group(18.6%) was higher than that in sequential chemotherapy group(7.7%)(χ2=5.079,P=0.024). Among patients with midpiece esophageal cancer, CR rate in concurrent chemotherapy group was higher than that in sequential chemotherapy group(20.0%vs6.7%, χ2=4.498,P=0.034). The CR rate in patients with TNM<Ⅳ and midpiece esophageal cancer was higher in concurrent chemotherapy group(21.2%) than that in sequential chemotherapy group(2.2%)(χ2=7.459,P=0.006). Results of Kaplan-Meier survival analysis showed that the total survival time(4.0 years) and the 3-year survival rate(73.2%) in concurrent chemotherapy group were higher than those in sequential chemotherapy group(2.8 years, 24.2%)(P<0.000 1,P<0.000 1). Logistic regression analysis showed that patients with TNM<Ⅳ and midpiece esophageal cancer(OR=11.85) had higher CR rate in group with concurrent chemoradiotherapy(P<0.05). Cox regression analysis showed that concurrent chemoradiotherapy was a protective factor for death in patients with esophageal cancer(HR=0.448,P<0.000 1). Concurrent chemoradiotherapy was also a protective factor for death in patients with TNM<Ⅳ(HR=0.286), with midpiece esophageal cancer(HR=0.499), with midpiece esophageal cancer and TNM<Ⅳ(HR=0.218), with non-esophageal involvement all around(HR=0.384), with TNM<Ⅳ and non-esophageal involvement all around(HR=0.197), and with TNM<Ⅳ and non-ulcer type(HR=0.266,P<0.05). Conclusion Compared to patients with esophageal cancer treated with sequential chemoradiotherapy, patients treated with concurrent chemoradiotherapy have higher CR rate and long-term survival rate. Patients with TNM<Ⅳ, midpiece tumor location, non-esophageal involvement all around and non-ulcer type esophageal are the superior population for concurrent chemoradiotherapy.

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Objective To investigate the risk factors affecting the occurrence of complications in patients with vaginal bleeding after in vitro fertilization/intracytoplasmic sperm injection embryo transfer(IVF/ICSI-ET) and the construction of nomogram prediction model. Methods A total of 272 patients with threatened abortion after IVF/ICSI-ET were retrospectively analyzed in this study. They were divided into the live birth group and abortion group according to the final outcome of pregnancy. Patient characteristics were evaluated using the chi-square test, independent-samples Student's t-test or Wilcoxon rank sum test. A nomogram was created to predict the pregnancy outcome of women with threatened abortion who received IVF/ICSI using multivariate logistic regression coefficients. Results There was no significant difference in the basic data, percentage of frozen embryos, treatment method, number of embryos transferred, high-quality embryo rate, and embryo implantation rate of the live birth group and abortion group(P>0.05). There were significant differences in body mass index, the onset of vaginal bleeding time after transplantation, serum levels of hCG and progesterone on 14 th day after embryo-transfer, and the number of gestational sacs between the two groups(P<0.05). After multivariate logistic regression analysis, the onset of vaginal bleeding time after transplantation and serum hCG levels on 14 th day after transfer were statistically significant(P<0.05). The nomogram was established based on the above indicators, with an area under the curve of 0.710 for the nomogram. The area under the ROC curve of our nomogram was better than the area under the ROC curve of a single risk factor(AUC of bleeding time after embryo-transfer: 0.644, AUC of serum hCG14:0.625). Conclusion The nomogram model established based on the onset of vaginal bleeding time after embryo-transfer and serum hCG value on 14 th day after embryo-transfer can better predict pregnancy outcome of patients with threatened abortion after IVF/ICSI-ET.