Abstract:Objective:　To observe the effect of curcumin on autophagic flux in Neuro-2a mouse brain neuroma cells of expressing APP695swe stably (N2a/APP695swe) and explore the potential mechanism. Methods:　Firstly, cells were divided into 4 groups: wild type group (WT), blank control group (Control), solvent control group (Sham) and curcumin group (Curcumin). Transmission electron microscopy (TEM) was performed to observe the morphological characteristics of autophagosomes in each group, Western blot was used to test the expressions of p62 and LC3Ⅱproteins. Meanwhile, Western blot and immunofluorescence staining were performed to detect the expression of Rab7 protein, and quantitative real-time PCR (qRT-PCR) was carried out to analyze the mRNA level of Rab7. Then cells were divided into 5 groups after transfection of pcDNA3.1, pcDNA3.1-Rab7, NC-siRNA and Rab7-siRNA respectively: Control group, pcDNA3.1 group, pcDNA3.1-Rab7 group, NC-siRNA group and Rab7-siRNA group. The morphological characteristics of autophagosomes were observed by transmission electron microscopy, and the levels of p62 and LC3Ⅱproteins were detected by Western blot, too. Results:　After treatment of curcumin, autolysosomes with monolayer were observed by TEM. And the expression of LC3Ⅱprotein was increased (P=0.000), while the expression of p62 was decreased (P=0.000). Meanwhile, the expressions of Rab7 were increased at both protein and mRNA levels (P=0.000, 0.000). After over-expression of Rab7, more autolysosomes were observed, and the expression of p62 protein was reduced (P=0.000), while the expression of LC3Ⅱprotein did not change significantly (P=0.669). After transfection of Rab7-siRNA in N2a/APP695swe cells, autophagosomes with double membranes were observed visibly. The expression of p62 protein was elevated (P=0.000), while the expression of LC3Ⅱprotein did not change prominently (P=0.622). Conclusion:　Curcumin stimulates autophagy flux in N2a/APP695swe cells, and the mechanism may be related to the enhancement of Rab7.

Abstract:Objective:　To investigate the effect of Aβ on lysosomes in neurons and microglia of Alzheimer’s disease (AD) model mice. Methods:　Six 6-month-old APP/PS1 double transgenic AD mice and six wild type (WT) mice were selected to observe the expression of neurons and microglia, as well as the co-expression of neurons, microglia and lysosomes by immunofluorescence staining. Western blot was used to detect the expression of autophagy related proteins. After SH-SY5Y cells were treated with Aβ25-35, adenovirus overexpressed transcription factor EB (TFEB) was intervened for 72 h. The levels of TFEB and lysosomal markers were detected by Western blot. Results:　Compared with wild-type mice, the expression of LC3 and p62 in AD transgenic mice at 6 months old increased (t=4.976, P=0.008; t=4.463, P=0.011), and the lysosomal marker protein LAMP1 decreased significantly (t=14.91, P=0.000 1). In AD model group, neurons were significantly lost, microglia were significantly proliferated and lysosome expression was significantly decreased. Compared with wild-type mice, the expression of p-mammalian target of rapamycin (p-mTOR) /mTOR protein in AD transgenic mice was significantly increased (t=5.501, P=0.005), and the expression of TFEB protein was significantly decreased (t=5.899, P=0.004). Aβ25-35 down regulated the protein levels of TFEB and Lamp1 in SH-SY5Y cells (t=10.94, P<0.001; t=17.22, P<0.001). Overexpression of TFEB significantly increased the protein level of lysosomal marker Lamp1 (t=5.136, P<0.01). Conclusion:　Aβ can decrease lysosomes in neurons and microglia of AD model mice, which may act by inhibiting mTOR/TFEB signaling pathway.

Abstract:Objective:　To study the regulatory effect of SIRT3/MnSOD/ROS signaling pathway on osteogenic differentiation of adipose derived stem cells (ADSC). Methods:　In this study, we first cultured ADSC isolated from rat inguinal fat pads, and ADSC were identified by surface markers (CD29, CD44, CD34, CD45) and they could be induced to osteogenic and adipogenic differentiation. During osteogenic differentiation induction in vitro, expressions of SIRT3 were studied by western blotting. We used Lenti-SIRT3shRNA transfection to knockdown SIRT3 within ADSC, osteogenic capacities were identified by ARS staining, osteogenic differentiation-related genes Runx2, ALP, OCN mRNA expressions were assessed by qPCR, and the activity of MnSOD and reactive oxygen species (ROS) levels within ADSC were tested using kits. Results:　The expression of SIRT3 mRNA (7 d: 1.71±0.03, 15.21±0.17, P=0.014, n=5; 14 d: 1.44±0.04, 9.70±0.18, P=0.002, n=5) and protein (7 d: 0.70±0.01, 2.99±0.07, P=0.012, n=5; 14 d: 1.00±0.08, 2.74±0.50, P=0.009, n=5) increased during osteogenic differentiation of ADSC. Lenti-SIRT3 shRNA transfection could inhibit SIRT3 mRNA expression (7 d: 1.02±0.05, 0.36±0.01, P=0.011, n=5; 14 d: 1.04±0.08, 0.30±0.02, P=0.002, n=5) and protein expression (7 d: 0.88±0.04, 0.29±0.01, P=0.034, n=5; 14 d: 1.18±0.03, 0.43±0.01, P=0.014, n=5), with decreased MnSOD activities (0.91±0.13, 0.29±0.01, P=0.002, n=5) and increased ROS levels (7 d: 1.00±0.10, 2.66±0.32, P=0.013, n=5; 14 d: 1.24±0.10, 2.59±0.31, P=0.014, n=5). The expression of corresponding target genes Runx2, ALP and OCN related to osteogenesis decreased (Runx2: 1.07±0.02, 0.19±0.01, P=0.001, n=5; ALP: 1.09±0.02, 0.53±0.01, P=0.001, n=5; OCN: 0.95±0.02, 0.33±0.01, P=0.004, n=5). Inhibition of osteogenic differentiation, induced by knockdown of SIRT3, could be reversed by pretreatment of NAC (ROS: 2.63±0.20, 1.07±0.19, P=0.010, n=5). Conclusion:　SIRT3 within mitochondria plays vital roles in promoting osteogenic differentiation of ADSC, by mediating enhancement of MnSOD activities and reduction of intracellular ROS levels.

Abstract:Objective:　To observe the efficacy of programmed cell death 1 (PD-1) immunotherapy combined with bevacizumab for double-target anti-angiogenic therapy on stageⅣlung adenocarcinoma, and to analyze its influence on cellular immune function, short-term prognosis and adverse reactions. Methods:　A total of 67 patients with stageⅣlung adenocarcinoma admitted to our hospital between December 2018 and August 2020 were selected in this study and they were divided into control group (n=33) and combined group (n=34) according to different treatment methods. Control group were treated with chemotherapy+PD-1 inhibitor, and combined group were treated with chemotherapy+PD-1 inhibitor+bevacizumab. The short-term treatment efficacy, changes of cellular immune function and Karnofsky performance status (KPS) score before and after treatment, adverse reactions and progression-free survival time were compared between the two groups. Results:　The total effective rate of treatment and disease control rate of combined group were 29.41%and 79.41%respectively, which were significantly higher than 9.09%and 54.55%of control group (P<0.05). After treatment, the CD3⁺, CD4⁺and CD4⁺/CD8⁺in combined group were significantly higher than those in control group (P<0.05), and there was no significant difference in CD8⁺compared to control group (P>0.05). The increase and stable rate of KPS score of patients in combined group was significantly higher than that in control group (73.53%vs.45.45%) (P<0.05). There were no significant differences in the incidence of adverse reactions such as bone marrow suppression, gastrointestinal reactions, blood toxicity, liver-kidney functional damage and peripheral neurotoxicity in combined group compared with those in control group (P>0.05). The median progression-free survival time were 9.73 months in combined group and were 6.05 months in control group, with significant differences (P<0.05). Conclusion:　PD-1 immunotherapy combined with bevacizumab for double-target anti-angiogenic therapy of stageⅣlung adenocarcinoma can significantly enhance the short-term treatment efficacy, improve the immune function, promote the quality of life and prolong the progression-free survival time.

Abstract:Objective:　To investigate the effect and mechanism of fibroblast growth factor 21 (FGF21) on the proliferation and activation of stellate cells in the inflammatory environment of pancreatic cancer cells. Methods:　The appropriate time to establish an inflammatory microenvironment was determined by RT-qPCR to detect interleukin (IL) -6 and IL-8 mRNA expression level of cholecystokinin (CCK) on human pancreatic cancer cells (PCC) such as Capan-1 and MIA PaCa-2. PCC (Capan-1, MIA PaCa-2) CCK supernatant and PCC (Capan-1, MIA PaCa-2) CCK+FGF21 supernatant were acted on human pancreatic stellate cells (PSC). CCK-8 and Western blot were used to detect the effect of each group’s supernatant on the activity of pancreatic stellate cells and the expression of α-smooth muscle protein (α-SMA), a marker of activation. RT-qPCR and Western blot were used to detect the effect of FGF21 on the expression of PCC EGR1 and PDGFB mRNA and protein after CCK treatment. Western blot further detected the effect of AMPK inhibitor (compound C) added to PCC CCK group and PCC CCK+FGF21 group on the expression of p-AMPK and EGR1 protein in AMPK-κB signaling pathway. Results:　The relative expressions of IL-8 mRNA in Capan-1 6 h group and in MIAPaCa-2 24 h group treated with CCK were significantly higher than those in other groups (P<0.05). Compared with the PCC CCK supernatant group, the PCC CCK+FGF21 supernatant group had significantly inhibited the proliferation of PSC cells (P<0.05), the expression of α-SMA protein was significantly reduced (P<0.001). Intervention with FGF21, EGR1, PDGFB mRNA and protein expressions were significantly lower than those in CCK group (P<0.05). After compound C was added to the PCC CCK+FGF21 group, the expression level of p-AMPK protein decreased (P<0.01), and the expression level of EGR1 protein expression increased (P<0.05). Conclusion:　FGF21 may prevent tumor by inhibiting the proliferation and activation of stellate cells in the inflammatory environment of pancreatic cancer cells and the mechanism may be related to its activation of the AMPK pathway of pancreatic cancer cells in the inflammatory environment, down-regulation of EGR1 expression and reduction of PDGFB expression.

Abstract:Objective:　To explore the role of osteopontin (OPN) in inhibiting fibrosis after intrauterine adhesions (IUA) in mice. Methods:　Primary bone marrow mesenchymal stem cells (MSCs) of C57 BL/6 mice and OPN^-/-C57 BL/6 mice were isolated and cultured, and the surface antigens of separated cells were detected by flow cytometry. Thirty female mice were randomized into sham operation group, model group, OPN^-/-MSCs group, MSCs group, and solvent group, with 6 mice in each group. One week after modeling, MSCs and OPN^-/-MSCs were injected into the left and right uterine horns of the mice respectively. Two weeks after the treatment, two-dimensional ultrasound was conducted for the diagnosis of mouse uterus morphology, HE staining and Masson staining for the detection of the morphological changes and fibrosis of the endometrium of the uterus, immunohistochemical staining for the detection of the expression of α-smooth muscle actin (α-SMA), and Western blot for the detection of the protein expression of transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), typeⅠcollagen α1 (COL1A1), and typeⅢcollagen α1 (COL3A1). Results:　MSCs and OPN^-/-MSCs cells were successfully isolated and cultured, and expression of CD29, CD44 and CD90 were positive and expression of CD34 was negative in both groups. Compared with the model group, the morphological changes of the uterus in the OPN^-/-MSCs group and the MSCs group were improved, the number of glands increased in varying degrees, the area of fibrosis decreased, and the positive expression of α-SMA decreased significantly (P=0.000). TGF-β1, VEGF, COL1A1 and COL3A1 protein expression levels were significantly decreased (all P=0.000). Compared with the OPN^-/-MSCs group, the uterine morphology of the MSCs group improved more markedly, the number of glands increased, and the area of fibrosis decreased, the positive expression of α-SMA decreased significantly (P=0.000), and the expression levels of TGF-β1, VEGF, COL1A1 and COL3A1 proteins also decreased significantly (all P=0.000). Conclusion:　MSCs are more effective than OPN^-/-MSCs in inhibiting intrauterine adhesions and endometrial fibrosis in mice, indicating that OPN has the effect of regulating bone marrow mesenchymal stem cells to inhibit endometrial fibrosis of intrauterine adhesions in mice.

Abstract:Objective:　To observe the influence of proanthocyanidins (OPC) on serum cardiac troponinⅠ (cTn-Ⅰ), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-px) on the expression level of RIHD rats, as wells as their possible effects and mechanism of action. Methods:　Sixty male sprague dawley (SD) rats were randomized into 6 groups, with 10 rats in each group, namely normal control group, simple irradiation group, OPC low dose (50 mg/kg) group, OPC middle dose (100 mg/kg) group, OPC high-dose (150 mg/kg) group, and blueberry (200 mg/kg) group. In addition to the normal control group, the rats in the other groups were given 6 MV X-ray single high-dose irradiation to the heart, with a total dose (Dt) of 20 Gy, and the rats in the OPC group and blueberry group were intragastrically administered the corresponding drugs. Four weeks after the radiotherapy, hematoxylin eosin (HE) staining and Masson staining were used to observe the pathological changes of myocardial tissue in rats, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of serum cTn-Ⅰ, SOD, MDA, GSH-px. Results:　Compared with the normal control group, the pathological scores and myocardial collagen volume fraction (CVF) scores of the simple irradiationgroup, the OPC group, and the blueberry group increased (P<0.05); compared with the simple irradiation group, the pathological scores and CVF scores of the OPC group and the blueberry group increased (P<0.05), with significant differences. Compared with the normal control group, the expression levels of cTn-Ⅰand MDA in the simple irradiation group, OPC group, and blueberry group increased, while the expression levels of SOD and GSH-px decreased; compared with the simple irradiation group, the expression levels of cTn-Ⅰand MDA in the OPC group and blueberry group increased, while the expression levels of SOD and GSH-px decreased, with significant differences (P<0.05). Conclusion:　OPC can inhibit the oxidative stress, down-regulate the expression levels of serum cTn-Ⅰand MDA in RIHD rats, and up-regulate the expression levels of serum SOD and GSH-px, thus inhibiting the myocardial damage in RIHD rats, with a better curative effect than the blueberry.

Abstract:Objective:　To investigate the effect of irisin on the function and apoptosis of pancreatic β cells induced by palmitic acid (PA). Methods:　Firstly, INS-1 cells were cultivated and treated with different concentration gradients of irisin. The optimal concentration of 1 500 ng/mL was selected according to the protein results of Western blot. Secondly, INS-1 cells were divided into the normal group, the PA group, the palmitate acid+1 500 ng/mL irisin group, the palmitate acid+1 500 ng/mL irisin+rapamycin group and the palmitate acid+1 500 ng/mL irisin+3-MA group. The glucose-stimulated insulin secretion (GSIS) test (with glucose levels of 3.3 mmol/L and 16.7 mmol/L) was performed to determine the insulin level in each group. Western blot was used to determine the levels of mamlian target of rapamycin (mTOR), p-mTOR, p62, LC3Ⅱand LC3Ⅰ; mRFP-GFP-LC3 and flow cytometry were used to determine the autophagic flow and cell apoptosis, respectively. SPSS 20.0 was used to conduct data analysis. Results were presented as the mean±SD, one-way analysis of variance followed by Turkey HSD was used to calculate differences among the various study groups. Results:　①Compared with the normal group, the ratio of LC3Ⅱ/Ⅰin the PA group was higher (0.745±0.035 vs.1.015±0.012, P=0.000). With the increase of irisin concentration, the level of LC3Ⅱ/Ⅰwas gradually increased and had statistical significance (I1: 1.465±0.028, I2: 2.269±0.063, I3: 1.858±0.074, I4: 2.355±0.074; P=0.000). When irisin concentration was 1 500 ng/mL, the level of LC3Ⅱ/Ⅰwas the highest.②GSIS levels in the INS-1 cell supernatant in the PA group was significantly reduced for stimulation of 3.3 mmol/L and 16.7 mmol/L of glucose when compared with the normal group [(0.447±0.067) ng/mL vs. (0.138±0.012) ng/mL, P=0.000; (0.625±0.041) ng/mL vs. (0.245±0.043) ng/mL, P=0.000], while in the irisin treated group were increased when compared with the PA group [(0.138±0.012) ng/mL vs. (0.322±0.041) ng/mL, P=0.001; (0.245±0.043) ng/mL vs. (0.414±0.036) ng/mL, P=0.005].③The number of autophagosome in the PA group was significantly higher than that in the normal group (2.33±1.53 vs.10.33±1.53, P=0.000), but there was no obvious change in the number of autophagolysosome (2.33±1.53 vs.3.33±1.53, P=0.939). In the irisin group, the autolysosome number and autolysosome number was increased when compared with PA group (10.33±1.53 vs.15.67±1.53, P=0.007; 3.33±1.53 vs.12.00±2.00, P=0.001). Treated with PA for 24 hours, the apoptosis rate of INS-1 cells was increased when compared with the normal group [(11.417±1.297) %vs. (46.507±0.525) %, P=0.000], while the irisin-treated group had a significantly reduced apoptosis rate when compared with the PA group [(46.507±0.525) %vs. (33.767±1.083) %, P=0.000]. Conclusion:　Irisin ameliorates apoptosis and improves secretion function of INS-1 cells induced by PA through autophagy.

Abstract:Objective:　To investigate the potential molecular mechanism of fine particulate matter (PM_2.5) on activation of NLRP3 inflammasome and apoptosis in rat alveolar macrophages. Methods:　Rat alveolar macrophages were treated with PM_2.5suspension collected by medium flow atmospheric sampler. After action of the suspension on rat alveolar macrophages (NR8383), MTT assay was used to detect the cell survival rate. Western blot was performed to detect the expression of NLRP3, Cathepsin-B and apoptosis-related proteins (Bax, Bcl-2, Caspase-3). The level of Caspase-1, interleukin-18 (IL-18) and interleukin-1β (IL-1β) in the cell supernatant induced by PM_2.5were measured by ELISA method. Results:　With the increase of PM_2.5concentration and the extension of stimulation time, the survival rate of NR8383 decreased (24 h: F=17.253, P=0.000; 48 h: F=18.678, P=0.000; 72 h: F=256.104, P=0.000); PM_2.5accelerated the expression of NLRP3 (F=437.166, P=0.000) and Cathepsin-B (F=42.062, P=0.000) in NR8383; PM_2.5 up-regulated the expression of pro-apoptotic protein Bax (F=72.827, P=0.000), down-regulated the expression of anti-apoptotic protein Bcl-2 (F=390.322, P=0.000), and down-regulated the expression of Caspase-3 (F=169.833, P=0.000); PM_2.5increased the level of Caspase-1 (F=629.866, P=0.000), IL-18 (F=587.165, P=0.000) and IL-1β (F=68.472, P=0.000) in cell culture medium. Conclusion:　PM_2.5can activate NLRP3 inflammasome in alveolar macrophages through the lysosomal model, promote the secretion of IL-18 and IL-1β, and cause apoptosis of alveolar macrophages.

Abstract:Objective:　To investigate the effect of vitamin D₃on inflammation, helper T cell 1/helper T cell 2 (Th1/Th2) and monocyte chemoattractant protein-1/cell surface chemokine receptor 2 (MCP-1/CCR2) signal in experimental autoimmune thyroiditis (EAT) rats. Methods:　Lewis rats were randomly divided into the control group (n=10), the model group (n=15) and the treatment group (n=15). The rat model of EAT was established by immunizing porcine thyroglobulin (pTG). The treatment group received intraperitoneal injection of 1, 25- (OH) ₂vitamin D₃at the dose of 5 mg/kg, once every other day for consecutive four weeks, and the other two groups were given normal saline of equal volume. The morphological changes of thyroid tissue in rats were observed by hematoxylineosin (HE) staining. Serum thyroid stimulating hormone (TSH), anti-thyroglobulin antibody (TGAb), anti-thyroid peroxidase antibody (TPOAb) and cytokines interferon-γ (IFN-γ), interleukin-12 (IL-12), interleukin-14 (IL-4) and interleukin-10 (IL-10) were detected by enzyme-linked immunosorbent assay (ELISA). The relationship between TSH and autoantibodies and cytokines was analyzed by stepwise linear regression. NF-κB p65 in thyroid tissue was detected by immunohistochemistry. MCP-1/CCR2 signal axis in thyroid tissue was analyzed by Western blot. Results:　In the model group, thyroid follicles were damaged and inflammatory cells were infiltrated, while the pathological changes of thyroid in the treatment group were significantly improved and tended to the control group. The levels of TSH, TGAb and TPOAb in the treatment group were significantly lower than those in the model group (t=4.000, 2.603, 5.279; P=0.000, 0.015, 0.000), but were still significantly higher than those in the control group (t=2.400, 2.216, 7.392; P=0.025, 0.037, 0.000). Compared with the model group, the levels of IL-4 and IL-10 were significantly increased in the treatment group (t=3.522, 2.432; P=0.001, 0.022), and the levels of IFN-γ, IL-12 and IFN-γ/IL-4 and IL-12/IL-10 were significantly decreased in the treatment group (t=2.940, 4.700, 5.416, 8.178; P=0.007, 0.000, 0.000, 0.000), which tended to the levels of the control group. Stepwise regression analysis showed that TSH was positively correlated with TGAb, TPOAb, IFN-γ and IL-12, and negatively correlated with IL-4 and IL-10 (r=0.872, 0.868, 0.731, 0.706, -0.557, -0.236, all P<0.001), among which TGAb, TPOAb and IL-12 had the most significant effect on TSH. Compared with the model group, the levels of NF-κB p65, MCP-1 and CCR2 in the thyroid tissues of rats in the treatment group were decreased, with statistically significant differences (t=2.432, 2.267, 5.143; P=0.000, 0.031, 0.000). Conclusion:　Vitamin D₃may inhibit NF-κB pathway to down-regulate Th1 cytokines and up-regulate Th2 cytokines, thereby reducing Th1/Th2 ratio. Or, it reduces thyroiditis in EAT rats, improves thyroid function, inhibits thyroid antibody production and protects thyroid by inhibiting MCP-1/CCR2 signaling axis.

Abstract:Objective:　To investigate the effect of aldehyde dehydrogenase 2 (ALDH2) on monocyte (THP-1) -human aortic endothelial cells (HAECs) adhesion and its mechanisms. Methods:　HAECs was divided into the Control group, ALDH2 NC group, ALDH2 group, siRNA-ALDH2 NC group, siRNA-ALDH2-1, -2, -3 groups. The expression of ALDH2 protein and mRNA was detected by Western blot and real-time PCR. The adhesive ability between THP-1 and HAECs was detected by cell adhesion assay. The transcription of nuclear factor-kappa B (NF-κB) was detected by Western blot and immunofluorescence. Results:　Compared with the Control group and the ALDH2 NC group, the expression of ALDH2 was up-regulated in the ALDH2 group (P<0.05). Compared with the Control group and the siRNA-ALDH2 NC group, the expression of ALDH2 was down-regulated in the siRNA-ALDH2-1, -2, -3 groups (P<0.05), and the expression of ALDH2 was the lowest in the siRNA-ALDH2-2 group. Compared with Control group and ALDH2 NC group, cell adhesive ability, the expression of NF-κB in nucleus and the red fluorescence was decreased (P<0.05), and the expression of NF-κB in cytoplasm increased in ALDH2 group (P<0.05). Compared with Control group and siRNA-ALDH2 NC group, cell adhesive ability, the expression of NF-κB in nucleus and the red fluorescence was increased (P<0.05), the expression of NF-κB in cytoplasm decreased in siRNA-ALDH2 group (P<0.05). Conclusion:　ALDH2 could inhibit the adhesion between THP-1-HAECs by inhibiting NF-κB nuclear transcription, and finally alleviate atherosclerosis.

Abstract:Objective:　To investigate the effect of miR-503 on migration and invasion in human melanoma cells A375 and its mechanism. Methods:　The real-time quantitative PCR (qRT-PCR) was used to detect the expression level of miR-503 in melanoma tissues and cell lines; miR-503 mimics or miR-503 inhibitors were used to perform transient transfection to A375 cells, and Transwell assay was used to test cell migration and invasion abilities. The potential binding site of miR-503 on the CXCL9 was predicted by using online bioinformative databases, and the result was verified by double luciferase assay. The migration and invasive ability of A375 cells were detected after transfected with CXCL9 alone or in combination with miR-503. Results:　Expression of miR-503 was significantly downregulated in melanoma tissues when compared with adjacent normal tissues (t=6.602, P=0.000). In addition, expression of miR-503 in the three melanoma cell lines (A375, SK-MEL-1 and SK-MEL-5) was significantly lower than that in the HEM cells (F=19.445, P=0.000). Migration and invasion abilities in A375 cells after transfection of miR-503 mimics were significantly inhibited (P=0.017, P=0.049), while in A375 cells after transfection of miR-503 inhibitors were promoted (P=0.004, P=0.000). CXCL9 was the direct target gene of miR-503 and promoted the migration and invasion of A375 cells (P=0.000, P=0.009), of which effectiveness was able to be reversed by miR-503. Conclusion:　The miR-503 suppresses migration and invasion of melanoma cells by targeted-regulating CXCL9.

Abstract:Objective:　To reveal the whole branches and distribution pattern of facial and anterior cervical cutaneous nerves visible to the naked eye via improved Sihler’s staining. Methods:　Chinese formalin-fixed cadavers, including 12 adults and 6 children, were used. Skin specimens containing subcutaneous fat were taken from the facial and anterior cervical regions and were stained with modified Sihler’s staining. Results:　It was found that the facial skin was not only innervated by trigeminal nerve, but also by branches of facial nerve. The anterior cervical skin was innervated by branches of nerve from muscles besides the anterior cervical cutaneous nerve. After Sihler’s staining of the facial and anterior cervical specimens, the overall distribution pattern of cutaneous nerve branches, which was not able to be shown by gross anatomy, was visible to the naked eye. The passage of each cutaneous nerve, arborized branches and distribution range were clearly visible. In frontotemporal junction area, infraorbital area and mental area, close link between facial nerve branches and trigeminal nerve was obvious. At the midline, there existed links between left and right nerve branches. The distribution range of transverse cervical nerve and supraclavicular nerve was relatively large, and both of them were linked. The transverse cervical nerve was able to innervate the lower lip area. Conclusion:　These results can provide morphological guidance for maxillofacial surgery, plastic surgery and cosmetic surgery to avoid cutaneous nerve injury, for material selection and matching of sensory reconstruction, and local anesthesia blocking.

Abstract:Objective:　To observe the effect of ursolic acid (UA) on the biological activity of A549 cells derived from human pulmonary epithelial cells under lipopolysaccharide (LPS) and its potential mechanism. Methods:　The CCK-8 method was used to detect the effect of UA (10, 50, 100, 150 and 200 μg/mL) at different concentrations on the proliferation of A549 cells. In addition, the effect of 10 ug/mL UA on the proliferation of A549 cells at different time points was also detected. The qPCR was used to detect the mRNA expression of interleukin (IL) -1β, IL-6 and tumor necrosis factor-α (TNF-α) in A549 cells under LPS. The effect of UA on the expression of IL-1β in A549 cells under LPS was detected by ELISA. Results:　UA was able to significantly inhibit the activity of A549 cells. Results of CCK-8 showed that 10 μg/mL UA was able to significantly inhibit the activity of A549 cells after 24 hours of UA treatment (P<0.05). In addition, UA was able to significantly inhibit the mRNA expression of TNF-α, IL-1β and IL-6 induced by LPS (P<0.05). Among them, the inhibition effect of LPS-induced IL-1β was the most obvious. Results of ELISA showed that UA to inhibit inflammation was not influenced by ERK and JNK signaling pathway inhibitors, but p38 signaling pathway inhibitors was able to significantly reverse the inhibitory effect of UA on LPS-induced IL-1β (P<0.05). However, p38 signal pathway activator was able to reverse the inhibition effect (P<0.05). Conclusion:　UA can inhibit the LPS-induced IL-1β and can exerts function through p38 pathway.

Abstract:Objective:　To investigate the association of fragmented QRS complex (fQRS) with cardiac autonomic nerve function and left ventricular remodeling in patients with acute ST-segment elevation myocardial infarction (STEMI). Methods:　A retrospective analysis was performed on 170 STEMI patients hospitalized in the cardiology department of our hospital. According to the presence of fQRS on the ECG, patients were divided into nfQRS group (n=94) and fQRS group (n=76). Basic clinical data, 24 h dynamic ECG indicators and echocardiography data were analyzed to compare the differences between the patients of the two groups. Results:　Compared with the nfQRS group, the values of standard deviation of NN intervals (SDNN), standard deviation of all 5-min average NN intervals (SDANN), very low frequency (VLF), low frequency (LF), ratio of low frequency to high frequency power (LF/HF) of heart rate variability (HRV) in the fQRS group were significantly decreased (P<0.05); the left ventricular ejection fraction (LVEF) of left ventricular function of the fQRS group was significantly lower than that of the nfQRS group, while the left ventricular end-diastolic dimension (LVEDD) and N-terminal pro brain natriuretic peptide (NT-proBNP) values were significantly higher than those of nfQRS group (P<0.01). Multiple logistic regression analysis showed that after adjusting for age and gender, reduced SDNN, SDANN, VLF, LF, LF/HF, LVEF and increased NT-proBNP were risk factors for fQRS in STEMI patients. Spearman correlation analysis showed that SDNN, SDANN, VLF, LF, LF/HF, LVEF were significantly negatively correlated with fQRS in STEMI patients, while LVEDD, NT-proBNP were significantly positively correlated with fQRS (P<0.01). Conclusion:　In STEMI patients with fQRS on ECG, myocardial ischemia is serious, cardiac autonomic nerve function damage is obvious, and left ventricular remodeling is significant.

Abstract:Objective:　To explore the diagnostic value of prediction model established on high resolution computed tomography (HRCT) imaging features in elderly hip fracture patients with early acute lung injury (ALI). Methods:　We retrospectively collected the data from 301 elderly patients with hip fracture. All patients were divided into two groups according to the presence of ALI, including ALI group (n=121) and non-ALI group (n=180). Univariate and multivariate analyses were used to compare the sex, age, and chest HRCT data between the two groups. The HRCT performance of the ALI group was observed, and the prediction model of HRCT was established for diagnosis of elderly hip fracture patients with early ALI. Results:　The HRCT signs of patch shadow, intense parenchymal opacification (IPO), diffuse ground glass opacity (DGGO) and interlobular septal thickening were significantly more common in the ALI group than in the non-ALI group (P<0.05). The prediction model of HRCT was established using multivariate logistic regression, on the basis of following independent risk factors: IPO, DGGO, and interlobular septal thickening (OR>1, P<0.05). For diagnosis of elderly hip fracture patients with early ALI, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this model were 93.4%, 85.6%, 88.7%, 81.3%, 95.1%, respectively. Conclusion:　The prediction model based on HRCT imaging characteristics is helpful to diagnose elderly hip fracture patients with early ALI, providing direct and reliable imaging information for clinical diagnosis and treatment.

Abstract:Objective:　To investigate the effect and prognosis of preservation of the subvalvular apparatus on left ventricular functions in mitral valve replacement. Methods:　Retrospective analysis was made among 92 patients who underwent mitral valve replacement in The First Affiliated Hospital of Chongqing Medical University from March 2016 to September 2019. Those patients were divided into two groups, among which, 48 cases preserved the posterior and subvalvular apparatus of mitral valve (experimental group), while the other 44 cases underwent traditional mitral valve replacement (control group). Parameters of left ventricular function of echocardiographic measurements were compared between experimental group and control group at preoperative, early postoperative period (7 days after surgery) and late postoperative period (3 months, 6 months after surgery), and perioperative conditions, etc. Results:　The application time of vasoactive drugs in the experimental group was significantly shorter than that in the control group [(4.25±1.19) days vs. (5.64±1.63) days, P<0.05], and the incidence of complications in the experimental group (8.34%) was significantly lower than that in the control group (25.00%) (P=0.031). Six months after the operation, the left atrial diameter (LAD) [(32.64±3.76) mm], left ventricular end diastolic diameter (LVEDD) [(43.60±3.68) mm]and left ventricular end systolic diameter (LVESD) [(31.81±4.73) mm]were significantly lower in the experimental group than those in the control group. The left ventricular ejection fraction (LVEF) [(64.86±4.23) %]and left ventricular fraction shortening (LVFS) [(36.04±2.90) %]were significantly higher in the experimental group than in the control group (P<0.05). Conclusion:　Mitral valve replacement with preservation of subvalvular apparatus is beneficial for renewing cardiac function and a short-term improvement of left ventricular function.

Abstract:Objective:　To investigate the influence of apolipoprotein AⅠ (apoAⅠ) at admission on the prognosis of patients with pulmonary embolism (PE). Methods:　A total of 161 patients who were hospitalized with initial diagnosis of PE in our hospital from August 1, 2013 to August 15, 2019 were retrospectively screened. The receiver operating characteristic (ROC) curve was used to calculate the optimal cut-off value of apoA I and patients were divided respectively into 2 groups which contained the low level group and the high level group. Follow-up was started with the admission time, and the deadline was September 15, 2019. The end-event was all-cause death. Kaplan-Meier method was applied to draw the survival curves and log-rank test was used to compare two curves. Univariate and multivariate Cox proportional hazards regression model analysis was used to analyze variables included apoAⅠwhich potentially influenced the prognosis. Results:　During the follow-up period, 39 patients died by all-cause, with fatality rate of 24.22%. The optimal cut-off of ROC was 0.965 g/L and the area under the curve (AUC) was 0.716 (95%CI=0.616-0.815). The cases of dead patients in the high level apoAⅠgroup (≥0.965 g/L, n=112), and low level apoAⅠgroup (<0.965 g/L, n=49) were 15 and 24, respectively. The fatality rate of patients in the low level group was 48.98%and in the high level group was 13.39%. Univariate Cox proportional hazards regression model analysis showed that age, total cholesterol, malignant tumor, the use of statins and the level of apoAⅠhad significance to prognosis of patients (P<0.2). Multivariate Cox proportional hazards regression model analysis concluded that age (P=0.001), the use of statins (P=0.003), malignant tumor (P=0.001) and level of apoAⅠ (P=0.004) were independent factors influencing PE prognosis, indicating that apoAⅠ<0.965 g/L was of predictive value to the prognosis of patients with PE. Log-rank test indicated that survival rate in the low level group was significantly lower than that in the high level group, with statistically significant difference (P=0.000). Conclusion:　The decrease of apoAⅠlevel detected in admission is related with the increase of mortality risk, which can predict the prognosis of patients with PE.

Abstract:Objective:　To study the difference in the cuspal incline angle of normal and cracked teeth by cone beam computer tomography (CBCT), providing a theoretical basis for the rapid and non-invasive diagnosis of cracked teeth. Methods:　According to the case inclusion criteria, 22 patients were included in the cracked teeth group (11 maxillary first molars and 11 mandibular first molars) and 44 patients were included in the normal teeth group (22 maximum first molars and 22 mandibular first molars). After 3D reconstruction by CBCT scans, oblique angles of the mesial, buccal, lingual and mesial angles of the maxillary first molars were measured. Results:　Oblique angles of the mesial and buccal tongue tips of the maxillary first molars in two groups were 106.70°±7.69°and 119.50°±7.97°, respectively, and of the distal and middle buccal and tongue tips were 109.30°±7.15°and 115.00°±7.48°. Oblique angles of the mesial and buccal tongue tips of the mandibular first molars were 104.40°±6.81°and 117.20°±9.25°, and of the distal and middle buccal and tongue tips were 105.60°±7.78°and 114.70°±8.35°. Oblique angle of the cusp in the cracked tooth group was smaller than that in the normal group (P<0.05). Conclusion:　After detecting the oblique angle, it is found that the smaller the angle, the greater possibility of the occurrence of cracked teeth is, which provides a theoretical basis for the early detection of tooth cracks by CBCT.

Abstract:Objective:　To explore the clinical application value of endoscopic nasobiliary drainage (ENBD) in the treatment of left hepatolithiasis complicated with choledocholithiasis under simultaneous triscopy (laparoscopy, choledochoscopy and duodenoscopy). Methods:　A total of 49 patients with left hepatolithiasis complicated with choledocholithiasis with or without cholecystolithiasis treated in the Department of Hepatopancreatobiliary Surgery of The Second People’s Hospital of Chengdu from January 2014 to July 2018 were analyzed retrospectively. All patients underwent laparoscopic regular hepatic lobectomy and laparoscopic choledocholithotomy. Among them, 22 cases were treated with ENBD (observation group), and 27 cases were treated with T-tube drainage (control group). The clinical effects of these two ways were comparatively analyzed. The observation indicators were perioperative period and the follow-up situations. The follow-up was made by telephone. SPSS 20.0 statistical software was used for the data analysis. Results:　The postoperative exhaust time in the observation group was earlier than that in the control group, which was (57.4±13.4) h and (67.9±12.7) h, respectively, with statistical significance (t=-2.818, t=0.007, all P<0.05). The postoperative tube-taking time of the observation group was (4-12) d, which was significantly shorter than that of the control group (75-95) d, with statistical significance (Z=6.022, Z=0.000, all P<0.05). Conclusion:　In view of the limited case study in our hospital, ENBD carried out in patients with left intrahepatic hepatolithiasis complicated with choledocholithiasis has the advantages of accelerating the recovery time of gastrointestinal function and shortening the tube-taking time, indicating that it’s a safe and effective surgical method.

Abstract:Objective:　To evaluate the value of ultrasound (US) combined with magnetic resonance imaging (MRI) in diagnosing fetal chest and abdomen abnormalities. To propose and discuss the clinical significance of structural malformation score (SMS) based on chromosome examination results. Methods:　A retrospective study was conducted on 77 fetuses who were examined by US and MRI from March 2016 to September 2019 and met the inclusion criteria. The indications of US combined with MRI were discussed, and the sensitivity, specificity, positive predictive value and negative predictive value of ultrasound, MRI and ultrasound combined with MRI were calculated. The receiver operating characteristics (ROC) curve was used to analyze the value of ultrasound and MRI physicians in the diagnosis of fetal chest and abdomen abnormalities. According to the chromosome results, the SMS for the chest and abdomen of the fetus was determined by the percentage of chromosomal positive cases. Results:　①The limitation of ultrasound application was the main reason for increasing MRI examination.②Follow-up of 77 pregnant women showed normal (negative) fetuses in 27 cases (35.1%) and abnormal (positive) fetuses in 50 cases (64.9%), 25 cases (32.5%) were negative and 52 cases (67.5%) were positive for US, and 28 cases (36.4%) were negative and 49 cases (63.6%) were positive for MRI. The sensitivity, specificity, positive predictive value, and negative predictive value of US combined with MRI were 96.0%, 92.6%, 96.0%and 92.6%, respectively, which were significantly higher than those of US alone and MRI alone. The area under the ROC curve (AUC) for US diagnosis was 0.792, the AUC for MRI diagnosis was 0.819, and AUC for US combined MRI diagnosis was 0.943. Compared with single US and single MRI, the difference was statistically significant (Z=2.23, P=0.026; Z=1.97, P=0.049). ③According to the calculation of the positive percentage of chromosome, SMS was divided into 5 grades, namely 0≤1 score<25%, 25%≤2 scores<50%, 50%≤3 scores<75%, 75%≤4 scores<100%, and 100%=5 scores. When 4 scores≤SMS<10 scores, clinical chromosome or gene testing was recommended, and when SMS≥10 scores, termination of pregnancy was recommended if clinically necessary. Conclusion:　Joint diagnosis of ultrasound and MRI has the highest comprehensive diagnosing efficiency, and precise diagnosis and accurate assessment scoring play an important role in guiding clinical decision-making.

Abstract:Objective:　To study the surgical effect of radiofrequency ablation combined with left atrial appendage closure in treating valvular disease complicated with atrial fibrillation. Methods:　A total of 120 patients with cardiac mitral valve disease and atrial fibrillation in our hospital were collected and divided into the study group-patients after mitral valve surgery with concomitant radiofrequency surgical ablation and left atrial appendage closure (56 cases), and the control group-patients undergoing only mitral valve surgery (64 cases) according to the type of operation. Operation indicators, sinus rhythm restoration rates, cardiac function, correlation factors in blood and complications were observed. Results:　The operation time and cardiopulmonary bypass time in the study group were longer than those in the control group (t=3.129, P=0.002; t=5.873, P=0.000), and other indexes were not different. The sinus rhythm restoration rate in the study group was 83.3%at the end of the operation, 57.4%at the 3rd day after operation, 59.3%before discharging hospital, 72.2%at the 3rd month after operation, 74.1%at 6th months after operation and 75.9%at one year after operation; the sinus rhythm restoration in the control group were 14.5%at the end of the operation, 11.3%at the 3rd day after operation, 11.3%before discharging hospital, 8.1%at the 3rd month after operation, 9.7%at the 6th month after operation and 8.1%at one year after operation, with statistically significant differences (P=0.000). The cardiac function and factors in blood in the study group were better than those in the control group. According to follow-up, the incidence of complications (cerebral infarction and hemorrhage) in the study group was lower than that of the control group (χ²=4.551, P=0.033; χ²=5.716, P=0.017). Conclusion:　Radiofrequency ablation combined with left atrial appendage closure in treating valvular disease complicated with atrial fibrillation is safe, with significance in preventing thromboembolism.

Abstract:Objective:　To investigate the correlation between the expression of serum miR-125b-5p and homocysteine (Hcy), hypersensitive C-reactive protein (Hs-CRP) and matrix metalloproteinase-9 (MMP-9) in elderly patients with acute ischemic stroke (AIS), and to provide evidence for the diagnosis and treatment of AIS. Methods:　The study involved in 174 cases of AIS who admitted to the Third People’s Hospital of Hainan Province from January 2016 to May 2019. The National Institutes of Health Stroke Scale (NIHSS) score were used to divide the patients into mild group (n=51, NIHSS score<5 points), moderate group (n=79, 5 points≤NIHSS score≤20 points), and severe group (n=44, NIHSS score>20 points). Another 65 healthy subjects were selected as control group. Real-time fluorescent quantitative PCR was used to detect the expression of serum miR-125b-5p in each group. The diagnostic value of serum levels of miR-125b-5p, Hcy, Hs-CRP and MMP-9 for AIS patients were analyzed by ROC curve. Pearson correlation analysis was conducted to analyze the correlation of serum levels of miR-125b-5p with Hcy, Hs-CRP and MMP-9 in AIS patients. Results:　The levels of serum miR-125b-5p [(2.74±0.95) vs. (0.68±0.13)], Hcy [(19.26±5.37) μmol/L vs. (5.84±1.20) μmol/L], Hs-CRP [(6.85±1.73) mg/L vs. (1.04±0.28) mg/L]and MMP-9 [(150.75±30.82) μg/L vs. (78.62±11.40) μg/L]in AIS group were significantly higher than those in control group (t=14.217, P=0.000; t=13.475, P=0.000; t=10.624, P=0.000; t=9.806, P=0.000). The serum levels of miR-125b-5 [(4.10±1.18) vs. (2.62±0.84) and (1.70±0.53)], Hcy [(28.35±6.80) μmol/L vs. (18.42±4.93) μmol/L and (11.50±3.27) μmol/L], Hs-CRP [(11.92±3.25) mg/L vs. (6.14±1.57) mg/L and (2.76±0.63) mg/L]and MMP-9 [(225.80±47.63) μg/L vs. (142.52±26.80) μg/L and (86.50±12.62) μg/L]in severe group were significantly higher than those in moderate group and mild group (F=10.845, P=0.000, F=8.716, P=0.000, F=5.184, P=0.003, F=4.972, P=0.010). The AUC (95%CI) of serum miR-125b-5p in diagnosis of elderly AIS was 0.846 (0.787-0.903), which was significantly higher than that of Hcy[0.754 (0.693-0.812)], Hs-CRP[0.728 (0.672-0.788)] and MMP-9[0.695 (0.634-0.753)]. When the optimal critical value of miR-125b-5p was 2.10, the sensitivity and specificity were 88.0%and 80.7%. Relevant analysis showed that serum levels of miR-125b-5p were positively correlated with levels of Hcy, Hs-CRP and MMP-9 in elderly patients with AIS (r=0.836, 0.745, 0.702, P<0.01). Conclusion:　Serum miR-125b-5p expression level is significantly increased in AIS patients, and correlated with Hcy, Hs-CRP and MMP-9 levels, which may be used as biomarkers for the diagnosis of AIS in elderly patients.

Abstract:Objective:　To investigate the effect of different concentrations of remifentanil on the effective concentration 50% (EC₅₀) and the anesthesia quality in patients receiving target controlled infusion (TCI) of propofol. Methods:　One hundred and fifty patients who received endoscopic mucosal resection (EMR), with ASA physical statusⅠ/Ⅱand ages of (59.11±10.08) years old, were randomly divided into four groups: C group (propofol group), R1 group (0.5 ng/mL of remifentanil combined with propofol), R2 group (1.0 ng/mL of remifentanil combined with propofol) and R3 group (1.5 ng/mL of remifentanil combined with propofol). The Dixon’s sequential method was used to determine the EC₅₀of propofol concentration, with initial plasma effect-side concentration of 5.0 μg/mL via TCI and step size of 1 μg/mL. Concentration of propofol was able to be increased for next one patient when deglutition reflex or body movement in gastroscope placement for one patient, whereas the concentration was going to be decreased. The EC₅₀ and 95%confidence interval (CI) of propofol in different groups calculated by Probit regression analysis. Adverse events of operative time, anesthesia and resuscitation time, intraoperative coughing and respiratory and heart rate depression were recorded. Results:　EC₅₀of propofol in C group, R1 group, R2 group and R3 group were 6.686 μg/mL, 6.476 μg/mL, 5.970 μg/mL and 5.640 μg/mL, respectively. The operation and anesthesia duration among four groups had no statistically significant differences (P>0.05). For the resuscitation time, there was no statistically significant difference between C group and R1 group (P>0.05), but there were statistically significant differences between R2 group and R3 group and C group (P<0.05). For heart rate (HR) during placing gastroscope, there was no statistically significant difference between C group and R1 group (P>0.05), but there were statistically significant differences between R2 group and R3 group and C group (P<0.05). For mean arterial pressure (MAP), there was no statistically significant difference between C group and R1 group (P>0.05), but there were statistically significant differences between R2 group and R3 group and C group (P<0.05). For respiratory depression during anesthesia, there was no statistically significant difference between C group and R1 group (P>0.05), but there were statistically significant differences between R2 group and R3 group and C group (P<0.05). Conclusion:　In endoscopic mucosal resection, remifentanil concentration of 1.0 ng/mL combined with the propofol TCI is safer and more effective, without less coughing, respiratory depression or bradycardia.

Abstract:Objective:　To investigate the value of blood lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and creatine kinase isoenzyme (CK-MB) in the diagnosis of severe adenovirus pneumonia (SAP) in children. Methods:　A total of 159 children diagnosed with adenovirus pneumonia in the respiratory department of our hospital from January 1, 2018 to December 31, 2019, were divided into the ordinary adenovirus pneumonia group and the SAP group according to the severity of their condition. The levels of LDH, AST and CK-MB in blood were compared between the two groups, and the receiver operating curve (ROC curve) was used to compare the test efficiency of the three for SAP. Results:　Compared with children with ordinary adenovirus pneumonia, the serum levels of LDH, AST and CK-MB in severe children were higher, and the differences were statistically significant (P<0.05). The ROC curve showed that LDH had the best diagnostic efficiency for SAP. Conclusion:　Increased serum levels of LDH, AST and CK-MB are of great help to diagnose SAP, especially the changes in blood LDH.

Abstract: