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Objective To investigate the role of scavenger receptor A member 3(Scara3) in the phagocytosis of multi-walled carbon nanotubes(MWCNT) and cytokine production. Methods Under addition or without serum, expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and IL-6, and phagocytosis of MWCNT were compared between the control(NC) and knockdown group(Scara3 k/d) cells. Results Compared with the presence of serum, when there was without serum, the expression of TNF-α, IL-1β, and IL-6 and the uptake of MWCNT in the NC group and Scara3 k/d group all decreased after MWCNT stimulation. Compared with NC group, whether with serum or without serum, Scara3 k/d significantly reduced the expression of TNF-α, IL-1β and IL-6 induced by MWCNT and the uptake of MWCNT. Conclusion Scara3 mediates phagocytosis of MWCNT and cytokine production in macrophages.

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Objective To construct eukaryotic expression vector and to explore the subcellular localization of GPR107 in eukaryotic cells, human G protein coupled receptor 107(GPR107) gene is cloned. Methods Firstly, the cDNA of human GPR107 gene was cloned by molecular cloning technique, and the recombinant vector pCMV-Tag2 B-GPR107 was constructed by eukaryotic expression vector pCMV-Tag2 B. The expression of GPR107 was detected by Western blot, and the localization of GPR107 in 293 T cells was observed by immunofluorescence labeling and laser confocal microscope. Results The cDNA of human GPR107 was confirmed by gene sequencing and the eukaryotic expression vector pCMV-Tag2 B-GPR107 was constructed successfully. The results of Western blot showed that compared with the non-transfection group, the expression of GPR107 in the transfection group was significantly up-regulated. Laser confocal microscopy showed that GPR107 was mainly expressed in the cytoplasm. Conclusion GPR107 is overexpressed in 293 T cells and localized in the cytoplasm.

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Objective To investigate the effect of lncRNA(DGUOK-AS1) on proliferation and apoptosis of lung squamous cell carcinoma. Methods LncRNA DGUOK-AS1 was found to be different in tumor tissues and normal tissues by TCGA database.SiRNA interference technology was used to decrease the expression of DGUOK-AS1 in lung squamous cell lines NCI-H520 and SK-MES-1. Cell proliferation was detected by CCK-8 method, and flow cytometry was used to detect changes in cell apoptosis, then Western blot was used to detect related proteins changes in cell proliferation, apoptosis and Wnt/β-Cantenin pathway. Results Compared with human normal lung epithelial cells(BEAS-2 B),the expression of DGUOK-AS1 increased in lung squamous cell carcinoma NCI-H520 and SK-MES-1 cells(P<0.001). The results of CCK-8 assay showed that decreased DGUOK-AS1 expression in NCI-H520 and SK-MES-1 could inhibit the proliferation ability of the cells compared with the negative control(P<0.01). Flow cytometry apoptosis detection results showed that reduced expression of DGUOK-AS1 could promote apoptosis with the negative control in NCI-H520 and SK-MES-1 cells(P<0.01).The results of Western blot showed that decreased expression of DGUOK-AS1 could reduce the expression of PCNA, Bcl-2, β-Cantenin, c-myc and Cyclin D1 proteins, and increase the expression of Bax proteins. RNA plasmid separation experiments showed that DGUOK-AS1 was mainly located in the cytoplasm. Conclusion The expression of long non-coding RNA DGUOK-AS1 increased in lung squamous cell carcinoma cells. The highly expressed DGUOK-AS1 may promote cell proliferation and inhibit cell apoptosis by affecting the Wnt/β-Cantenin pathway, and DGUOK-AS1 may be an important molecular target for the treatment of lung squamous cell carcinoma.

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Objective To investigate the effect of morphine pretreatment on the expression level of Caspase-3 and apoptosis of myocardial cells after ischemia reperfusion injury in rats. Methods A total of 51 male adult SD rats were randomly divided into 3 groups: the control group(Sham), ischemia-reperfusion group(IR) and morphine preconditioning group(1 μmol/L, MP). The Sham group was perfused with K-H solution for 195 min. In IR group,K-H solution was infused for 45 min to prepare in vitro whole heart ischemia for 30 min,and 10,60 and120 min post-ischemia reperfusion models were made. In MP group,K-H solution with morphine was perfused and K-H solution without morphine was perfused for 5 min each before ischemia,with a total of 3 cycles. Infarct volume( IS),area at risk( AAR) and IS/AAR were determined by 2,3,5-triphenyl-tetrazolium( TTC) staining. Lactic dehydrogenase( LDH) activity in coronary artery outflow was measured by chemical colorimetry. Caspase-3 and cardiomyocyte apoptosis were detected after 10,60 and 120 min of reperfusion via Western blot and TUNEL staining,respectively. Results Compared with sham group,the percentage of infarct volume at the end of reperfusion increased in IR group,LDH activity,Caspase-3 protein expression and cardiomyocyte apoptosis ratio significantly increased in IR group at each time point after reperfusion( P<0. 05). Compared with IR group,morphine pretreatment significantly decreased IS/AAR ratio,LDH active Caspase-3 protein level and myocardial apoptosis ratio( P<0. 05). LDH level at 10 min reperfusion,Caspase-3 protein expression at 60 min reperfusion and apoptosis rate at120 min reperfusion were the highest in IR group and MP group. Conclusion The protective effect of morphine pretreatment on myocardium is associated with decreased expression of Caspase-3 protein at the early stage of reperfusion and decreased apoptosis of myocardial cells after reperfusion.

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Objective To study the pathway of noradrenalin(NA) effects on two type neurons(sleep-promoting neurons, interneurons) in the ventrolateral preoptic area(VLPO). Methods The whole cell patch clamp technique and pharmacological methods were used to analyze the pathway of NA excitating or inhibiting effects on the two type neurons in the VLPO. The types of NMDA receptor expressions in VLPO were identified by RT-PCR. Results Electrophysiological results showed that the excitatory effects of NA(100 μmol/L) on VLPO interneurons were blocked by NMDA receptor blocker(AP5)(P<0.05,n=8), but not by AMPA receptor(CNQX) and GABA receptor blocker. The inhibitory effects of NA(100 μmol/L) on the sleep-promoting neurons of VLPO were not blocked by AP5, CNQX or flumazenil. RT-PCR results showed that three subtypes of NMDA receptor(GluN2 A, GluN2 B, GluN1) were expressed in VLPO. Conclusion The excitatory effects of NA on VLPO interneurons is an indirect effects caused by glutamatergic neurons releasing glutamate, which is mediated by NMDA receptor of VLPO interneurons.

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Objective To observe the effect of melatonin(MLT) on diabetic nephropathy(DN) rats, and to explore the mechanism of MLT delaying the progression of DN in rats. Methods Fifteen male SD rats were randomly divided into the normal group, model group and MLT group with 5 rats in each group. Rats in the model group and the MLT group were given 55 mg/kg of streptozotocin(STZ) by intraperitoneal injection in order to establish a DN rat model. After the model was successfully established, rats in the MLT group were given 10 mg/(kg·d)of MLT by intraperitoneal injection, and rats in the normal group and model group were given the equal volume of normal saline. Abdominal aortic blood and kidney tissue of rats were taken 16 weeks after they were given, blood urea nitrogen(BUN) and serum creatinine(SCR) in serum were detected; massion staining was used to observe morphological changes of kidney tissue; β-galactosidase staining(SA-β-gal) was used to detect changes in kidney cell aging; Western blot was used to detect the expression levels of autophagy markers(p62, LC3 B), myosin light chain kinase(MLCK) and cyclin-dependent kinase(CDK) inhibitors(p16, p53, and p21). Results Compared with the normal group, the levels of BUN and SCR in serum in the model group increased, renal interstitial fibrosis was obvious, the autophagy level of kidney tissue decreased, and the expression levels of MLCK, p16, p53, and p21 increased. Compared with the model group, the levels of BUN and SCR in serum in the MLT group decreased, and the aging of the kidney tissue was delayed. After MLT was administered, the pathological damage was significantly reduced, and the autophagy level of kidney tissue was also improved. The expression levels of MLCK, p16, p53 and p21 decreased. Conclusion MLT can improve the progress of DN in DN rats by increasing autophagy and delaying aging.

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Objective To investigate the effect of Mitoquinone(MitoQ) on the migration and apoptosis of human lung adenocarcinoma A549 cells through NIX-mediated mitochondrial autophagy. Methods A549 cells were exposed to various concentrations of MitoQ for 12 hours, the Cell Counting Kit-8(CCK-8) assay was used to detect cell viability. Cell migration was measured with scratch assay. Western blot analysis was used to detect the expression levels of the mitochondrial autophagy and apoptosis related proteins NIX, p62 and Bax. Results Compared with that in the control group, MitoQ groups showed significant inhibition of proliferation and migrate. Accompanied by the occurrence of apoptosis, the expression of autophagy-promoting related proteins(NIX) was up-regulated, the expression of autophagy-inhibiting related proteins(p62) was down-regulated, the expression of apoptosis-promoting related proteins(Bax) was up-regulated, and the difference increased with elevating MitoQ concentration(P<0.05); compared to the control group, MitoQ groups exhibited a decreased capacity for cell migrate and proliferation, and an increased levels of mitochondrial autophagy and apoptosis. Conclusion MitoQ can inhibit A549 cells migration and proliferation, and promote A549 cells apoptosis. The mechanism may be related to the MitoQ regulating key molecules on the mitochondrial autophagy pathway and inducing autophagy in A549 cells, and the key molecule of mitochondrial autophagy may be the NIX protein.

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Objective To explore the pathological characteristics of myogenic contracture and its possible mechanism in rabbit model of knee extension contracture. Methods According to the immobilization time, thirty New Zealand white rabbits were randomly divided into 5 groups(n=6): control group(I-0, without any treatment), 1-week immobilization group(I-1, with left knee joint complete extending immobilization for 1 week), 2-week immobilization group(I-2, with left knee joint complete extending immobilization for 2 weeks), 4-week immobilization group(I-4, with left knee joint complete extending immobilization for 4 weeks) and 8-week immobilization group(I-8, with left knee joint complete extending immobilization for 8 weeks). At the end of each immobilization time point, the rabbits were killed, and then the myogenic contracture and arthrogenic contracture were analyzed by detecting the angle of knee joint contracture after immobilization. The cross-sectional area of rectus femoris muscle fiber was analyzed by Masson staining. The protein expression levels of hypoxia inducible factor-1α(HIF-1α) and transforming growth factor-β1(TGF-β1) were analyzed by Western blot. Results During the 8 weeks, the angle of myogenic contracture in the immobilization group increased with the immobilization time within 4 weeks, but there was no significant difference between I-4 group and I-8 group after 4 weeks. Masson staining results showed that the cross-sectional area of rectus femoris decreased continuously within 4 weeks of immobilization, while the proportion of collagen fiber deposition increased continuously within 4 weeks after immobilization. However, there was no significant difference in the reduction of cross-sectional area of rectus femoris and the increase of collagen fiber deposition between I-8 group and I-4 group after 4 weeks of immobilization. The expression level of HIF-1α protein increased continuously within 4 weeks after immobilization, but there was no significant difference in the expression level of HIF-1α protein between I-4 group and I-8 group after 4 weeks immobilization; the expression of TGF-β1 protein increased significantly within 2 weeks, but there was no significant difference between the each group after 2 weeks immobilization. Conclusion The muscle fibrosis and atrophy are mainly pathological features of myogenic contracture in rabbit model of knee extension contracture, which is time-dependent change, and the mechanism may be related to the high expression of HIF-1α and TGF-β1.

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Objective To establish lncRNA-SNHG4 overexpressing gastric cancer cell lines; to examine the role of SNHG4 on cell growth, proliferation and the sensitivity to cisplatin in gastric cancer cells; to compare the expression levels of SNHG4 in gastric cancer and adjacent normal tissues, and to analyze the correlation between SNHG4 expression levels and pathological characteristics in gastric cancer patients. Methods The expression levels of SNHG4 in cell lines and tissues were detected by RT-qPCR; cell viabilities, cell proliferation and growth ability were detected by MTT and 3-D cell culture assay respectively; the correlation between SNHG4 expression levels and pathological characteristics in gastric cancer patients was analyzed by chi square test. Results MTT and 3-D cell culture experiments showed that overexpression of SNHG4 promoted the proliferation and cisplatin resistance in gastric cancer cell lines MKN-45 and AGS(P<0.05); the expression levels of SNHG4 in gastric cancer tissues were significantly higher than that in adjacent normal tissues; the high expression levels of SNHG4 in gastric cancer tissues were significantly correlated with tumor volume, lymph node metastasis and pathological grade in gastric cancer patients(P<0.05), but not with patients′age, gender or clinical stage. Conclusion SNHG4 promotes cell proliferation, growth and cisplatin resistance in gastric cancer cells. The high expression levels of SNHG4 are correlated with pathological characteristics in gastric cancer patients. SNHG4 has important clinical significance in gastric cancer.

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Objective To explore the inhibitory effect of sophocarpine on lung cancer cellsin vitroas well as its possible mechanism. Methods A549 cells were treated with different concentrations of sophocarpine, the IC50of sophocarpine to A549 was detected by MTT. The sophocarpine group was treated with proper concentration and the untreated A549 cells were used as the control group. Cell proliferation was detected by clone formation and BrdU, cell apoptosis was detected by TUNEL and flow cytometry, the expression of proliferating cell nuclear antigen(PCNA), MYC, Bax, Bcl-2, lipocalin type prostaglandin-D-synthase(L-PTGDS)and PTGDR2 was detected by Western blot. Besides, the PGD2 group was treated with 5 ng/ml PGD2 activator, clone formation, TUNEL and Western blot were performed to detect the effect of PGD2 on proliferation and apoptosis of A549 cells. Results The IC50of sophocarpine to A549 was 5.196 mmol/L, clone formation and BrdU experiment showed that the proliferation of A549 cells in the 5 mmol/L sophocarpine treated group was lower than that in the control group while the apoptosis of A549 cells was higher than that in the control group. The expression of PCNA, MYC, Bax, Bcl-2 was lower than that in the control group, while the expression of Bax was higher than that in the control group, all of the differences were statistically significant(P<0.05). The protein expression of L-PTGDS and PTGDR2 in sophocarpine group was higher than that in control group(P<0.05). The proliferation of A549 cells treated with 5 ng/ml PGD2 activator was inhibited, and apoptosis significantly increased(P<0.05). Conclusion Sophocarpine can inhibit the proliferation and promote the apoptosis of lung cancer cells, as well as activating PGD2/PTGDR2 pathway, thus inhibiting the occurrence of lung cancer.

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Objective To evaluate the therapeutic value of JTE-907(cannabinoid CB2 receptor reverse agonists) in a model of neutrophilic asthma, and to determine the potential therapeutic mechanism of CB2 receptor activation by observing its effect on the differentiation of CD4+na6 ve T cells. Methods In order to study the effect of JTE907 on the differentiation of T cell subtypes, used anin vitrosystem of Th lineage-specific differentiation of na6 ve CD4+T lymphocytes isolated from the rat spleen. The expression of Tbx21, Gata3, interleukin(IL)-9, ROR-C, FoxP3, CNR2 mRNA was detected by qRT-PCR. 40 rats were divided randomly into four experimental groups with 10 animals each: normal control group, model group, JTE-907 low-dose group(JTE-907-L, intraperitoneal injection of 5 mg/kg) and JTE-907 high Dose group(JTE-907-H, 20 mg/kg). Except for the normal control group, rats at other three groups were established neutrophil asthma models. The concentration of serum OVA-specific IgE and BALF cytokine was detected by ELISA. The percentage of Th17 and Treg cells in spleen was analyzed by flow cytometry, and the expression of RORγt, Foxp3 and STAT5 protein in lung tissue was detected by Western blot. Results qRT-PCR analysis showed that JTE907 induced the expression of FoxP3 and CB2 receptors at all the concentrations tested. After OVA sensitization and LPS stimulation, the level of OVA-sIgE in the model group increased(P<0.05), and the number of neutrophils and the levels of interferon(IFN)-γ, tumor necrosis factors(TNF)-α, IL-17 and IL-5 in BALF significant increased(P<0.05). JTE-907 treatment significantly reversed the above changes(P<0.05). In addition, compared with the model group, the ratio of Th17/Treg in spleen of JTE-907 treatment group significantly decreased(P<0.05), and the level of RORγt protein in lung tissue significantly decreased(P<0.05), while the levels of Foxp3 and p-STAT5 significantly increased(P<0.05). Conclusion The activation of CB2 receptor has a protective effect in rat with Th-17 mediated neutrophilic asthma. The mechanism of JTE907 effect involves improving Th17/Treg balance, as well as regulating their respective cytokine levels.

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Objective To explore the effect of silencing human epididymal protein 4(HE4) on the proliferation, invasion, apoptosis and JAK/STAT3 signaling pathway of colon cancer cells. Methods 124 patients with colon cancer and 40 healthy people were selected. Western blot and immunohistochemical staining were used to detect the expression of HE4 in colon cancer and adjacent tissues. Enzyme linked immunosorbent assay(ELISA) was used to detect the expression of HE4 in serum of patients with colon cancer and healthy people. Western blot was used to detect the expression of HE4 in colon cancer cell lines Caco-2, SW480, HT-29, HCT-116 and hcoepic. HT-29 cells were transfected with HE4 si-RNA or si-control respectively. Cell proliferation was detected by CCK-8 method, invasion ability was detected by Transwell assay, and apoptosis was detected by Annexin-V-FITC/PI flow cytometry. Western blot was used to detect the protein expression levels of JAK1, p-JAK1, JAK2, p-JAK2, STAT3 and p-STAT3. Results The relative expression of HE4 protein in colon cancer tissues was higher than that in adjacent tissues(P<0.05). The expression of HE4 was positive in colon cancer tissues, of which 78 cases(62.90%) were highly expressed, while only 24 cases(19.35%) were overexpressed in the adjacent tissues(P<0.05). The serum HE4 concentration in patients with colon cancer was higher than that in normal subjects(P<0.05). The relative expression of HE4 protein in Caco-2, SW480, HT-29 and HCT-116 cells was higher than that in hcoepic cells(allP<0.05). The expression level of HE4 was not related to gender, age and tumor size, but related to differentiation degree, TNM stage, lymph node metastasis and distant metastasis(P<0.05). After silencing HE4 gene, the proliferation and invasion ability of HT-29 cells decreased, while the apoptosis rate increased(P<0.05). Silencing HE4 gene had no effect on the total protein of JAK1, JAK2 and STAT3, but could down regulate the expression of p-JAK1, p-JAK2 and p-STAT3(allP<0.05). Conclusion Silencing HE4 can inhibit colon cancer cell proliferation, invasion, and apoptosis, and down-regulate the expression of related proteins in the JAK/STAT3 signaling pathway.

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Objective To explore the effect of overexpression of miR-302 on the pro-inflammatory level of colon cancer cells,the level of mitochondrial oxidative stress and tumor formation in nude mice. Methods Colon cancer cells SW480 were randomly divided into a blank control group( Control),a negative control group( miR-NC),and an overexpression group( miR-302 mimic); the expression of miR-302 after transfection was detected by qRTPCR; inflammatory factors interleukin( IL)-6 and inducible nitric oxide synthase( i NOS) were tested with enzyme linked immunosorbent assay( ELISA); IL-1 β and tumor necrosis factor α( TNF-α) mRNA expression were detectd by qRT-PCR detection; the changes in mitochondrial membrane potential were detected by flow cytometry;oxidative stress markers malondialdehyde( MDA) and lactate dehydrogenase( LDH) were detected by kits; mitochondrial damage-related proteins B-cell lymphoma/leukemia 2( Bcl-2) associated X( Bax),Bcl-2,cleaved cysteinyl aspartate specific proteinase( Cleaved cas)-3 and cas3,c-Myc,and their relative levels( Bax/Bcl2,Cleaved cas3/cas3) were detected by Western blot; a nude mouse xenograft model was established to detect tumor tissue weight; caspase 3 positive expression was detected by immunohistochemistry. Results Compared with the miR-NC group,the miR-302 expression,IL-6 and i NOS protein concentrations,IL-1β and TNF-α mRNA levels,oxidative stress marker levels( MDA,LDH),mitochondrial damage-related protein expression( Bax/Bcl2,Cleaved cas3/cas3) and the positive expression of caspase 3 in tumor tissue significantly increased( P<0. 05); compared with miR-NC group,miR-302 Mimic group mitochondrial membrane potential,c-Myc expression and tumor tissue weight significantly reduced( P<0. 05). Conclusion Overexpression of miR-302 induces increased levels of inflammatory factors in colon cancer cells,mitochondrial oxidative stress,and inhibits tumor formation in nude mice.

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Objective To investigate the effect and mechanism of down-regulation of volume-activated chloride channel protein ClC-3 on H9 c2 cardiomyocytes. Methods H9 c2 cardiomyocytes were transfected with siRNA to construct a cell model of down-regulated ClC-3; immunofluorescence staining was used to observe the surface area of H9 c2 cells; qRT-PCR was used to detect ANP, BNP and β-MHC of H9 c2 cells; the level of mitochondrial membrane potential was detected by JC-1 fluorescent probe; the image analysis system was used to study the regulatory volume decrease(RVD) of H9 c2 cardiomyocytes; the change of chloride current on H9 c2 cell membrane was detected by whole cell patch clamp technology. Results Compared with the control group, siRNA transfection of H9 c2 cells for 72 h could reduce the expression levels of ClC-3 mRNA and protein; and the down-regulation of ClC-3 expression was similar to isoproterenol, which could increase the surface area of H9 c2 cardiomyocytes and the iconic factors. Besides, it decreased the mitochondrial membrane potential of H9 c2 cells, reduced the capacity of volume regulation, and inhibited the activation of volume-activated chloride ion current. Conclusion The down-regulation of ClC-3 expression induces H9 c2 cardiomyocyte hypertrophy. Its mechanism may be related to its damage to mitochondrial function, reduction of cell volume regulation and inhibition of volume-activated chloride current activation.

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Objective To study the mechanism of saxagliptin in alleviating insulin resistance in diabetic rats by miR-15 a-5 p/insulin receptor(INSR) axis. Methods Firstly the intersection of miRNA that might target INSR was sellected from Targetscan, miRDB and miRWalk by inputing the transcript of INSR ENST00000341500. Then dual luciferase reporter assay was used to verify the targeting relationship. Subsequently the insulin resistance-type 2 diabetes mellitus(IR-T2 DM) models were constructed. Then, IR-T2 DM rats were treated with miR-15 a-5 p mimic, miR-15 a-5 p inhibitor or saxagliptin. Rats were sacrificed after 8 weeks to obtain quadriceps femoris muscles and peripheral blood. Blood pressure, blood glucose, lipid metabolic parameters, homeostasis model assessment(HOMA) insulin resistance index(IRI) as well as insulin sensitivity index(ISI) were measured in each group, respectively. Quantitative PCR was used to detect the expression of intersection miRNA and IRS-1, IRS1, GLUT4 mRNA while Western blot was adopted to test the protein expression of INSR, p-IRS-1 and GLUT4 in skeletal muscle of rats in each group. Results miR-15 a-5 p was selected as the targeting miRNA of INSR. Compared with negative control group, the expression of INSR, IRS-1 and GLUT4 in skeletal muscle as well as the concentration of HDL-C, BW and ISI in peripheral blood of IR-T2 DM rats treated with miR-15 a-5 p inhibitor or saxagliptin markedly increased( P<0. 5),but the concentration of hemoglobin A1 c( Hb A1 c),triglyceride( TG) and low-density li Poprotein cholesterol( LDL-C),systolic blood pressure( SP),diastolic blood pressure( DP) and IRI in peripheral blood significantly increased in the above two groups( P<0. 05). The trend of the above indexes in miR-15 a-5 p mimic group was completely opposite to that in miR-15 a-5 p inhibitor and saxagliptin group( P<0. 05). In addition,saxagliptin combined with miR-15 a-5 p inhibitor had more effictive capacity in relieving insulin resistance than treating the diabetic rats with saxagliptin or miR-15 a-5 p inhibitor alone( P<0. 05). Conclusion Based on the results observed during this study,the paper shows that saxagliptin has distinct effect on relieving insulin resistance by down regulating miR-15 a-5 p to up regulate INSR expression in diabetic rats.

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Objective To investigate the expression of miR-513 a-3 p in colorectal cancer tissues and adjacent normal tissues, and to explore the mechanism of miR-513 a-3 p inducing colorectal cancer(CRC) cell growth and migration. Methods The total of 30 patients with colon cancer were selected as the research objects. The expression levels of miR-513 a-3 p in colon cancer tissues and adjacent tissues were detected by in situ hybridization and real-time fluorescence quantitative PCR. CRC cell lines HT-29 and SW480 were cultured and transfected with miR-513 a-3 p analogues as the expression promoting group. The negative control group was set up. The cell viability was detected by tetramethyl azo salt(MTT) colorimetry, the cell invasion was detected by Transwell method and cell migration was detected by cell scratch test. The expression of NF-κB and Wnt/β-Catenin pathway related proteins was detected by Western blot. Results The results of situ hybridization showed that the positive expression of miR-513 a-3 p in colon cancer tissues was higher than that in adjacent tissues. The results of qRT-PCR showed that the relative expression of mir-513 a-3 p was(0.37±0.09) in colon cancer tissues and(1.01±0.16) in adjacent tissues, and the difference was statistically significant(P<0.01). Compared with the negative control group, the cell viability of the promoted expression group increased in 3 and 4 d(P<0.01), the invasion rate of HT-29 and SW480 cells in the promoting expression group increased(P<0.01), and the cell migration ability of HT-29 and SW480 in the promoting expression group was enhanced. The results of Western blot showed that compared with the negative control group, the relative expressions of p-p65, p-IκBα, Wnt3 a, Wnt5 a and β-Catenin protein in HT-29 cells and SW480 cells significantly increased(P<0.01). Conclusion The overexpression of miR-513 a-3 p promotes the growth, migration and invasion of colon cancer cells. The possible mechanism is related to the activation of NF-κB pathway and Wnt/β-Catenin pathway.

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Objective To probe into the synergistic mechanism ofCurcumaelongae RhizomaandPaeoniae RadixAlba in the treatment of rheumatoid arthritis(RA) by network pharmacology. Methods The active components of the drug pair and predicted targets were collected from TCMSP public database and literature. The “drug-component-target” network was constructed by Cytoscape 3.7.1 software. Disease targets were obtained by searching “Rheumatoid Arthritis” in GeneCards, OMIM and CTD databases. The PPI network was built in STRING database, and the GO function annotation and KEGG signal pathway enrichment analysis were performed by ClueGo plugin. The drug-component-target-disease network of the pair was constructed, and the network feature analysis was made by network analyzer of the Cytoscape 3.7.1 software. Results Totally 13 components and 445 potential targets in the pair and 2 060 targets relating to RA were excavated, and 53 common targets of the pair were anti-RA targets. The top three targets were AKT1, TNF and PTGS2 in the PPI network by degree. The GO function annotation was 225. KEGG pathways of anti-RA target fromCurcumaelongae Rhizomawas 28, whilePaeoniae RadixAlba was 74 which they shared 24 pathways in conmmon. Conclusion Curcumaelongae RhizomaandPaeoniae RadixAlba may regulate AKT1, TNF or PTGS2 so as to antioxidise, inhibit the inflammatory response, regulate immune function and adjust apoptosis to treat rheumatoid arthritis synergistically.

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Objective To investigate the effects of CD98 gene silencing on proliferation, apoptosis and PI3 K/AKT pathway of melanoma B16-F10 cells. Methods shRNA CD98 vector was constructed and transfected into B16-F10 cells. The cells were randomly divided into three groups: Control group, shRNA-NC group and CD98-shRNA3 group for subsequent experiments. Cell proliferation was detected by clone formation assay. Flow cytometry was used to detect apoptosis. Transwell was used to detect cell invasion. Western blot was used to detect the protein expression levels of CD98, Ki67, PCNA, Bax, Bcl-2, Caspase-3, PI3 K, p-PI3 K, AKT, and p-AKT. Results Compared with Control group, group CD98-shRNA3 CD98 protein expression level decreased significantly(P<0.05), clone formation rate decreased significantly(P<0.05), Ki67, PCNA protein expression level decreased significantly(P<0.05), the apoptosis rate increased significantly(P<0.05), Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 ratio were significantly higher(P<0.05), the number of invasive cells significantly reduced(P<0.05), The ratio of p-PI3 K/PI3 K and p-AKT/AKT significantly reduced(P<0.05). Conclusion CD98 silencing may inhibit the proliferation and invasion of melanoma B16-F10 cells and promote apoptosis by inhibiting the activation of PI3 K/ATK signaling pathway.

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Objective To explore the effect of silencing APE-1 on transcriptome expression and TNF signaling pathway in hepatoma cell Hep 3 B. Methods A hepatocellular carcinoma Hep 3 B stable cell line silencing APE-1 expression was subjected to transcriptome sequencing. The experimental group was cells with silencing APE-1 expression, and the control group was cells transfected with blank plasmid(n=3). Bioinformatics analysis of differentially expressed genes, and functional enrichment of GO, KEGG, and DO, and protein interaction networks of differential genes were conducted. The key genes of TNF signaling pathway, TNFAIP3 and MAP2 K6 were detected by real-time PCR in the experimental/control group cells. Ten pairs of liver cancer/cancer adjacent clinical samples were collected and their protein expression was detected by immunohistochemistry. Based on data from 374 patients with liver cancer in the TCGA database, the correlation between APE-1 and TNFAIP3, APE-1 and MAP2 K6 expression was analyzed. Results Silencing APE-1 resulted in a up-regulation of 84 genes and down-regulation of 39 genes in hepatoma cell Hep 3 B. The top 10 differential genes were APE-1, CEMIP, MAP2 K6, TAF11 L11, CNGB1, COL5 A3, OTULINL, DNAJC12, FOLR1, and ACKR3. RT-PCR verified that TNFAIP3 and MAP2 K6 were down-regulated in hepatoma cell Hep 3 B silencing APE-1. TNFAIP3 and MAP2 K6 were highly expressed in clinical liver cancer tissue samples, and there was a significant positive correlation between APE-1 and TNFAIP3, APE-1 and MAP2 K6 expression(P<0.001). Conclusion APE-1 is a key anti-tumor gene in the development of liver cancer, which may play a role in affecting the TNF signaling pathway.

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Objective To investigate the effect of knocking out hsa_circRNA_102958 on the proliferation, oxidative stress and mitochondrial function of pancreatic cancer PANC-1 cells. Methods Pancreatic cancer PANC-1 cells were transfected with shRNA-NC and three different circ_102958 shRNA series. RT-PCR was used to detect the expression of circRNA_102958. The best interference effect was selected for circ_102958 shRNA1 for follow-up experiments. CCK8 was used to detect cell proliferation multiples; Hoechst staining was used to observe cell morphology changes; total superoxide dismutase(SOD) activity detection kit, malondialdehyde(MDA) detection kit and DCFH-DA kit were used to measure SOD, MDA, and active oxygen(ROS)content respectively; flow cytometry was used to detect JC-1 red/green fluorescence ratio; Western blot was used to detect Bax/Bcl-2, cleaved cas9/cas9, cleaved cas3/cas3, c-Myc protein expression. Results Compared with the Control group, interfering with the expression of circRNA_102958 inhibited the proliferation of pancreatic cancer PANC-1 cells(P<0.05), the cell morphology tended to be disordered, apoptotic cells increased, and the contents of MDA and ROS were up-regulated(P<0.05). SOD activity decreased(P<0.05), the ratio of red/green fluorescence of JC-1 was down-regulated(P<0.05), and the protein expression of Bax/Bcl-2, cleaved cas9/cas9 and cleaved cas3/cas3 increased(P<0.05). Conclusion Interfering with the expression of circRNA_102958 can inhibit the proliferation and mitochondrial function of pancreatic cancer PANC-1 cells, and promote cell apoptosis and oxidative stress.

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Objective To investigate the effect of sodium selenite on the biological behavior of lung cancer cells and the specific mechanism of its effect on lung cancer apoptosis through the NF-κB signaling pathway. Methods CCK-8 cell proliferation assay and cell scratch assay were used to observe the proliferation and migration of lung cancer cells. Flow cytometry, qPCR and Western blot were used to detect the apoptosis of lung cancer cells and the expression of Bcl-2, Bax, NF-κB, p-NF-κB, IkBα, p-IkBα in lung cancer cells respectively. The distribution of p-NF-κB in lung cells was observed by immunofluorescence assay. Results The results of CCK-8 cell proliferation assay and cell scratch assay showed that lung cancer cell proliferation and migration were significantly inhibited by sodium selenite. The results of Flow cytometry showed that the apoptosis of lung cancer cells significantly increased after sodium selenium treatment. Compared with the control group, the expression levels of p-NF-κB, p-IkBα, Bcl-2 protein and Bcl-2 mRNA in the sodium selenite group were down-regulated, while the expression of Bax protein and mRNA were up-regulated. The results of immunofluorescence experiments showed that sodium selenite could inhibit the phosphorylation level of NF-κB. The qPCR results showed that sodium selenite and BAY11-7082(NF-κB inhibitor) could down-regulate the expression level of Bcl-2 mRNA, while up-regulate the expression of Bax mRNA. Conclusion Sodium selenite may inhibit proliferation and migration of lung cancer cells and induce apoptosis by inhibiting the NF-κB signaling pathway.

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Objective To investigate the influence of gonadotropin releasing hormone agonist(GnRH-a) on pregnancy outcomes in frozen-thawed embryo transfer cycles for patients with endometriosis and infertility in normal serum CA125. Methods A retrospective analysis was performed on 146 FET cycles in patients who didn′t accept GnRH-a with EMs in normal serum CA125 before FET(0-35 U/ml), the ROC curve and the best statistical cutoff values were obtained. According to the best statistical cutoff values and whether GnRH-a was applied before FET,228 patients were divided into Group A( CA125 was 0-16. 79 U/ml without GnRH-a),group B( CA125 was16. 79-35 U/ml without GnRH-a) and Group C( CA125 was 16. 79-35 U/ml with GnRH-a),and the general condition and pregnancy outcome were compared among the three groups. Results There were no statistically significant differences between the three groups in age,years of infertility,body mass index,number of transplanted embryos and high quality embryo rate. There were statistically significant differences in embryo implantation rate and clinical pregnancy rate between Group B,C and Group A,and there were statistically significant differences in embryo implantation rate and clinical pregnancy rate between group B and Group C( P<0. 05). There were no statistically significant differences in endometrial thickness,ectopic pregnancy rate and live birth rate among the three groups. Conclusion Serum CA125≥16. 79 U/ml before FET in EMs patients can predict the adverse pregnancy outcome,and applying GnRH-a before FET can improve the adverse pregnancy outcome in EMs patients.

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Objective To investigate the expression level of soluble mannose receptor(sMR) in hepatocellular carcinoma(HCC) patients and its clinical significance. Methods The serum sMR levels of patients with hepatocellular carcinoma, chronic hepatitis B(CHB) and healthy controls(HC) were detected by enzyme-linked immunosorbent assay(ELISA). The general data were collected, and the serum sMR levels in patients with different stages and pathological characteristics were compared. The ROC curve was drawn to evaluate the diagnostic efficacy of sMR and AFP for hepatocellular cancer. Results The serum sMR level of hepatocellular cancer patients was related to tumor stage, size and number. The combined diagnostic efficacy of serum sMR and AFP was significantly higher than that of a single indicator. Conclusion Serum sMR level is significantly increased in patients with hepatocellular cancer. sMR detection has important clinical significance and application value in the diagnosis of hepatocellular cancer.

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Objective To investigate the effects of probiotics on the functional metabolism of intestinal flora and clinical diseases in the neonatal intensive care unit( NICU) premature infants. Methods A total of 40 premature infants from the NICU were randomly divided into the probiotics group and the control group. Feces samples of two groups were collected on the 10 th day, and intestinal flora was detected and analyzed by 16 S rRNA high-throughput sequencing. And the differences in the incidence of clinical diseases were also compared between the two groups. Results Compared with the control group, the abundance of bifidobacterium and lactobacillus in intestinal flora of the probiotics group on the 10 th day was higher(P<0.05), while clostridioides, staphylococcus and cupriavidus bacteria were lower(P<0.05). In terms of functional analysis, the functions of the pentose phosphate pathway and nicotinate and nicotinamide metabolism in the probiotics group were higher than those in the control group(P<0.05), while the necroptosis was lower(P<0.05). In addition, the function gene expression levels of human pathogen sepsis(P<0.01), pneumonia, gastroenteritis and diarrhea(P<0.05) were also lower in the probiotics group. Moreover, the bilirubin and hemoglobin on the 10 th day(P<0.01) and the incidence of diarrhea and feeding intolerance(P<0.05) in the probiotics group were lower than those in the control group. And the incidence of sepsis and necrotizing enterocolitis( NEC) was also lower, but the difference was not significant. Conclusion This study shows that probiotics may have a significant effect on the metabolic function of intestinal flora and clinical disease in NICU premature infants, and explain the mechanism of probiotics to reduce the risk of clinical disease from the perspective of function.

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Objective To investigate the correlation between subcortical ischemic vascular disease(SIVD) and paraoxonase 1(PON1) gene polymorphism. Methods Sixty-five SIVD patients and 35 medical examiners in the same period were included as SIVD group and control group, respectively. The Fazekas classification and the number of luminal infarcts(LI) were evaluated according to the MRI findings of the head, and the SIVD group was divided into the WML group and the LI group. According to the degree of brain damage, they were divided into mild disease group, moderate disease group and severe disease group. Sanger sequencing method was used to determine the expression of the subject′s PON1 gene at locus 192 and 55 respectively. Results (1) The average age was higher than that of the control group(P<0.05), and the high-density lipoprotein cholesterol(HDL-C) level was lower than that of the control group(P<0.05).There was a statistically significant difference in smoking status between the SIVD group and the control group(P<0.05);(2) The distribution of Q192 R and L55 M genotypes in the SIVD group and the control group conformed to the Hardy-Weinberg equilibrium law;(3) The difference of PON1 gene Q192 R and L55 M genotype distribution between the SIVD group and the control group was statistically significant(χ2=8.780,P<0.05; χ2=4.239,P<0.05). Both the R allele frequency and M allele frequency in the SIVD group were higher than those in the control group(χ2=8.765,P<0.01; χ2=4.926,P<0.05);(4) In the WML group and the LI group, the R allele frequency of the severe disease group was higher than that of the moderate disease group and the mild disease group(P<0.016 7), and the M allele frequency of the moderate disease group and the severe disease group were both higher than that of mild disease group(P<0.016 7);(5) Multivariate Logistic regression analysis showed that age, smoking, and carrying PON1 gene R and M alleles were all independent risk factors for the risk of SIVD(P<0.05). Conclusion The pathogenesis of SIVD may be related to the difference in the expression of the PON1 gene at locus 192 and 55. Carrying R and M alleles at this locus may promote the pathogenesis of SIVD. These findings provide a certain theoretical basis for finding new targets for the prevention and treatment of SIVD.

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Objective To investigate the correlation between the expression of SPNS2 in hepatocellular carcinoma and its epithelial-mesenchymal transition(EMT). Methods 10 fresh clinical specimens of HCC and 70 paraffin sections of HCC surgical specimens(including cancerous tissues and corresponding adjacent tissues) and clinical related pathological data were collected,and the two main markers of SPNS2 and EMT,N-cadherin( N-cadherin),E-cadherin( E-cadherin) expression in cancer and adjacent tissues were detected,and the correlation between SPNS2 and clinicopathological features and EMT was analyzed. Results The expression level of SPNS2,N-cadherin in cancer tissues were higher than those in adjacent tissues( P<0. 01),and the expression of E-cadherin was lower than that of adjacent tissues( P<0. 01). The analysis showed that SPNS2 was positively correlated with Ncadherin and negatively correlated with E-cadherin. The expression of SPNS2 in HCC was not associated with the patient's age,gender,and degree of differentiation,but was closely related to TNM grade and microvascular invasion. Conclusion SPNS2 is differentially expressed in HCC,and it may promote the occurrence of HCC by regulating EMT transcription factors. SPNS2 is closely related to TNM grade and microvascular invasion,and can be used for clinical prognosis assessment.

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Objective To investigate the effect of neutrophil/lymphocyte(NLR), platelet/lymphocyte(PLR), monocyte/lymphocyte(MLR) and C-reactive protein/albumin(CAR) on the prognosis of patients with idiopathic inflammatory myopathy associated interstitial pneumonia(IIM-ILD). Methods A total of 117 patients with IIM-ILD were selected. The clinical and laboratory characteristics in survivors and non-survivors were compared. The NLR, MLR, PLR and CAR optimal cut-off values of the predicted survival rate were calculated by using the receiver operating characteristic curves(ROC). Cox proportional hazard model was used for multivariate analysis to determine the factors affecting prognosis. Results Among 117 patients with IIM-ILD, 37 patients(31.62%) died during the follow-up period. Multivariate analysis suggested that age, high-dose glucocorticoid, hypoproteinemia, anti MDA5 antibody positive and high MLR(>0.599) were independent predictors of poor prognosis in patients with IIM-ILD. Conclusion High MLR can be used to assess the prognosis of the IIM-ILD patients together with other indicators, which may have the value of evaluating the prognosis of IIM-ILD patients.

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To establish a C57 BL/6 J mice model of diarrhea-predominant irritable bowel syndrome(D-IBS) induced by wrap restrain stress(WRS), and to evaluate the feasibility and characteristics of this model. The D-IBS model of C57 BL/6 J mice was established by WRS. The general condition, fecal water content, body weight and intestinal transport function of mice were observed, and the pathological changes of colon were observed. Compared with the control group, the fecal water content in the D-IBS model group increased, the body weight decreased, the intestinal transport speed accelerated, and the colon length shortened. The colonic mucosa of mice in the model group showed inflammatory changes involving the lamina propria and mainly lymphocytosis. The D-IBS animal model of C57 BL/6 J mice can be prepared by WRS, and the symptoms and histopathological changes of intestinal mucosa in mice molded are more similar to those in human patient with D-IBS.

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To investigate the effect of generalized joint laxity(GJL) and two types of grafts(autologous hamstring tendon and allograft tendon) on anterior cruciate ligament(ACL) reconstruction. 138 patients with ACL reconstruction were followed up for 24-36 months. Four study groups were divided according to the Beighton score and the type of graft used from 138 patients who received ACL reconstruction. Preoperative and postoperative knee joint function assessment was conducted, including KT-2000 examination, IKDC subjective score and Lysholm knee joint score. Follow-up results of 2 to 3 years after surgery showed that in patients with GJL, the efficacy of autologous hamstring tendon transplantation was worse than that of allograft tendon transplantation, and over time, patients with GJL had a worse prognosis than those without GJL, regardless of which graft was selected. As a conclusion, Allograft tendons are recommended for ACL injury patients with GJL.

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The clinical data of 82 patients with trachea lesion in thyroid diseases were retrospectively analyzed, including 32 cases with unilateral or bilateral giant nodular goiter, 5 cases with hyperthyroidism accompanied with nodular goiter, 4 cases with subacute thyroiditis, 10 cases with thyroid lymphoma, 4 cases with anaplastic thyroid carcinoma, 24 cases with papillary thyroid carcinoma and 3 cases with recurrent papillary thyroid carcinoma. The effective treatment of trachea lesion in thyroid diseases should be performed differently according to the nature of lesions, the extent and scope of lesions involving trachea and the complications of the patients themselves, so as to maxizime the safety of operation.

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To evaluate the diagnostic and differential ability of RV1737 peptide antigens in tuberculosis and the peptide immunoreactivity which was based on the cellular immune simulated interferon gamma(IFN-γ) release test(IGRA) method. After the action of peptides A, B and C, the IFN-γ level of the latent tuberculosis(LTBI) group was significantly higher than that of the comparative Tuberculosis(TB) group(P<0.05). The sensitivity of the combination group A+B+C was significantly higher than that of peptides A, B, C and fusion D(P<0.05). The antibody of tuberculosis IgG was detected by indirect ELISA based on polypeptide. Better reactivity in TB group serum, the absorbance of IgG antibody of polypeptide A in the TB group was significantly greater than that of polypeptides B and C(P<0.05). The area of polypeptide A was the largest when ROC was drawn between groups, and when combined with A+B+C polypeptide, the curve area increased and the difference was statistically significant(P<0.05). RV1737 polypeptides may be candidate antigens for the diagnosis of latent tuberculosis, ELISA may be established to detect peripheral blood TB IgG antibody by polypeptide antigen. In particular, polypeptide A may be the new antigen for screening serological experimental tuberculosis.