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Objective To investigate the cytotoxic effects of doxorubicin(Dox)-loaded nanoparticles based on hollow copper sulfide(CuS) on the human pancreatic cancer cell line BxPC-3,to achieve the combination of chemotherapeutic drugs and photothermal therapy, and to enhance the killing effect of chemotherapy on pancreatic cancer. Methods Hollow CuS nanoparticles, PEG-modified nanoparticles and DOX@PEG-CuS nanoparticles were synthesized and characterized. BxPC-3 cells were divided into four groups,BxPC-3 cells+PBS,BxPC-3 cells+Free DOX, BxPC-3 cells+PEG-CuS, BxPC-3 cells+DOX@PEG-CuS. Inductively coupled plasma mass spectrometry was used to detect the cellular uptake of BxPC-3 cells. MTT assay and dead and alive staining assay were used to detect the killing effect of each experimental group on pancreatic cancer cell line BxPC-3. Results CuS nanoparticles with a size about 356.3 nm were successfully prepared, and after surface modification with PEG, the particle size of the PEG-CuS nanoparticles was about 209.8 nm. CuS nanoparticles modified by PEG are stable in 10% serum. The results of ICP-MS showed that PEG-CuS uptake rate of pancreatic cancer cells increased with the incubation time at 2 h(12.95%), 4 h(20.20%) and 6 h(32.98%)(P<0.05,t=4.083;P<0.01,t=5.889). The results of MTT method showed after combined photothermal therapy and chemotherapy, the doxorubicin-loaded CuS had the best killing effect, and the killing rate of pancreatic cancer cells was up to 88.3%, which was better than 52.04% of the CuS treatment group alone(F=711.6,P<0.000 1) and 45.58% of the free Dox group(F=711.6,P<0.000 1). Conclusion The hollow copper sulfide particles modified by PEG can be stably present in 10% serum. The uptake rate of CuS nanoparticles increases with time after PEG modification. Photothermal therapy combined with chemotherapy can achieve a higher killing effect on pancreatic cancer cell line BxPC-3.

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Objective To investigate the expression of fusion protein HSPE1-MOB4 in heat shock protein family member 1(HSPE1) and murine human-like alpha-chemokine analog1(MOB4) in different cells. Methods Reverse transcription polymerase-chain reaction(RT-PCR) was used to detect the expression of fusion protein HSPE1-MOB4 in different cells. Sanger gene sequencing was used to explore different subtypes of fusion protein. Protein position in cells was detected by immunofluorescence technique and laser confocal microscopy.The expression of fusion protein HSPE1-MOB4 in normal cells and tumor cells was detected by immunoblotting. Results The fusion protein HSPE1-MOB4 was expressed in normal cells and tumor cells. The results of gene sequencing showed that the fusion protein HSPE1-MOB4 had two different subtypes, and the expression positions of the two subtypes were the same. Compared with normal cells, the amount of fusion protein expressed in tumor cells was higher. Conclusion The fusion protein HSPE1-MOB4 has two subtypes and the reported one is highly expressed in tumor cells. This may provide new ideas for finding new tumor biomarkers.

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Objective To clarify the regulatory role of miRNA-26 b(miR-26 b) in the pluripotency of mouse embryonic stem cells(mESCs). Methods Real-time quantitative PCR was used to detect the expression of miR-26 cluster precursors in mESCs and MEF.Real-time quantitative PCR was used to detect the expression of miR-26 b during the formation of embryoid body(EB).CCK-8 assay was used to detect the effect of miR-26 b on the proliferation ability of mESCs.Flow cytometry was used to detect the regulatory effect of miR-26 b on cell cycle and apoptosis of mESCs.Alkaline phosphatase staining kit was used to detect the regulatory effect of miR-26 b on ALP activity in mESCs.Real-time quantitative PCR was used to detect the effect of miR-26 b on the mRNA expression of pluripotent transcription factors(Oct4, Sox2 and Nanog) in mESCs.Western blot was used to detect the effects of miR-26 b on the protein expression of three transcription factors in mESCs.The morphology of mESCs after transfecting pcDNA-pre-miR-26 bin vitrowas observed. Results In mESCs, the expression of pre-miR-26 b was highest among all members in miR-26 clusters. All miR-26 precursors were up-regulated in differentiated mouse embryonic fibroblasts(MEF), pre-miR-26 b showed the most significant increase among the three members(about 10-fold increase).Mature miR-26 b was significantly up-regulated from day 2 to day 6 during embryonic body(EB) formation, and the expression level of mature miR-26 b in MEF was significantly higher than that in mESCs.MiR-26 b overexpression repressed mESCs proliferation and arrested cell cycle in G1 phase. MiR-26 b did not affect mESCs apoptosis.Alkaline phosphatase staining showed that miR-26 b decreased the activity of ALP in mESCs.MiR-26 b overexpressing mESCs reduced expression of pluripotency markers(Oct4, Sox2 and Nanog).The effect of miR-26 b on mESCs differentiation was studied using anin vitrodifferentiation model. Differentiation was observed as early as day 4 of miR-26 b overexpression. Conclusion This study confirms the regulatory effects of miR-26 b on mESCs pluripotency.

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Objective To explore the regulation of miR-373-3 p on proliferation and metastasis of colon cancer SW480 cells by targeting Rab22 a. Methods The expression of miR-373-3 p in colon cancer tissues and cells was detected by RT-PCR. Luciferase reporter assay was used for testing the targeting relationship between miR-373-3 p and Rab22 a. Cells were divided into Control(SW480), Negative control(mimic NC), miR-373-3 p mimic, pLV-Rab22 a, pLV-Rab22 a+miR-373-3 p groups, each group was transfected with miR-373-3 p or Rab22 a recombinant lentivirus. RT-PCR was used to measure the relative transcript levels of miR-373-3 p and Rab22 a, cell proliferation was detected by CCK-8, wound scratch way was performed for detecting the migration of cells, the invasion of cells were detected by Transwell assay, the protein expressions of Rab22 a, Ki67, E-cadherin, N-cadherin were determined by Western blot. Results Compared with normal colon tissues, the expression of miR-373-3 p in colon cancer tissues notably decreased. Compared with human normal colon cancer cell NCM460, the expression of miR-373-3 p in colon cancer cells markedly decreased. In luciferase reporter assay, compared with Rab22 a WT+mimic NC, the luciferase activity in Rab22 a WT+miR-373-3 p mimic decreased. In cytological experiments, compared with SW480 group, the gene expression of miR-373-3 p and protein expression of E-cadherin increased in miR-373-3 p mimic group, and the gene and protein expression of Rab22 a, cell growth level, migration and invasion ability, protein expression of Ki67, N-cadherin decreased, meanwhile, the cell growth level, migration and invasion ability, protein expressions of Rab22 a, Ki67, N-cadherin increased in pLV-Rab22 a group, the E-cadherin protein expression decreased; Compared with pLV-Rab22 a group, the cell growth level, migration and invasion ability, protein expressions of Rab22 a, Ki67, N-cadherin decreased in pLV-Rab22 a+miR-373-3 p group, and the E-cadherin protein expression increased. Conclusion miR-373-3 p can target Rab22 a, and inhibit the proliferation and metastasis of colon cancer SW480 cells.

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Objective To establish a TaqMan real-time fluorescent quantitative PCR of spotted fever group rickettsiae(SFGR). Methods According to theompAgene sequence ofRickettsia.japonica,the specific primers and probes were designed to develop the TaqMan-based Real-time PCR method. The specificity, sensitivity and repeatability of the established method were verified. The established TaqMan real-time PCR method was used to detect SPGR infection in 80 clinic blood samples collected in Anhui Province and compared with the nested PCR. Results The real-time quantitative PCR method for detecting SPGR was successfully established. The cyclic threshold of the standard curve and the template copy number showed a good linear relationship(r>0.99). This method had strong specificity, high sensitivity and good repeatability in the detection of the spotted heat rickettsial nucleic acid samples. Conclusion This study establishes a real-time quantitative PCR assay based on TaqMan probe to detect SPGR, which provides a rapid and effective technical means for rapid diagnosis in clinical laboratories.

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Objective To observe and analyze the effect of interfering VIRMA gene on the migration ability of gastric cancer cells, and preliminarily explore the mechanism. Methods The MGC803 and SGC7901 gastric cancer cells were transfected with lentivirus to interfere the expression of VIRMA. The cell proliferation in each group was detected by CCK-8. The migration ability of cells was detected by wound healing assay and Transwell assay. The changes of VIRMA and FN1 expression were detected by qRT-PCR and Western blot. Results qRT-PCR and Western blot showed that lentivirus effectively affected VIRMA gene expression in gastric cancer cells(P<0.05). Interfering VIRMA gene inhibited the proliferation of gastric cancer cells effectively(P<0.05). The 24 h wound healing distance of gastric cancer cells in the interference group was less, and the number of transmembrane cells was also less than that of the control group(P<0.05). After interfering with VIRMA gene, the expression of FN1 decreased(P<0.05). Conclusion Lentivious-mediated knockdown of VIRMA can inhibit the migration ability of gastric cancer MGC803 and SGC7901 cells, and the mechanism may be related to the regulation of FN1 expression.

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Objective To study whether dexmedetomidine could affect the proliferation and migration of Huh-7 human hepatocellular carcinoma cells and to investigate the potential molecular mechanism. Methods The experiment was divided into two parts: cells and animals. Firstly, Huh-7 cells were randomly classified into six groups: normal control(group CON), dexmedetomidine 0.1 nmol/L(group D1), dexmedetomidine 1 nmol/L(group D2), dexmedetomidine 10 nmol/L(group D3), group OGD/R, OGD/R with dexmedetomidine 0.1 nmol/L(group D+OGD/R). Cells proliferation was measured by MTT assay. SIRT1 protein expression was assessed by Western blot. Transwell migration assay was used to detect the migration of Huh-7 cells. Secondly, the mouse xenograft model of hepatocellular carcinoma was established, and then randomly divided into 2 groups: group DEX and group CON. Normal saline or dexmedetomidine(0.5 mg/kg) was daily injected(s.c.) to the mice for six days in group CON or group DEX, respectively. Mice were euthanized after 30 days and the tumor size was recorded. Results Dexmedetomidine alone increased the cell viability of Huh-7 cells, and further improved the cell viability induced by OGD/R injury. On the other hand, the ability of migration was significantly increased by dexmedetomidine. Dexmedetomidine increased the expression of SIRT1 in normal condition and in OGD/R injury. However, there was no difference in tumor burden between group DEX and group CON. Conclusion Dexmedetomidine can affect the proliferation and migration of Huh-7 human hepatocellular carcinoma cells, in which SIRT1 may participate.

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Objective To explore the effects of exercise therapy on pathological injury and expression of skeletal muscle forkhead box transcription factor O1(FOXO1) and glucose transporter 4(GLUT4) in diabetes rats induced by streptozotocin(STZ). Methods A total of 75 male Wistar rats were enrolled and 60 cases of them were randomly selected and made into diabetes model, and they were divided into model group, low-intensity, middle-intensity and high-intensity exercise groups.The remaining 15 rats were enrolled as healthy control group.HE-staining was applied to observe gastrocnemius muscle.The expression of cysteinyl aspartate specific proteinase-3(Caspase-3),cysteinyl aspartate specific proteinase-9(Caspase-9),FOXO1 and GLUT4 proteins was detected by Western blot.The levels of blood glucose, total cholesterol(TC), triglyceride(TG), high density lipoprotein-cholesterol(HDL) and low density lipoprotein-cholesterol(LDL) were detected by automatic biochemical analyzer. The enzyme-linked immunosorbent assay was applied to detect levels of interleukin-6(IL-6), tumor necrosis factor-α ( TNF-α) and interleukin-10( IL-10). The biochemical methods were applied to determine activity levels of superoxide dismutase( SOD),malondialdehyde( MDA) and lactate dehydrogenase( LDH) in tissue homogenate of aortic vessel. The expression levels of FOXO1 and GLUT4 mRNA were detected by RT-PCR. Results Compared with healthy control group,expression of Caspase-3,Caspase-9 and FOXO1 protein,expression levels of TC,TG,LDL,IL-6,TNF-α and FOXO1 mRNA,activity of LDH and MDA were increased in model group( P<0. 05),while expression of GLUT4 protein,expression levels of HDL,IL-10,SOD and GLUT4 mRNA were decreased( P<0. 05). Compared with model group,expression of Caspase-3,Caspase-9 and FOXO1 protein,expression levels of TC,TG,LDL,IL-6,TNF-α and FOXO1 mRNA,activity of LDH and MDA were decreased in all exercise groups( P<0. 05). And the above indexes were the lowest in medium-intensity exercise group,while GLUT4 protein expression,expression levels of HDL,IL-10,SOD and GLUT4 mRNA were increased( P<0. 05). And the above indexes were the highest in medium-intensity exercise group. Conclusion Exercise therapy can alleviate pathological injury of diabetes rats by increasing expression levels of skeletal muscle GLUT4 protein and mRNA,and decreasing expression levels of skeletal muscle FOXO1 protein and mRNA. The effect of middle-intensity exercise is the best.

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Objective To investigate the role of CXCL12 on hepG2 cells apoptosis and its molecular mechanism. Methods hepG2 cells apoptosis was detected by flow cytometry(FCM), protein expression involving hepG2 apoptosis was detected by Western blot and laser scanning confocal microscopy(LSCM), protein expression of PI3 K/AKT signaling pathway was detected by Western blot. Results 100 ng/ml CXCL12 had an ability in inhibiting hepG2 cells apoptosis, down-regulating the protein expression of Bax, up-regulating the protein expression of Bcl-2, p-p85 and p-AKT(ser473), while had no effect on the protein expression of p110β, p85 and AKT. The inhibitor of PI3 K(LY294002) blocked the anti-apoptosis effect of CXCL12, increased the protein expression of Bax, decreased the protein expression of Bcl-2, p-p85 and p-AKT(ser473), while had no effect on the protein expression of p110β, p85 and AKT. Conclusion CXCL12 can increase the ratio of Bcl-2/Bax, inhibit hepG2 cells apoptosis, and the potential mechanism may be related to the activation of PI3 K/AKT signaling pathway.

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Objective To generate a G protein-coupled receptor kinase 2(GRK2) heterozygous(GRK2+/-) mice by CRISPR/Cas9 for investigating the pathogenic role and therapeutic implication of GRK2.Methods GRK2 targeting vector was constructed and the mixture of single guide RNA and Case9 mRNA was microinjected into zygotes of female C57 BL/6 mouse to generate gene mutation in Exon3. We got both 13 bp-deletion and 19 bp-deletion GRK2+/-miceaccording to genotyping performed through PCR and gene sequencing. Since GRK2 homozygous are embryonic lethality,we cross-bred 19 bp-deletion F0 GRK2+/-mice with C57 BL/6 to obtain stable 19 bp-deletion F1 generation of GRK2+/-mice for propagation and the following experiments. Results The genotype of GRK2+/-mouse was steady with significant lower body weight compared with C57 BL/6 mouse at the same age. Western blot results showed that almost half of the GRK2 protein expression was reduced in immune organs like spleen and thymus as well as other tissues. Conclusion This GRK2 targeted genetically engineered mouse model will provide a promising approach for understanding the role of GRK2 in the development of diverse organs and cells as well as its pathogenic role and therapeutic implications in chronic diseases.

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Objective To investigate the effect of interference with AKAP12 on the proliferation ability of human breast cancer MCF-7 cells and to explore its mechanism. Methods MCF-7 cells were exogenously transfected lentivirus with shRNA-AKAP12 and shRNA-NC, respectively. Western blot was conducted to examine the expression of AKAP12 protein in transfected cells. The proliferation ability of transfected cells were measured by CCK-8 assay and cell plate cloning assay. Western blot was used to detect the protein expression levels of cyclin-dependent kinase(CDK) inhibitors(p21, p27) and cell cycle progression-related CyclinD1. Results Compared to shRNA-NC cells, the expression of AKAP12 protein in shRNA-AKAP12 cells decreased. CCK-8 assay and colony formation assay showed that the cell proliferation ability of shRNA-AKAP12 cells increased significantly(P<0.000 1,P<0.001). Compared to the shRNA-NC cells, the expression of the cyclin-dependent kinase(CDK) inhibitors(p21, p27) were down-regulated in the shRNA-AKAP12 cells, while the cell cycle progression protein(CyclinD1) was up-regulated. Conclusion The study indicated that interference with AKAP12 gene could increase the proliferation ability of MCF-7 cells. It accelerated the G1-S phase transition of MCF-7 cells after interference with AKAP12 gene. Thus it is available to clinical treatment and prognosis evaluation of estrogen receptor-positive breast cancer.

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Objective To investigate the protective effect and mechanism of miR-155 on nucleus pulposus cells of rats. Methods Rat nucleus pulposus cells were cultured. miR-155 expression was detected after interleukin-1β(IL-1β) treatment and transfection. Cells were divided into Control group, IL-1β group(model group), IL-1β+miR-155 mimic group and IL-1β+miR-155 inhibitor group. Apoptosis rate was detected by flow cytometry. The expression levels of interleukin-6(IL-6), inducible nitric oxide synthase(iNOS) and interleukin-10(IL-10) were detected by ELISA. The contents of lactate dehydrogenase(LDH), superoxide dismutase(SOD), malondialdehyde(MDA) and glutathione(GSH) were detected by the kit. Western blot was used to detect the protein expression levels of Caspase-3, Caspase-9, p65, p-p65 and tumor necrosis factor α(TNF-α). Results IL-1β inhibited the expression of miR-155. Compared with the control group, the apoptotic rate of nucleus pulposus cells, Caspase-3, Caspase-9, IL-6, iNOS, LDH, MDA, NF-κB p65 and TNF-α expression in the model group all increased significantly, while IL-10, SOD and GSH all decreased significantly. Compared with the model group, miR-155 mimic reversely regulated the above indicators. Conclusion MiR-155 inhibits apoptosis, inflammation and oxidative stress in nucleus pulposus of rats, which may be related to the inhibition of NF-κB signaling pathway activation.

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Objective To study the effects of different iodine intake on thyroid function and the methylation of 5′-CpG island in the promoter region of sodium iodine transporter(NIS) gene in rats, and to explore whether iodine affects the expression level of NIS through regulating the methylation of NIS gene. Methods Forty female SD rats were randomly divided into five groups(8 rats in each group). The rats in NI group(control) were fed with normal content of iodine, and the rats in LI group, 5 HI group,10 HI Group and 20 HI group were fed with different amounts of potassium iodide. These rats were sacrificed 3 months after the feeding and all serum samples were tested for free triiodothyronine(FT3),free thyroxine(FT4) and thyroid stimulating hormone(TSH) by using chemiluminescent detection technology. The expression of NIS protein in the rats' thyroid tissues was determined by immunohistochemical staining. The status of 5′-CpG island promoter methylation of NIS gene in the rats' thyroid tissues wasdetected by bisulfite sequencing PCR(BSP). Results ① Compared with the iodine-adapted group, the serum FT3 concentration of the low-iodine group and the high-iodine group decreased, and the difference was significant(P<0.05). Compared with the iodine-adapted group, the serum FT4 in the low-iodine group and the high-iodine group slightly increased, but the difference was not statistically significant(P>0.05). There was no significant difference in TSH level between groups(P>0.05).② The expression level of NIS in thyroid tissues of the LI group was higher than that of the NI group, with statistically significant difference(P<0.05), while that of the 5 HI group was lower than that of the NI group, with no statistically significant difference(P>0.05), while those of the 10 HI group and the 20 HI group were lower than that of the NI group(P<0.01);③ There was no significant difference in CpG1 methylation ratio and CpG2 methylation ratio between the groups(P>0.05). Further study on the sequence of-420 to-110 nucleotides in the NIS promoter region(CpG3) showed that compared with the normal iodine group, the methylation ratio of CpG3 in the low-iodine group and the high-iodine group decreased, but the difference was not statistically significant(P>0.05). Conclusion Iodine overdose or iodine deficiency can cause iodine nutritional disorders, but it does not affect the methylation status of the 5′-CpG island promoter methylation of sodium iodide symporter gene in rats. Iodine may affect the expression level of NIS through other ways.

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Objective To investigate the effect and mechanism of miR-17 on non-small cell lung cancer(NSCLC) by regulating fatty acid metabolism. Methods The expression of miR-17 mRNA in human non-small cell lung cancer cell lines(H1703, A549, H1299, L78, PGCL3, H460) and normal lung cells(BEAS-2 B) was detected by RT-qPCR. The growth of A549 cells was detected by colony formation assay. The migration ability of A549 cells was detected by scratch test. The invasion ability of A549 cells was detected by Transwell method. The phospholipid and triglyceride content in A549 cells was detected by kit. The expression levels of cluster of differentiation 36(CD36),fatty acid synthase(FASN), acetyl-CoA carboxylase(ACC1) and fatty acid binding protein 5(FABP5) in A549 cells were detected by Western blot. The targeting relationship between miR-17 and stearoyl-CoA desaturase(SCD) was verified by a dual luciferase report. Results In non-small cell lung cancer cell lines(H1703, A549, H1299, L78, PGCL3, H460), the expression of miR-17 mRNA was significantly down-regulated(P<0.01). In the miR-17 mimic group, the number of cell clones significantly decreased(P<0.01), the scratch closure rate significantly decreased(P<0.01), the number of invasive cells significantly decreased(P<0.01), the content of phospholipids and triglycerides significantly decreased(P<0.01), and the expression of FASN, ACC1, FABP5 and CD36 protein was significantly down-regulated(P<0.01). Up regulation of CD36 reversed the regulation of miR-17 on growth, metastasis and fatty acid metabolism in non-small cell lung cancer cells. miR-17 regulated the expression of CD36 by targeting SCD. Conclusion MiR-17 inhibits the growth and metastasis of non-small cell lung cancer cells by targeting SCD to regulate CD36-mediated fatty acid metabolism.

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Objective To investigate the effects of curcumin(CUR) on lipopolysaccharide(LPS)-induced depression in mice and its mechanism. Methods Thirty male ICR mice were randomly divided into control group, LPS group and CUR group, ten mice in each group. The depression model was established by intraperitoneal injection of LPS 1 mg/kg. The depressive-like behaviors of mice were evaluated by sucrose preference test(SPT), open field test(OFT) and forced swim test(FST). The changes of Nissl body in neurons of prefrontal cortex(PFC) were observed by Nissl staining. C-Jun N-terminal kinase(JNK), phosphorylated JNK, Bax(Bcl-2 associated X protein), Bcl-2, caspase-3 in PFC area were detected by Western blot. Results The percentage of sweet water preference, total distance in OFT and crossing number in CUR group were higher than those in LPS group(P<0.05), and immobility time in FST was lower than that in LPS group(P<0.05). The results of Nissl staining showed that number of nerve cells in CUR group was more than that in LPS group(P<0.05).Western blot showed that the expression levels of p-JNK, Bax and caspase-3 were decreased in PFC area of CUR group while Bcl-2 were increased compared with the LPS group(P<0.05). Conclusion Curcumin can improve the depressive behavior induced by LPS and reduce the damage of neurons, which may be related to the inhibition of JNK phosphorylation, the decrease of Bax and Caspase-3, and the increase of Bcl-2 expression.

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Objective To investigate the inhibitory effect of N-acetylcysteine(NAC) on the expression of inflammatory cytokines in human kidney proximal tubular epithelial cell induced by angiotensin Ⅱ(Ang Ⅱ). Methods HK2 cells were divided into four groups(control group, Ang Ⅱgroup, NAC group and Ang Ⅱ+NAC group). HK2 cells were treated with NAC(5 mmol/L), Ang Ⅱ(1 mmol/L) for 48 h. The levels of tumor necrosis factor α(TNF-α) and interleukin-6(IL-6) mRNA were detected by RT-PCR. The expression of TNF-α,monocyte chemotactic protein 1(MCP-1) and interleukin-1β(IL-1β) protein was detected by Western blot. The distributions of TNF-α were detected by immunofluorescence assay. Results Compared with the control group, the protein expression of inflammatory cytokines such as TNF-α, MCP-1 and IL-1β and mRNA expression(TNF-α, IL-6) increased in HK-2 cells when treated with Ang Ⅱ infusion. After NAC treatment, the above changes reduced in ang Ⅱ + NAC group, and the same results were showed by immunofluorescence technology. Conclusion N-acetylcysteine can inhibit the Ang Ⅱ-induced inflammation-related cytokines expression in human kidney proximal tubular epithelial cell.

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Objective To investigate the effect of crocin on the growth, exercise and multi-organ injury of melanoma A375 cells, as well as the expression of proteins related to the AKT and STAT3 signaling pathways.Methods A subcutaneously transplanted tumor model of A375 nude mice was established. The mice were randomly divided into 4 groups, and the tumor was injected with 0, 20, 50, 100 mg/kg of crocin and normal saline respectively, and 0.1 ml each time to observe the changes of the tumor. Immunohistochemistry was used to detect the levels of Ki67,Caspase-3 and the expression of VEGF and Vimetin. The expression of Survivin and Bcl-2/Bax genes was detected by RT-PCR. The histopathological morphology of liver,lung and kidney was observed by HE staining. Western blot was used to detect the expression of motility and EMT markers E-cadherin,N-cadherin and Fibronectin,as well as phosphorylation of AKT and STAT3. Results Compared with the control group,the volume and weight of melanomas in the low,medium and high dose groups of Crocin decreased( P<0. 05); the number of Ki67,VEGF and Vimentin positive cells decreased( P<0. 05),while the number of Caspase-3 positive cells increased( P<0. 05); the expression of Survivin mRNA decreased( P<0. 05); the expression of Fibronectinprotein decreased( P<0. 05).Compared with the control group,the expression of Bcl-2/Bax mRNA in the middle and high dose groups decreased( P<0. 05); the expression levels of E-cadherin and N-cadherin decreased( P<0. 05); the phosphorylation levels of AKT and STAT3 decreased( P<0. 05); Crocin alleviated the tissue damage of liver,lung and kidney. Conclusion By inhibiting the phosphorylation of AKT and STAT3,Crocin can reduce the pathomorphological changes of liver,lung and kidney tissues,and inhibit the growth and movement of A375 melanoma,suggesting that saffron has a relieving effect on A375 melanoma. Saffron can be used as an effective and safe non-toxic regulator to prevent and treat melanoma.

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Objective To investigate the effect of TRX-1 overexpression on apoptosis in Parkinson's disease PC12 cells by regulating Nrf2/HO-1 pathway. Methods PC12 cells were induced by 1-methyl-4-phenyl-pyridine(MPP+) to establish Parkinson's disease cell model. The plasmid pcDNA-hTRX-1 was transfected into PC12 cells, and the expression of TRX-1 was detected by qRT-PCR. MTT assay was used to detect cell proliferation, flow cytometry was used to detect apoptotic rate and intracellular reactive oxygen species level. Then Western blot was used to detect the expression of apoptotic related proteins Bax, Bcl-2, Cyt-c, cleaved Caspase-3 and oxidative stress related proteins Nrf2 and HO-1. Results TRX-1 was low expressed in Parkinson's disease PC12 cells, and over-expression of TRX-1 could improve the viability of Parkinson's disease PC12 cells, inhibit cell apoptosis, and significantly activate Nrf2/HO-1 pathway. Conclusion Over-expression of TRX-1 can inhibit the apoptosis of PC12 cells in Parkinson's disease by activating Nrf2/HO-1 pathway.

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Objective To compare the effects of five single drugs of xiaorusanjietang-radix rehmanniae praeparata, paeonia lactiflora, fructus gardeniae, sculellaria barbata and fritillaria thunbergii on the sensitivity of drug resistance to adriamycin resistant human breast cancer cell line MCF-7/ADR. Methods Radix rehmanniae praeparata, paeonia lactiflora, fructus gardeniae, sculellaria barbata and fritillaria thunbergii were prepared and distilled. The freeze-dried powder of each medicine was dissolved into 1 mg/ml liquid storage solution with phosphate buffer saline(PBS). The methyl thiazolyl tetrazolium(MTT) assay was used to determine the non-toxic and lethal dose of each drug to MCF-7/ADR cells. After pretreatment of MCF-7/ADR cells with non-toxic dose as the reverse concentration of drug resistance, adriamycin was added and MTT method was used to detect whether the single drug had a reverse effect on cell resistance. Results MCF-7/ADR was treated with non-toxic low dose(1 μg/ml) and high dose(10 μg/ml), respectively, and no single drug radix rehmanniae praeparata, paeonia lactiflora, fructus gardeniae and sculellaria barbata were observed to reverse cell resistance. However, when treated with fritillaria thunbergii alone, adriamycin had a killing effect(P<0.05), and the dose-dependent reversal effect was observed when combined with adriamycin of different concentrations. Conclusion The single drug fritillaria thunbergii in xiaorusanjietang has a reversal effect on the drug resistance of MCF-7/ADR cells, and the reversal ability is dose-dependent.

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Objective To investigate the effect of exosomes secreted by human mesenchymal stem cells(hMSCs) on the proliferation and secretion of monocytes. Methods hMSCs were cultured by centrifugation method, and the supernatant of hMSCs was collected. Exosomes were extracted by kit and observed by SEM. CD63 and CD81 levels were tested by Western blot.Monocytes were isolated by density gradient centrifugation. Lipopolysaccharide(LPS) was used as a stimulant to induce proliferation and the synthesis of inflammatory cytokines. After treated by exosomes, MTT viability assay and apoptosis analysis by flow cytometry(FCM) were used to study the proliferative ability. Tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6) and interleukin-8(IL-8) in the culture supernatant were measured by enzyme-linked immunosorbent assay(ELISA). Results Exosomes secreted by hMSCs were small ball-shaped under SEM. CD63 and CD81 were positively expressed by Western blot. MTT showed that LPS could promote the proliferation of monocytes, while exosomes inhibited the proliferation of monocytes. However, after treated by exosomes, the apoptosis of monocytes significantly increased. ELISA showed that exosomes could inhibit the secretion of TNF-α, IL-1β, IL-6 and IL-8 by monocytes. Conclusion Exosomes secreted by hMSCs can inhibit the proliferation of monocytes and the secretion of TNF-α, IL-1β, IL-6 and IL-8 by monocytes while promote the apoptosis. It is preliminarily proved that hMSC-derived exosomes have immune-regulatory function.

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Objective To investigate the inhibitory effect and mechanism of phillyrin(PHN) on inflammatory response induced by lipopolysaccharide(LPS). Methods An LPS-induced mouse macrophage(RAW264.7) inflammatory model was established to study the secretion of inflammatory cytokines and the expression of related proteins. The effects of PHN at different concentrations on the activity of mouse macrophages were detected by MTT assay. The nitric oxide(NO) release was detected by Griess method. The expression levels of tumor necrosis factor α(TNF-α) and interleukin-6(IL-6) were detected by ELISA assay. The expression level of TLR4 signaling pathway-associated protein was detected by Western blot. Binding affinity was detected by molecular docking. Furthermore, an acute lung injury(ALI) model induced by LPSin vivowas established, and the protective effect of PHN was studied by observing histopathological changes. Results PHN had no significant inhibitory effect on cell proliferation in the concentration range below 30 μmol/L. Furthermore, PHN could inhibit the secretion of NO, TNF-α and IL-6 by LPS-induced macrophage cells. Further studies had shown that PHN inhibited the secretion of cytokines by inhibiting the TLR4 signaling pathway, and molecular docking showed that PHN had a strong affinity with TLR4 protein. In addition, PHN showed good function in vivo anti-inflammatory activity in an LPS-induced ALI model. Conclusion PHN can effectively regulate LPS-induced inflammation, which may be through blocking the TLR4 signaling pathway to reduce the expression of inflammatory cytokines.

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Objective To investigate the effect of the aqueous extracts of mulberry on adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) stimulated by H2O2. Methods BMSCs were isolated from the bone marrow of rats and identified by flow cytometry. The aqueous extracts of mulberry were prepared by vacuum freeze-drying. CCK-8 method was performed to detect the toxicity of mulberry aqueous extract(15, 30, 60 mg/L) to BMSCs. Oil red O staining was used to identify the lipid droplets. Immunofluorescence and Western blot were used to detect the expression of PPARγ and C/EBPα in BMSCs treated by the aqueous extracts of mulberry(15, 30, 60 mg/L). Results CCK-8 assay showed that the aqueous extracts of mulberry did not affect the growth of BMSCs(P>0.05). Oil red O staining showed that the aqueous extracts of mulberry inhibited the formation of lipid droplets in BMSCs induced by H2O2, showing a dose-effect relationship(P<0.01). The aqueous extracts of mulberry(60 mg/L) significantly inhibited the expression of PPARγ and C/EBPα in BMSCs stimulated by H2O2(P<0.05). Conclusion The aqueous extracts of mulberry can inhibit the adipogenic differentiation of BMSCs induced by H2O2trough PPARγ and C/EBPα signaling pathway.

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Objective To observe the difference of human GSE1 coiled-coil protein(GSE1) expression in hepatocellular carcinoma(HCC) and adjacent non-tumor tissues,and then explore the clinical role and clinicopathological significance of GSE1 in the development and progression of HCC. Methods qRT-PCR,Western blot and immunohistochemistry were used to detect the expression of GSE1 protein in HCC and adjacent non-tumor tissues. The results were analyzed by SPSS 20.0 software. The relationship between GSE1 mRNA expression and the clinicopathological characteristics of HCC was discussed. Results The results of qRT-PCR showed that the expression level of GSE1 mRNA in 30 groups of fresh HCC was higher than that in adjacent non-tumor tissues, and the difference was statistically significant(P<0.000 1). The results of Western blot showed that GSE1 was highly expressed in HCC tissues, but low expressed in adjacent non-tumor tissues.The results of immunohistochemistry showed that the positive rate of GSE1 protein was 50.70%(36/71) in HCC tissues and 19.51%(8/41) in 41 adjacent non-tumor tissues. There was a significant difference in the expression of GSE1 between HCC and adjacent non-tumor tissues(P<0.05).The expression of GSE1 protein in HCC and adjacent non-tumor tissues was correlated with clinical pathological stage(P<0.01),but not with gender,age,vascular invasion and Edmondson histological grade. Conclusion GSE1 is highly expressed in HCC, and it may be closely related to the development of HCC.

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Objective To investigate the correlation between diabetic kidney disease and sweat function by measuring urine albumin to creatinine ratio(UACR) and the skin electrical conductivity(ESC). Methods A total of 300 type 2 diabetes mellitus(T2 DM) patients were enrolled in this study. UACR and Sudoscan were done for each patient. The conductivity analyzer Sudoscan was used to measure the skin electrical conductivity(ESC) of hands and feet. All patients were divided into two groups according to the hands ESC(HESC) and feet ESC(FESC), i.e. patients with normal sweat function(HESC>60 μs and FESC>70 μs, NSF group,n=125) and patients with abnormal sweat function(HESC≤60 μs or FESC≤70 μs, ASF group,n=175). The relevance of UACR to sweat function was later analyzed. Results Age, the duration of diabetes and UACR were significantly higher in the ASF group compared with the NSF group, while HESC and FESC were significantly lower(P<0. 05 orP<0. 01). Spearman correlation analysis showed that both HESC and FESC negatively correlated with UACR(rs=-0.16,-0.17,P<0.01), the duration of diabetes(rs=-0.18,-0.15,P<0.01) and Scr(rs=-0.11,-0.16,P<0.05). Multivariate stepwise linear regression analysis showed that the duration of diabetes and UACR were independent risk factors for HESC. The duration of diabetes, UACR and Scr were independent risk factors for FESC. When UACR was used as the criterion for diagnosing diabetic kidney disease, the area under curve(AUC) of HESC and FESC was 58.7% and 59.8% respectively, the specificity was 64.3% and 70.5% respectively, the sensitivity was 57.2% and 65.6% respectively. Conclusion Sweat function test is a potential predictor for kidney disease in patients with T2 DM.

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Objective To investigate the expression and clinical significance of family with sequence similarity 72 member A(FAM72 A) in hepatocellular carcinoma(HCC).Methods FAM72 A mRNA expression overview in normal tissues was performed by HPA database.GEPIA and Oncomine databases were used to analyze the different expression of FAM72 A in HCC and normal liver tissues.LinkedOmics platform was used to analyze the relationship between the expression level of FAM72 A and the pathological characteristics of HCC.Relationship between expression level of FAM72 A and prognosis was analyzed through Kaplan-Meier Plotter. c Bio Portal and STRING databases were used to establish the FAM72 A co-expression molecular network. KEGG pathway enrichment analysis and GO enrichment analysis were done using DAVID. Results FAM72 A was low expressed in normal liver tissues.FAM72 A expression in HCC tissues was higher than that in normal liver tissues( P<0. 05). Expression of FAM72 A was correlated with age( P = 0. 006 8),pathological stage( P = 0. 018),T stage( P = 0. 022) and race( P= 0. 034). Patients with high expression of FAM72 A tended to have poorer prognosis( P = 0. 000 12). FAM72 A co-expression genes involved in biological process including DNA transcription,regulation of DNA transcription,cell division and nuclear division( FDR<0. 05). KEGG pathway enrichment analysis showed that these genes were involved in cell cycle,RNA transport,spliceosome,DNA replication,P53 signaling pathway,base excision and repair,and other related pathways( FDR<0. 05). FAM72 A co-expression molecular regulatory network was established. Conclusion Expression of FAM72 A in HCC is higher and the patients with high expression level of FAM72 A have poorer prognosis. Expression of FAM72 A is correlated with race,age and pathological stage.FAM72 A may be potential therapeutic target and potential prognostic biomarker of HCC.

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Objective To investigate the changes and clinical significance of interleukin-22(IL-22) and interleukin-22 binding protein(IL-22 BP) in peripheral blood of patients with type 2 diabetes mellitus(T2 DM). Methods A total of 90 patients with T2 DM were selected as the research objects of type 2 diabetes group, and another 40 normal healthy people were selected as the control group. The levels of IL-22 and IL-22 BP were measured by enzyme-linked immunosorbent assay(ELISA), and biochemical indexes and chronic complications were recorded. Results Compared with the normal control group, the level of IL-22 expression in T2 DM group was lower(P<0.001), while the level of IL-22 BP was higher(P<0.01). Correlation analysis showed that IL-22 was negatively correlated with IL-22 BP and ankle brachial index(ABI)(ρ=-0.273,-0.312,P<0.01), positively correlated with waist hip ratio(WHR), fasting plasma glucose(FPG), glycosylated hemoglobin(HbA1 C) and homeostasic model assessment of insulin resistance(HOMA-IR)(ρ=0.209, 0.282, 0.243, 0.241,P<0.05), IL-22 BP was negatively correlated with HbA1 C and low density lipoprotein cholesterol(LDL-C) to different degrees(ρ=-0.290,-0.208,P<0.05). Logistic regression analysis showed that IL-22 and IL-22 BP levels were related factors for the pathogenesis of T2 DM. Compared with non-diabetic nephropathy group, the expression of IL-22 in diabetic nephropathy group decreased(P<0.01), while the expression of IL-22 BP increased(P<0.05). Conclusion The level of IL-22 expression decreases and IL-22 BP expression increases in type 2 diabetic patients, which may be related to blood glucose control, T2 DM and diabetic nephropathy.

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Objective To investigate the expression of GPR110 in different pathological subtypes of stage IA lung adenocarcinoma and its relationship with clinicopathological features and prognosis. Methods 90 cases of stage IA lung adenocarcinoma were selected. There were 30 cases each in the wall growth group, the acinar/nipple group and the micronipple/solid group. Immunohistochemistry was used to detect the expression of GPR110 protein in each group. Results The expression of GPR110 was correlated with the pathological subtype(P<0.05), but not with the patients' age, gender and tumor size(P>0.05). COX regression analysis showed that pathological subtype had an impact on the 5-year survival rate of patients. It was a risk factor for prognosis(P<0.05). Conclusion The high and low expression of GPR110 is closely related to the pathological subtype of stage IA lung adenocarcinoma, which may be a new biomarker for the potential prevention and risk prediction of early lung adenocarcinoma patients after surgery operation.

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Objective To study the value of plasma exosome miRNA-188-5 p and miRNA-4741 in the diagnosis of endometriosis(EMT). Methods RT-qPCR was used to analyze the expressions of plasma exosome miRNA-188-5 p and miRNA-4741 in 50 patients with EMT and 50 healthy controls. ROC curve was used to evaluate the roles of miRNA-188-5 p and miRNA-4741 in the diagnosis of EMT. Results The expression levels of miRNA-188-5 p and miRNA-4741 in case group were lower than those in control group(P<0.05). ROC curve showed that for the diagnosis of endometriosis, the sensitivity and the specificity of miRNA-188-5 p were 77.6% and 83.5%, the sensitivity and the specificity of miR-4741 were 84.5% and 91.2%, and the sensitivity and the specificity of the combination of them were 87.1% and 93.8%. Conclusion Compared with healthy women, the expressions of plasma exosome miRNA-188-5 p and miRNA-4741 in EMT are lower, and their clinical diagnostic efficiency are higher, which may be potential biomarkers for the diagnosis of EMT.

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Objective To investigate the role of peripheral blood circular RNA hsacirc0002715 in the diagnosis and differential diagnosis of systemic lupus erythematosus(SLE) patients.Methods Peripheral blood samples were collected from 76 SLE patients, 33 healthy controls(HC) and 88 non-SLE patients including 56 rheumatoid arthritis(RA) patients and 32 ankylosing spondylitis(AS) patients. Expression of peripheral blood circular RNA hsacirc0002715 was detected by qRT-PCR. Differences in peripheral blood circular RNA hsacirc0002715 among SLE, HC and non-SLE groups were comparatively analyzed. Correlations of peripheral blood circular RNA hsacirc0002715 with several clinical indexes were analyzed.Results ① The expression of peripheral blood circular RNA hsacirc0002715 was significantly higher than that in HC(P<0.001) and non-SLE(P<0.001).② The expression of peripheral blood circular RNA hsacirc0002715 in SLE patients was negatively correlated with C3(rs=-0.453,P<0.001), C4(rs=-0.336,P=0.004), WBC(rs=-0.486,P<0.001), HGB(rs=-0.277,P<0.016), PLT(rs=-0.300,P=0.009). The expression of peripheral blood circular RNA hsacirc0002715 in SLE patients was positively correlated with anti-ds DNA( rs= 0. 253,P = 0. 040). The expression of peripheral blood circular RNA hsa＿circ＿0002715 in SLE patients with postive anti-nucleosome was significantly higher than that in SLE patients with negative anti-nucleosome( P<0. 001).(3) The area under ROC curve( AUC),sensibility and specificity of peripheral blood circular RNA hsa ＿ circ ＿0002715 between SLE patients and HC was 0. 772,63. 16%,81. 82%. The AUC,sensibility and specificity of peripheral blood circular RNA hsa＿circ＿0002715 between SLE patients and non-SLE was 0. 674,55. 26%,72. 73%. The AUC,sensibility and specificity of peripheral blood circular RNA hsa ＿ circ ＿0002715 between SLE patients and all controls(non-SLE + HC) was 0. 701,51. 32%,80. 17%.(4) The sensibility of peripheral blood circular RNA hsa＿circ＿0002715 for SLE patients was superior to anti-ds DNA,and the sensibility and sensibility of combination of hsa＿circ＿0002715-anti-ds DNA significantly increased. Conclusion The expression of peripheral blood circular RNA hsa＿circ＿0002715 significantly increases in SLE patients,and it is correlated with the activity of SLE and damage of hematological system. This study suggests that the peripheral blood circular RNA hsa＿circ＿0002715 may be a potential diagnosis and differential diagnosis biomarker for SLE.

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Objective To investigate the expression of serum SPINK1 in hepatocellular carcinoma(HCC) patients, analyze the diagnostic efficacy of SPINK1 alone and combined with alpha fetoprotein(AFP) detection in HCC, and explore the value of SPINK1 in the short-term prognosis of HCC. Methods Serum was collected from patients in the HCC group(87 cases), patients in the chronic liver disease group(73 cases), and volunteers in the healthy control group(45 cases). Kruskal-Wallis H was used to compare the expression of SPINK1 among different groups, and analyze its relationship with Child-Pugh grades in HCC patients; the ROC curve was drawn and used to analyze the diagnostic efficacy of SPINK1, AFP and the combined detection for HCC. Risk factors for the Disease-free survival(DFS) of HCC were assessed by Kaplan-Meier and Cox regression analysis. Results Serum levels of SPINK1 and AFP were higher in the HCC group than those in the chronic liver disease and health control groups(χ2= 87.28,72.55, allP<0.01). Serum SPINK1 levels in chronic liver disease group were similar to those in healthy control group(P>0.05). The sensitivity of SPINK1 in the diagnosis of HCC was higher than that of AFP. There was no difference in AUC between the two biomarkers(P>0.05). The specificity and AUC of the combined were higher than that of any single one(P<0.05). The AUC of SPINK1 in the diagnosis of AFP-negative HCC was 81.70%(95%CI: 0.74-0.88), with the specificity and sensitivity as 71.18% and 95%(when the cut-off value was 1.75 ng/ml). Serum SPINK1 levels were different among HCC patients with different Child-Pugh grades(χ2=10.17,P<0.05). Short-time DFS of HCC in the high SPINK1 group was lower than that of the low SPINK1 group(P<0.01), Serum SPINK1 level was an independent risk factor for short-time DFS(HR=9.92, 95%CI: 2.82-34.87,P<0.01) in HCC patients. Conclusion Serum SPINK1 has high diagnostic value for HCC, especially in the diagnosis of AFP-negative HCC. Higher levels of SPINK1 have a high predictive value for short-time poor prognosis of HCC.

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In order to analyze the correlation between age factors and the epidemiology, this study reviewed 115 fungemia cases in our hospital during the period 2012-2019. There were 30 strains of Candida albicans,29 stains of Candida tropicalis, 27 strains of Candia para psilosis, 16 strains of Candida glabrata, 4 strains of Candida krusei and 9 strains of others. The study found that fungemia frequently happened in male and young patient. The study also found that the percentage of patient in ICU and department of medicine, were positively correlated with age, while surgery and hematology were negatively correlated with age. For gender, ICU, hematology, glucocorticoids, respiratory distress, diabetes and utilization of drugs, the differences among age groups were statistically significant. For ICU, shock and internal medicine, the differences between prognosis groups were statistically significant. Therefore, clinical analysis should be based on different age and different factors to assist diagnosis and improve prognosis.

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A Graussian kernel SVM model was constructed based on identified risk factors(including number of surgical segments, preoperative/postoperative intervertebral height, postoperative L4segment lordosis, and postoperative PT, SS) related to cage subsidence. The AUC was 0.93. The results will help the development of precision medicine in spine surgery.