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Objective To explore the feasibility of CS-BC composite membrane as guided bone regeneration(GBR) membrane by preparing chitosan(CS)-bacterial cellulose(BC) composite membrane, and testing the mechanical strength of the composite membrane both in dry and wet states as well as evaluating its cytocompatibility. Methods The CS solution and BC solution were mixed in different weight ratios and were dispersed evenly by ultrasonic. Pure CS membrane and composite membranes with different weight ratios of CS and BC(10 ∶1, 10 ∶3, 10 ∶5, 10 ∶7 and 10 ∶9) were prepared by self-evaporation process. Thereafter, the fabricated membranes were immersed into sodium hydroxide ethanol solution to remove the acid. The tensile strength of the as-fabricated membranes under dry condition and under hydration were measuredviamechanical universal testing machine(n=6). The microstructures of the composite membrane with the highest tensile strength were observed by scanning electron microscopy(SEM) and transmission electron microscopy(TEM). To characterize the chemical composition of the composite membrane, Fourier transform infrared(FTIR) spectrometer and X-ray diffraction(XRD) were used. CCK-8 assay was carried out to evaluate the survival rate of cells in the control group(without any membrane), the pure CS membrane group and the composite membrane group with maximum tensile strength(n=5) after cocultured with rat bone marrow stem cells(RBMSCs) for 1, 4 and 7 d. Results The cross-section of the composite membrane displayed ordered layer structure after introducing BC into CS matrix. The results of FTIR and XRD indicated the existence of BC in the composite membrane. The tensile strength of the CS-BC composite membrane increased first and then decreased with the increase of BC ratio. The tensile strength of the composite membrane reached the highest in dry and wet states when the weight ratio of CS and BC is 10 ∶7, which were almost(204.7±63.0) MPa and(44.4±6.4) MPa respectively. After cocultured with RBMSCs for 1, 4 and 7 d, there was no significant difference in the number of cells among the pure CS group, the CS-BC composite membrane group and the blank control group. Conclusion When the weight ratio of CS and BC is 10 ∶7, the tensile strength of the composite membrane are the best both in dry and wet state and the cell compatibility is excellent.

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Objective To investigate the reversal of drug resistance of drug-resistant hepatoma cell line HepG2/DDP by emetine and its mechanism. Methods The drug-resistant hepatoma cell line HepG2/DDP was established by high-dose cisplatin(DDP) shock combined with low-dose continuous induction. Reverse virtual screening, molecular docking, surface plasmon resonance(SPR) and Western blot assays were employed to explore the target of emetine. The target was validated by transfection assayin vivo. The inhibitory effect of emetine combined with DDP on the proliferation of HepG2/DDP cells was detected by cytotoxicity assay. The sensitization effect of emetine was verified by flow cytometry, and the expression of the apoptosis related proteins BCL2, Bax and Cleaved-caspase-3 were analyzed by Western blot. Results Emetine enhanced the sensitivity of HepG2/DDP cells to DDP and reduced the resistance index(RI) from 3.69 to 0.93. Reverse virtual screening, molecule docking and SPR results showed that emetine can stably bind to PARP-1. Western blot assays showed that emetine had a potent enzymatic inhibitory activity against PARP-1in vivo. Furthermore, emetine's potentiation of DDP in HEPG2/DDP cells nearly disappeared when the cells were transfected with siRNAs against PARP-1. Flow cytometry showed that emetine could enhance the proapoptotic effect of DDP on HepG2/DDP cells. Conclusion Emetine can enhance the sensitivity of HepG2/DDP cells to DDP, and its mechanism may be related to its inhibition of PARP-1.

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Objective To investigate the regulatory effects and partial mechanisms of berberine(BBR) on podocyte epithelial mesenchymal transformation(EMT) and apoptosis induced by high glucose(HG). Methods Podocytes at exponential growth stage were divided into normal group, high glucose group and berberine group(30, 60 and 90 μmol/L). The rate of podocyte apoptosis was detected by flow cytometry. Transwell and scratch assay were used to detect podocyte migration. Western blot was used to detect the expression of apoptosis protein Bim, podocyte EMT-associated proteins nephrin, α-smooth muscle actin(α-SMA), vimentin and tyrosine kinase 2(JAK2)/signal transductor and transcriptional activator 3(STAT3) pathway-related proteins. Laser confocal method was used to detect the expression of EMT-related proteins nephrin, α-SMA and vimentin in podocytes. Results Flow cytometry results showed that BBR(30, 60 and 90 μmol/L) could reduce the rate of podocyte apoptosis in high glucose environment. Transwell and scratch test results showed that BBR(30, 60 and 90 μmol/L) could inhibit abnormal podocytes migration in high glucose environment. Western blot results showed that BBR(30, 60 and 90 μmol/L) could decrease the protein expression of p-JAK2, p-STAT3, α-SMA, vimentin and Bim, and increase the protein expression of nephrin in podocytes under high glucose condition. Laser confocal results showed that the BBR(30, 60, and 90 μmol/L) could decrease the expression of α-SMA and vimentin in podocyte cytoplasm, increase the expression of nephrin in podocyte nucleus and cytoplasm. Conclusion BBR may alleviate the EMT and apoptosis of podocytes induced by high glucose through JAK2/STAT3 signaling pathway.

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Objective To investigate the role and mechanisms of empagliflozin(EMPA) on myocardial ischemia/reperfusion(MI/R) injury. Methods Forty male SD rats were randomly divided into Control group, MI/R group,2. 5 μmol/L EMPA combined with MI/R group(EMPA + MI/R) as well as EMPA and 10 μmol/L SIRT1 inhibitor EX527 combined with MI/R group(EMPA + EX527 + MI/R). Cardiac function, myocardial fiber morphology and infarct size were detected. The content of malonaldehyde(MDA), the activities of superoxide dismutase(SOD)and succinodehydrogenase(SDH) in myocardium were determined by ELISA. The protein expressions of Cytochrome c, Cleaved caspase-3, SOD2, gp91phoxand SIRT1 were observed by Western blot. The ROS level was detected by DHE staining.Results Compared with Control group, MI/R group manifested the decreased cardiac function and myocardial fiber rupture. Myocardial infarction area, the expressions of Cytochrome c, Cleaved caspase-3 and gp91phox, as well as the MDA content and ROS level increased, while decreased the activities of SOD and SDH, and the expressions of SOD2 and SIRT1. Compared with MI/R group, EMPA + MI/R group improved cardiac function and inhibited myocardial fiber rupture, myocardial infarction area, the expressions of Cytochrome c and Cleaved caspase-3. The expression of gp91phox, the MDA content and ROS level were also downregulated,while the activities of SOD and SDH and the expressions of SOD2 and SIRT1 were upregulated. However, the protective effects of EMPA on MI/R heart were blocked by EX527.Conclusion EMPA alleviates MI/R injury by inhibiting mitochondrial oxidative stress and apoptosisviaactivating SIRT1.

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Objective To study the neuropathological changes and process brain astrocyte microglia and neuron injury in rats at different time after mild blast-related traumatic brain injury(bTBI), and to investigate the neuroprotective potential of dietary supplementation with fish oil on mild bTBI rats. Methods 54 newly weaned SD rats were randomly divided into control group(n=18), model group(n=18) and treatment group(n=18). The model group and treatment group were fed with ordinary diet and oil-rich diet for 33 days to establish the mild bTBI model by shock wave, respectively. The control group rats were fed with ordinary diet without shock wave injury. Results Compared with control group, the weight of rats in the model group and treatment group decreased to a certain extent within 2 days after injury, and then recovered to the level before injury. After bTBI injury for 6 h, 24 h and 3 d, the number of GFAP positive staining astrocytes and vertebral cells in hippocampal area decreased in both model group and treatment group, whereas the number of activated microglia and apoptotic neurons in the hippocampus increased in a time-dependent manner in model group and treatment group. In addition, compared with the model group, the treatment group with oil-rich diet increased the number of astrocytes and pyramidal cells in the hippocampus after injury, and decreased the number of activated microglia and apoptotic neurons in the lesions area. Conclusion Pre-injury dietary supplementation with fish oil shows neuroprotective benefits in alleviating neurons injury and inhibiting neuroinflammatory response in a rat model of mild bTBI.

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Objective To study the rapid antidepressant effects of cannabioiol(CBD) on depression-like mice and its possible mechanism. Methods Chronic restraint was used to establish a mouse depression model. The test mice were divided into 5 groups: normal control group, model group, positive control group, CBD low-dose group (25 mg/kg) and CBD high-dose group(50 mg/kg). The mice in each group were given intragastric administration one hour before the behavioral experiment. After the behavioral experiment, the hippocampus and prefrontal cortex specimens were collected, and brain-derived neurotrophic factor(BDNF), postsynaptic density protein 95(PSD-95), synaptophysin(SYP) and target of rapamycin(mTOR) were tested by ELISA.Results Compared with the model group, the central area activity distance percentage, the central area activity time percentage and total distance increased in the open field experiment in the CBD low-dose group. Compared with the model group, the percentage of immobility in the forced swimming experiment in the low-dose CBD group decreased. The ELISA test results showed that CBD could rapidly increase the concentration of BDNF and PSD-95 in the prefrontal cortex, as well as the concentration of SYP and mTOR in the hippocampus and prefrontal cortex.Conclusion CBD can rapidly improve the behavioral performance of depression-like mice, and rapidly up-regulate the level of BDNF and synaptic protein in the hippocampus or prefrontal cortex.

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Objective To investigate the interactional roles and mechanism of the chronic stress during pregnancy(CSDP) and chronic offspring stress(COS) exposures in inducing male offspring depression. Methods The pregnant mice were randomly divided into the normal control group and the chronic stress group. After the offspring were born, male offspring were randomly divided into the normal control+normal control group(NC+NC), the normal control+chronic offspring stress group(NC+COS), the chronic stress during pregnancy+normal control group(CSDP+NC) and the chronic stress during pregnancy+chronic offspring stress group(CSDP+COS). The depression related indices in male offspring mice were detected by forced swim test and elevated plus maze. Pathological changes in hippocampus CA3 area were assessed by Golgi staining and TUNEL staining. The activity of hippocampal mTOR was investigated by using the Western blot to quantitatively detect the expression of mTOR and p-mTOR(Ser2448). Results Chronic stress during pregnancy induced depressive symptoms in male offspring accompanied by neuronal damage and apoptosis in the hippocampus and reduced mTOR activity. The chronic offspring stress ameliorated the depression-like behavior and pathological damage of hippocampal CA3 region in male offspring mice that caused by chronic stress during pregnancy. And the chronic offspring stress elevated mTOR activity. Conclusion Chronic stress during pregnancy induced depressive symptoms in male offspring. The chronic stress could ameliorate the depression-like behavior, neuronal damage and neuronal apoptosis in hippocampal CA3 region of male offspring mice that caused by chronic stress during pregnancy. The chronic offspring stress may improve the depression-like behavior of offspring caused by chronic stress during pregnancy through regulating mTOR activity by corticotropin releasing hormone(CRH).

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Objective To investigate whether all-trans-retinoic acid(ATRA) treatment affects the survival ofgastric cancer patients after surgery, and to analyze whether ATRA affects the proliferation, migration, invasion and apoptosis of gastric cancer cells by regulating epithelial-mesenchymal transition(EMT). Methods A total of 198 patients with gastric cancer after surgery were selected and divided into control group and ATRA group, and the difference in survival time between the two groups was compared. MGC-803 gastric cancer cells were culturedin vitroand divided into control group and ATRA group. The proliferation ability was detected by CCK-8 experiment, the migration ability was detected by cell scratch experiment, the invasion ability was detected by Transwell experiment, and the apoptosis was detected by flow cytometry. The expressions of EMT-related genes(E-cadherin, N-cadherin and Vimentin) were detected by Western blot, RT-qPCR and immunofluorescence. Results ATRA could prolong the survival time of patients after gastric cancer surgery. After ATRA treatment, the proliferation, migration and invasion abilities of gastric cancer cells decreased, and the apoptosis of gastric cancer cells was promoted at the same time. Western blot, RT-qPCR and immunofluorescence results showed that ATRA down-regulated the expression levels of N-cadherin and Vimentin and up-regulated the expression level of E-cadherin in gastric cancer cells. Conclusion ATRA can prolong the survival time of gastric cancer patients after surgery, promote the apoptosis of gastric cancer cells, and inhibit the proliferation, migration and invasion of gastric cancer cells by regulating EMT.

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Objective To explore a simple, convenient and reproducible method for thein vitroextraction, culture and identification of primary glomerular mesangial cells(GMCs) from non-human primate. Methods The kidneys of macaca mulattas were removed under aseptic conditions, and the glomeruli were separated using two different pore sizes(between 100 and 200 mesh) and digested with type VI collagenase at different concentrations(0.1 mg/ml and 0.2 mg/ml), and different digestion times(10 min and 15 min) were set for the two concentrations. The growth of GMCs under different digestion conditions was observed in the bottles. The structure of GMCs was observed under microscope and the expression of α-smooth muscle actin(α-SMA) and Nephrin in GMCs was detected by immunofluorescence and Western blot. The biological properties of GMCs were observed after stimulation with tumor necrosis factor-α(TNF-α), and the viability and proliferation of GMCs were detected by CCK-8 and High Connotation Cell Imaging System, respectively andthe migration ability of GMCs was detected by Transwell and cell scratch assay. Results The glomeruli collected using 0.1 mg/ml type VI collagenase digestion for 10 min were more apposed and the glomeruli cells were in a better growth state. Glomeruli in primary culture were adherent at 3 d. Irregularly shaped or star-shaped cells began to migrate at 7 d. GMCs gradually spread to the bottom of the bottle after 21～35 d. GMCs were positive for α-SMA protein and negative for Nephrin protein. 10 ng/ml TNF-α in vitro stimulation significantly enhanced the proliferation and migration ability of GMCs. Conclusion The method of collagenase digestion(0.1 mg/ml type VI collagenase digestion for 10 min) was successfully established for the isolation and culture of GMCs, which can provide a suitable cell model to better simulate human kidney diseases in the future.

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Objective To investigate the effects effect of lncRNA CASC2 on inflammation, migration and invasion of rheumatoid arthritis(RA) synovial fibroblasts by regulating the expression of miR-218-5 p and its mechanism. Methods 45 cases of RA synovial tissue and 18 cases of normal synovial tissue were collected, and qRT-PCR was used to detect the expression of CASC2 and miR-218-5 p in the synovial tissue. ENCORI prediction and dual luciferase reporting experiments analyzed the targeting between CASC2 and miR-218-5 p. RA synovial fibroblast MH7 A were transfected with pcDNA3.1-CASC2 and miR-218-5 p mimic, the expression of CASC2 and miR-218-5 p was detected by qRT-PCR; CCK-8 was used to detect cell proliferation; ELISA was used to detect the levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) in the supernatant of cells; Transwell was used to detect migration and invasion of cells; Western blot was used to detect expression of matrix metalloenzymes MMP-2 and MMP-9. Results Compared with normal synovial tissue, RA synovial tissue showed significantly down-regulated expressions of CASC2(P<0.05)and up-regulated expressions of miR-1236-3 p(P<0.05). The dual luciferase reporter showed that CASC2 was target genes of miR-218-5 p. Overexpression of CASC2 significantly suppressed cell proliferation, reduced the levels of TNF-α, IL-1β and IL-6 in the supernatant and migration and invasion, lowered the levels of miR-218-5 p and protein expression of MMP-2 and MMP-9 in RA synovial fibroblast MH7 A. However, transfecting miR-218-5 p mimic could reverse the situation. Conclusion RA synovial tissue showed significantly down-regulated expressions of CASC2, overexpression of CASC2 suppressed inflammation, migration and invasion of RA fibroblasts by regulating the expression of miR-218-5 p.

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Objective To study the mechanism of long non-coding RNA(lncRNA) RNF185-AS1 affecting the proliferation and invasion of glioma cells by regulating microRNA-637(miR-637). Methods The GEPIA database was used to analyze the expression level of RNF185-AS1 in glioma tissues and adjacent tissues. qRT-PCR was used to detect the expression level of RNF185-AS1 in glioma cell lines(SNB-19, LN382, U87 MG, U251) and normal brain astrocytes HEB. U251 cells were used as the research object, and sh-Con control plasmid(sh-Con group) and sh-RNF185-AS1 plasmid(sh-RNF185-AS1 group) were transfected respectively. qRT-PCR verified the silencing efficiency of RNF185-AS1. MTT experiment and Transwell invasion experiment were used to detect the proliferation and invasion of U251 cells after silencing RNF185-AS1. qRT-PCR and dual luciferase reporter gene experiment were used to detect the targeting relationship between RNF185-AS1 and miR-637. qRT-PCR and Western blot were used to detect the expression of high mobility group A1(HMGA1) gene. Results The expression level of RNF185-AS1 in glioma tissue was significantly higher than that in adjacent tissues(P<0.01). The expression level of RNF185-AS1 in the glioma cell line was significantly higher than that in normal brain astrocytes(P<0.01), and the expression of RNF185-AS1 in U251 cells increased the most significantly(P<0.01). Compared with the sh-Con group, the expression level of RNF185-AS1 in U251 cells in the sh-RNF185-AS1 group significantly decreased(P<0.01), the proliferation activity of U251 cells was significantly reduced(P<0.05), and the number of invaded cells was significantly less(P<0.01). RNF185-AS1 and miR-637 had complementary nucleotide sequences(P<0.01). Compared with the sh-Con group, the expression level of miR-637 in U251 cells in the sh-RNF185-AS1 group significantly increased(P<0.01), and the expression level of HMGA1 gene was significantly reduced(P<0.01). Conclusion RNF185-AS1 is highly expressed in glioma tissues and cell lines. Silencing RNF185-AS1 can inhibit the proliferation and invasion of glioma U251 cells by targeting up-regulation of miR-637 expression and down-regulation of HMGA1 gene expression.

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Objective To explore the mechanism of bufalin on tumor metastasis mediated by cancer-associated fibroblasts(CAF). Methods The primary CAFs were extracted from the tissues of colon cancer patients, classified into CAF-1 and CAF-2, and their morphology and surface markers were observed under confocal microscopy. Western blot detected the expression of CAF surface markers fibroblast activated protein(FAP), α-smooth muscle actin(α-SMA), and Vimentin(VIM) to confirm the identity of CAF. The CAF conditioned medium was collected, and the interleukin-6(IL-6) secretion level in the conditioned medium was detected by Elisa experiment. HCT116 cells were divided into 2 groups, which was HCT116 group and HCT116 with CAF conditioned medium group. The expression of P-STAT3 and STAT3, proteins related to STAT3 pathway in HCT116, was detected by Western blot. Western blot was used to detect epithelial mesenchymal transformation(EMT) of HCT116 and the changes in the migration and invasion ability of HCT116 were confirmed by scratch experiment and Transwell experiment. Two groups of HCT116 cells, including a control group and a bufalin group, were both treated with CAF-conditioned medium to observe the activation of STAT3 pathway and the changes in migration and invasion capabilities. Results The extracted cells showed a long spindle shape and expressed CAF surface markers FAP, α-SMA and VIM. The Elisa experiment showed that CAF secreted IL-6; Western blot results showed that the conditioned medium of CAF activated the STAT3 signaling pathway of HCT116; Western blot, the scratch experiment and Transwell experiment also found that the migration and invasion ability of HCT116 increased. When bufalin was added, the migration promotion effect of CAF was inhibited. Conclusion CAF activates the STAT3 pathway of tumor cells by releasing IL-6, and promotes the invasion and metastasis of colorectal cancer. Bufalin blocks the process by inhibiting the activation of the STAT3 pathway on tumor cells.

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Objective To study the protective effect of mesenchymal stem cells(iPSC-MSCs) derived from pluripotent induced stem cells on the articular cartilage tissue of patients with knee osteoarthritis(KOA) in vitro and its partial mechanism.Methods With informed consent, the articular cartilage of KOA patients was collected, shredded and treated with IL-1β(10 ng/ml) to stimulate 96 h, and then different numbers of iPSC-MSCs(1 × 104, 1 ×105, 1 × 106) cells were added and cultured for 3 d in a 37 ℃, 5% CO2incubator. In addition, IL-1β(10 ng/ml) induction group and culture medium control group were set up. Chondroitin sulfate assay method was used to detect glycosaminoglycan content in cartilage tissue, immunohistochemical method was used to detect the expression of type Ⅰ collagen and type Ⅱ collagen in the tissue, and ELISA method was used to detect the level of MMP13,IL-6 and IL-10 in the co-culture supernatant, HE staining was used to detect the pathological changes of cartilage tissuein vitro.Results Compared with the control group, IL-1β could induce swelling and death of chondrocytes in the cartilage tissue of KOA, increase the proportion of inflammatory cells, increase the level of type I collagen,decrease the level of type Ⅱ collagen, and the level of MMP-13 and IL-6 in the culture supernatant significantly increased(P<0. 05), and the content of IL-10 and glycosaminoglycan was significantly reduced(P<0. 05); compared with the IL-1β induction group, the co-culture of iPSC-MSCs with different cell numbers could reduce the MMP-13, IL-6 level and type I collagen level in the supernatant, promote the expression of type Ⅱ collagen, increase glycosaminoglycan content and IL-10 level in cartilage tissue.Conclusion iPSC-MSCs can regulate the level of inflammatory factorsin vitro, inhibit the degradation of chondrocyte matrix, and have a protective effect on articular cartilage tissue.

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Objective To explore the role and mechanism of miR-146 a in acute pancreatitis. Methods The expression of miR-146 a, interleukin-1 receptor associated kinase 1(IRAK1),inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) was detected by qPCR and ELISA, and the expression of autophagy related molecules LC3 and p62 was detected by Western blot. miR-146 a and IRAK1 were overexpressed to detect the expression changes of IL-6, TNF-α, LC3 and p62. A mutant plasmid at the binding site of IRAK1 3'UTR and through luciferase reporter gene assay. Results The results showed that taurolithocholic acid 3-sulphate(TLCs) inhibited miR-146 a and IRAK1 expression in a concentration-dependent manner, promoted the expression of IL-6, TNF-α and LC3, and inhibited the expression of p62. Overexpression of miR-146 a could block the effect of TLCs to a certain extent. The luciferase reporter gene results showed that miR-146 a directly targeted IRAK1 and negatively regulated it. Conclusion These results suggested that miR-146 a inhibited inflammation and autophagy in TLCs-induced AR42 J cells by targeting IRAK1.

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Objective To explore the effect of connective tissue growth factor(CTGF) on aortic calcification and its mechanism. Methods In vitrorat vascular smooth muscle cells(A7 r5) were used for vascular calcification model construction, and the time of calcification was determined using Alizarin staining. The cells were randomly divided into 7 groups to determine the effect of CTGF on calcification. Cells were transfected with CTGF-siRNA plasmid, and the expression of CTGF in cells was detected by RT-PCR and Western blot. To determine the effect of CTGF on cell calcification, cells were randomly divided into 7 groups. Alizarin staining was used to analyze the effect of CTGF on calcification. ELISA was used to detect the alkaline phosphatase(ALP) activity. RT-PCR was used to determine the expression of OPG, RANK and RANKL in A7 r5 cells. Results The optimal time for calcification of A7 r5 was 48 h. The expression of CTGF was reduced in the cells of the CTGF-siRNA group compared to the Control group, indicating that A7 r5 were successfully transfected with the CTGF-siRNA. Compared with the model group, the expression of calcified nodules was reduced, the activity of alkaline phosphatase significantly decreased(P<0.05), and the expression of OPG, RANK and RANKL was also significantly downregulated in the CTGF-siRNA group(P<0.05). Conclusion CTGF-siRNA attenuated A7 r5 cell calcification and alkaline phosphatase activity induced by high phosphorus and calcium, and regulated the OPN/RANK/RANKL signaling pathway, so as to play a therapeutic role in aortic vascular calcification.

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Objective To investigate the effect of ZIF-8-Pt nanoparticles on the radiosensitization of breast cancer. Methods ZIF-8-Pt nanoparticles were synthesized by chelating ZIF-8 with chloroplatinic acid(H2PtCl6). The potential, particle size and serum stability of the nanoparticles were measured. The spatial structure of ZIF-8-Pt nanoparticles was observed under a transmission electron microscope. The ability to produce oxygen(O2) by reaction with H2O2solution was measured. The uptake of ZIF-8-Pt nanoparticles by breast cancer cells(4 T1) was determined by inductively coupled plasma-mass spectrometry(ICP-MS). The toxicity of ZIF-8-Pt nanoparticles was evaluated by using MTT assay. The killing effect of ZIF-8-Pt nanoparticles combined with radiotherapy and radiotherapy alone on tumor cells was observed with MTT assay, Colony formation assay and animal treatment experiments. Results ZIF-8-Pt nanoparticles with a potent of-14 mv and a size of about 160 nm were successfully prepared. It was confirmed that ZIF-8-Pt nanoparticles were stable in 10% serum. The ability of ZIF-8-Pt nanoparticles to produce oxygen(O2) by reaction with H2O2solution was dose-dependent with particle concentration. ICP-MS results showed that the uptake of ZIF-8-Pt nanoparticles by 4 T1 cells increased with time. The clone formation assay proved that the killing effect of nanoparticles combined with radiotherapy on 4 T1 cells was related to the concentration of the particles. MTT assay and animal treatment experiments verified that the killing effect of nanoparticles combined with radiotherapy on breast cancer was better than that of radiotherapy alone. Conclusion The killing effect of ZIF-8-Pt nanoparticles combined with radiotherapy on tumor tissues is better than that of radiotherapy alone.

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Objective To study the sensitivity of human cervical cancer cells to cisplatin which increased by the lactoferrin active hexapeptide(LfcinB 4-9) and to investigate its mechanism. Methods MTT was used to detect the proliferation inhibition of cisplatin(DDP) combined with different concentrations of LfcinB 4-9 on human cervical cancer Hela cell line and the Hela cell line with the p62 gene knocked out(Hela/KO p62). The plate cloning assay was performed to detect the effect of DDP combined with LfcinB 4-9 on the cloning ability of human cervical cancer cells. The effect of DDP combined with LfcinB 4-9 on the apoptosis of human cervical cancer cells was detected by flow cytometry. RT-qPCR and Western blot were used to detect the expression levels of genes and protein such as SIAH, PSMA, and β-catenin in cancer cells Hela and Hela/KO p62.Results The inhibitory rate of the combination of LfcinB 4-9 and DDP on cervical cancer cells was significantly higher than that of DDP alone or LfcinB 4-9 alone(P<0. 05 or P<0. 01). Compared with DDP group, the cloning ability of human ovarian cancer cells was significantly reduced, and apoptosis increased in combined treatment(P<0. 05 or P<0. 01). The qRTPCR and Western blot assay showed that when DDP combined with LfcinB 4-9, the expression of SIAH and PSMA1 increased, while the expression of β-catenin decreased.Conclusion The combined effect of LfcinB 4-9 and DDP significantly enhances the sensitivity of cervical cancer cells to DDP, which is achievedviathe proteasome pathway.

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Objective To investigate the effects of methylation expression of ADH1B gene on cell proliferation and cell apoptosis in ovarian cancer(OC) cells. Methods Bioinformatics analysis was performed to compare the relative expression of ADH1B in OC and normal tissue. Quantitative real-time PCR(qRT-PCR) was used to detect the ADH1B mRNA expression in the OC tissues or cells, ovary normal epithelium tissues or cells. Treated with different concentrations of 5-Aza-dc for 48 h, cell viability was determined by CCK-8 method. Cell apoptosis morphology was detected by Hoechst 33258 staining. The changes of promoter methylation of ADH1B, the level of ADH1B in the mRNA and protein expressions were measured by using methylation specific PCR(MSP), qRT-PCR and Western blot, respectively. Results Bioinformatics analysis and qRT-PCR showed that ADH1B expression in OC was lower than that in normal tissue. After treatment with medium and high concentration of 5-Aza-dc, the cell proliferation was inhibited in two cells(P<0.01). After treatment with high concentration of 5-Aza-dc, marked morphological changes of apoptosis was observed in two cells. ADH1B was completely methylated in two cells. ADH1B was partially reversed by high concentration of 5-Aza-dC, and ADH1B mRNA and protein expression both increased by 5-Aza-dC in two cells(P<0.01). Conclusion 5-Aza-dC can down-regulate the methylation level of tumor suppressor gene ADH1B promoter region and make it re-express in ovarian cancer cells, thus exert its function of enhancing the inhibitory effect and apoptosis of ovarian cancer cells.

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Objective To investigate the effect of superoxide dismutase(SOD) mimetic 4-Hydroxy-TEMPO(Tempol) on aortic vasodilation function in mice exposed to intermittent hypoxia. Methods Male C57 mice were randomly divided into normal oxygen(Control) group, intermittent hypoxia(IH) and IH+Tempol group. The mice in IH and IH+Tempol group were exposed to 4-weeks intermittent hypoxia, 8 h daily. In IH+Tempol treatment group, the mice were given Tempol(100 mg/kg) each day by the oral administration in the last week of intermittent hypoxia. The isolated thoracic aorta reactivity was studied using wire myography, the content of reactive oxygen species(ROS) was detected by dihydroethidium(DHE) staining, the concentration of nitric oxide(NO) was measured by ELISA kits. Results Compared with Control group, the endothelium-dependent vasodilation of thoracic aorta induced by acetylcholine(ACh) significantly decreased and cardiac hypertrophy, the level of aortic ROS significantly increased, the concentration of aortic NO was significantly reduced in the IH group mice. Treatment with Tempol could significantly improve the aortic endothelium-dependent vasodilation dysfunction and cardiac hypertrophy, and significantly inhibited the increase of ROS and decrease of NO in the thoracic aorta induced by intermittent hypoxia. Conclusion Tempol can improve intermittent hypoxia induced-cardiovascular dysfunction in mice by reducing ROS and increasing the bioavailability of NO.

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Objective To detect exosomes concentration changes in manual platelet with different storage time and further investigate the effects of exosomes on coagulation function. Methods After divided into storage bags with same volume, the platelets were stored for 0, 3, 5 days(D 0, D 3, D 5) in(22±2)℃. Exosomes were isolated from platelet samples by ultracentrifugation. Western blot、transmission electron microscope and nano-flow cytometry were adopted to characterize exosomes. Meanwhile, exosomes particles concentration was detected by nano-flow cytometry. Exosomes with different storage time were added to plasma, then the effects on the plasma prothrombin time(PT) and activated partial thromboplastin time(APTT)were detected by the automatic coagulation analyzer. Expression of coagulation factor VII on exosomes from platelet stored for different time were tested by Western blot. Exosomes samples of D 0, D 5 were incubated with fresh whole blood, respectively. Then the coagulation indexes of each samples were detected by Thromboelastography test(TEG). Results Test showed the presence of TSG 101, CD 9, which were exosome specific markers. And the exosomes showed a double-layer membrane and cup-like structure. Moreover, the particle size distribution of exosomes was mainly between 60～80 nm. The concentration of exosomes stored for D 0, D 3, and D 5 were(13.86±7.93)×1012,(23.69±12.80)×1012,(45.81±25.87)×1012particles/L, respectively. Compared with D 0, the concentration of exosomes stored for D 3 and D 5 increased significantly(P<0.01), with 1.8 times and 4.0 times, respectively. Compared with the control group, PT in the D 0 and D 5 exosomes group could be decreased by[(0.16±0.15) s, P<0.01]、[(0.36±0.18) s, P<0.001], APTT could be decreased by [(1.11±1.21)s, P<0.05]、[(2.10±1.14)s, P<0.001], respectively. Semi-quantitative analysis of coagulation factor VII was performed by Western blot and it showed statistically differences in coagulation factor Ⅶ concentration(P<0.05). TEG test showed that the coagulation time R value of control group, exosomes of D 0 and D 5 were(5.46±0.90) min,(5.00±0.67) min,(4.38±1.00) min, respectively, indicating that exosomes had procoagulant activity(P<0.05). Conclusion With the prolongation of storage time, the particles concentration of exosomes in manual platelets continuously increased and the blood procoagulant function of exosomes is significantly enhanced, platelet-derived exosomes during storage may be involved in the procoagulant process through the coagulation factor Ⅶ.

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Objective To express human glucose 6-phosphate dehydrogenase(G6 PD) and establish its inhibitor screening modelin vitro. Methods G6 PD cDNA was amplified by PCR with a couple of primers flanked withNdeI andXhoI site at their 5′ terminals, respectively. G6 PD cDNA and pET-28 a plasmids were digested byNdeI andXhoI, and then linked. The linked products were transformed into Escherichia coli(E.coli)Top10. Recombinant plasmids were identified and transformed intoE.coliBL21(DE3), the expression of G6 PD was induced by 0.4 mmol/L IPTG, recombinant G6 PD was purified by Ni2+affinity column. Glucose 6-phosphate and NADP+ were used as substrate and coenzyme of G6 PD, respectively. G6 PD's activity was observed by the shift of A340of NADPH. The model was optimized to give the greatest performing concentration of G6 PD and dehydroepiandrosterone(G6 PD's inhibitor). Results Recombinant plasmid pET-28 a-G6 PD was constructed, soluble G6 PD was expressed in BL21(DE3) with a yield of 2.5 mg/Lviathe induction of IPTG. The optimum G6 PD concentration was 900 μg/L. The high Z′ score of 0.88 was achieved using 20 μmol/L DHEA as positive control. Conclusion Active G6 PD is expressed inE.coli, and this assay is highly suitable for screening of G6 PD inhibitorin vitro.

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Objective To investigate the effects of Pizotifen on proliferation, migration and invasion of A549 and PC9 cells of human lung adenocarcinoma and its possible mechanisms. Methods The logarithmic phase of A549 and PC9 cells were used for experiments, which were divided into control groups(cell control group and DMSO solvent control group) and experimental groups(including 10,20,30 and 40 μmol/L Pizotifen, respectively).CCK-8 assay was used to evaluate the effects of Pizotifen on A549 and PC9 cells proliferation. Wound-healing assay andTranswell assay were then performed to investigate whether Pizotifen affected the migration and invasion properties of A549 and PC9 cells. Finally, ELISA was used to determine the expression levels of Wnt3a/β-catenin signaling pathway related proteins and epithelial-mesenchymal transition(EMT) related proteins such as E-cadherin(E-cad) and matrix metalloproteinase-9(MMP-9) in A549 and PC9 cells after Pizotifen treatment. Results Compared with the solvent control group, the assay of CCK-8 in experimental groups showed that the cell vitality gradually decreased after Pizotifen treatment for 24 h and 48 h, and the inhibition rate of cell vitality was directly proportional to the action time of Pizotifen(P<0.05). At the same treatment time, the cell viability of A549 and PC9 in the experimental groups decreased with the increase of Pizotifen concentrations(20, 30 and 40 μmol/L).The test of Wound-healing showed that Pizotifen had an inhibitory effect on the migration of A549 and PC9 cells, and the higher the concentration of Pizotifen was, the lower the cell migration rate was(P<0.05). The experiment of Transwell showed that Pizotifen had an inhibitory effect on the migration and invasion of A549 and PC9 cells in a concentration-dependent manner(P<0.05). ELISA showed a decrease in the level of Wnt3a, β-catenin and MMP-9 protein and an increase in E-cad protein in the experimental groups compared with the solvent control group. Conclusion Pizotifen may inhibit the proliferation, migration and invasion of human lung adenocarcinoma A549 and PC9 cells in vitro, with the inhibition strength dependent of the concentration and action time of Pizotifen. Its potential mechanisms may include block of the Wnt3a/β-catenin-EMT pathway in cells.

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Objective To study the incidence, clinical characteristics and treatment plans of artificial liver in the treatment of severe liver disease complicated with invasive fungal infection(IFI). Methods The clinical data of patients with severe liver disease were retrospectively analyzed, and they were divided into observation group and control group according to whether they were complicated with IFI. The observation group was divided into clinical diagnosis group and proposed diagnosis group according to diagnostic criteria, and the clinical characteristics of each group were compared. Results 228 patients were enrolled in the study, 22 in the observation group and 206 in the control group. The hospitalization days, use of glucocorticoid for more than 7 days and WBC count between the two groups were statistically significant(P<0.05). The incidence of IFI was 9.65%(22/228), including 12 cases in the clinical diagnosis group and 10 cases in the proposed diagnosis group. The common infection site was respiratory tract(22, 100%). Conclusion Voriconazole is safe and effective in the treatment of severe liver disease complicated with IFI. Patients with high-risk factors of IFI should be alert to concurrent IFI if TBil rebound that cannot be explained by basic diseases.

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Objective To study the influencing factors of atopic dermatitis(AD) patients with keratosis pilaris(KP) in Chinese Han population, and to provide a basis for the diagnosis and treatment of AD. Methods The data of AD patients collected from the hospital dermatology department and the national AD sample collection network were analyzed by statistical methods. Results A total of 2 956 AD patients were collected, of which 699(23.6%) were associated with KP. Analysis showed that in terms of gender, region, chronic repetitive dermatitis, family history of atopic diseases, xeroderma, palm prints, periorbital black halo, ichthyosis, itching during sweating, white scratches, disease severity, onset in terms of types, there was a difference in the incidence of AD with KP and without KP(P<0.05). In addition, geographic area, xeroderma, palm pattern, periorbital black halo, ichthyosis, white scratches, skin lesion type-hypertrophic type, disease severity-moderate were positively correlated with KP associated with AD(P<0.05). Among them, the region(northern region), ichthyosis, palm pattern, xeroderma were strongly correlated with AD associated with KP. Conclusion Northern areas, palm pattern, ichthyosis and xeroderma are risk factors for AD with KP.

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Objective To investigate the value of 2020 Chinese guidelines for ultrasound malignancy risk stratification of thyroid nodules(Chinese thyroid imaging reporting and data system, C-TIRADS) and the combination with elastography technology in the diagnosis of thyroid nodules. Methods The gray-scale and elastic sonographic features of 117 thyroid nodules confirmed by postoperative pathological diagnosed were analyzed retrospectively. C-TIRADS was used to classify and evaluate the gray-scale image, the ultrasonic elastic characteristics combined with the C-TIRADS results to establish a joint diagnosis of thyroid nodules. Based on related pathological data, the diagnostic value of C-TIRADS and the joint diagnosis for thyroid nodule was statistically compared. Results The sensitivity, specificity, accuracy and AUC of C-TIRADS and the joint diagnosis for diagnosing thyroid nodules were 82.2%, 88.6%, 84.6%, 0.854 and 95.9%, 86.4%, 92.3%, 0.911. Compared with C-TIRADS alone, there were consistent elevations of the sensitivity, accuracy and AUC in the joint diagnosis, while the specificity slightly decreased(P<0.05). Conclusion C-TIRADS have a satisfied diagnostic value for thyroid nodules. The joint diagnosis by combining with elastography can further enhance ultrasound diagnosis.

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Objective To investigate the influence of etiology and clinical features on prognosis of adult hemophagocytic lymphohistiocytosis(HLH). Methods The clinical data of 104 adult HLH patients diagnosed in 5 medical centers of Huaihai Lymphoma Working Group(HHLWG). MaxStat algorithm was used to determine the optimal cut-off values of continuous variables. Cox proportional hazard model was used for univariate and multivariate analysis on prognosis; the survival curve was drawn by Kaplan-Meier; the differences between groups were tested by Log-rank test. Results Among the 104 HLH patients, 52 cases(50%) were male, with a median survival time of 2.13 months. The most common etiology in 104 patients was EBV infection(35.7%), followed by lymphoma(18.2%). The optimal cut-off values of age, fibrinogen, platelet, triglyceride, albumin and serum creatinine by MaxStat algorithm were 50-y, 2.27 g/L, 150×109/L, 2.87 mmol/L, 30.3 g/L and 56 μmol/L respectively. The overall survival(OS) rates of HLH due to different etiologies were different, among which the overall survival rates of lymphoma associated hemophagocytic syndrome(LAHS) and EBV-HLH patients were 15.8% and 30.2%, respectively. Multivariate analysis showed that age, albumin, serum creatinine and fibrinogen were independent prognosis factors for adult HLH. Conclusion EBV infection is the common cause of adult HLH. Among the different cause of HLH, the patients with LAHS have the lowest OS and the worst prognosis.

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Objective To investigate the correlation between thyroid stimulating hormone(TSH) level and metabolic syndrome(MS) in subclinical hypothyroidism(SCH) in a district in Hefei City. Methods Using the method of cluster sampling survey, according to the inclusion and exclusion criteria, 2 628 permanent residents aged over 18 years(living time≥5 years) in a district of Hefei City were enrolled. Based on the the thyroid function, subjects were divided into SCH group and normal thyroid function group(control group); According to MS diagnostic criteria, they were divided into MS group and non-MS group; According to the trisection of TSH level, SCH group was divided into three subgroups: low TSH level, medium TSH level and high TSH level. The differences of clinical parameters between different groups were compared. Logistic regression analysis was used to investigate the risk of MS in different TSH levels of SCH patients and the independent influencing factors of MS. Receiver operating curve(ROC) was used to explore TSH as a potential biomarker for the diagnosis of MS in SCH patients. Results The detection rate of SCH was 19.29%(507/2 628) and that of MS was 29.45%(774/2 628). Compared with the control group, the detection rate of MS in SCH group was significantly higher(34.91% vs 27.99%,P=0.002); In SCH group, the detection rate of MS increased gradually with the increase of TSH level(trend χ2= 13.717,P=0.001). Compared with the control group, the risk of MS in SCH patients with high TSH level increased by 1.722 times(OR= 1.722, 95%CI1.702～2.885,P=0.009). Multiple logistic regression analysis found that TSH was an independent risk factor for MS(OR=1.850, 95%CI1.625～2.002). ROC analysis showed that the area under the curve(AUC) of TSH was 0.811(95%CI0.696～0.904), the optimal cutoff point of TSH was 8.76 mIU/L, and the sensitivity and specificity were 87.5% and 73.9% respectively. Conclusion The prevalence of MS in SCH patients increased gradually with the increase of TSH level in Hefei City. TSH is an independent risk factor for MS, and can be used as a diagnostic marker for MS in SCH patients.

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Objective To investigate the effect of nicorandil combined with Wenxin granules for intracoronary injection on the incidence of perioperative major adverse cardiovascular events(MACE) and related prognostic indicators in patients with ST segment elevation myocardial infarction(STEMI) after percutaneous coronary intervention(PCI). Methods 160 STEMI patients were selected as the research subjects, and they were randomly divided into control group, nicorandil group, Wenxin granule group and combination group, with 40 cases in each group. The TIMI blood flow grading, postoperative corrected TIMI blood flow frame number, cardiac function indexes(including 24-hour postoperative cTnI, CK-MB peak value) and the occurrence of perioperative(7 d) MACE events were compared among the four groups of patients condition. Results The incidence of MACE during hospitalization in the control group, nicorandil group, Wenxin granule group and combination group were 47.5%, 30.0%, 30.0% and 22.5%, respectively, and the incidence of postoperative perioperative reperfusion arrhythmia in the combination group was lower in the control group and nicorandil group(P<0.05). The incidence of no-reflow/slow-reflow and the peak value of cTn I in the combination group were lower than thatose in the control group and Wenxin granule group(P<0.05), the peak value of CK-MB was lower than that in the control group(P<0.05), and the cTFC was lower than that in the control group, Wenxin granule group and nicorandil group(P<0.05). Conclusion Intracoronary injection of nicorandil combined with Wenxin granules can reduce perioperative myocardial ischemia injury and effectively reduce the incidence of perioperative reperfusion arrhythmia in STEMI patients after PCI.

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Objective To analyze the distribution of virulence genes and drug resistance ofKlebsiella pneumoniae(KP), and to study the correlation between the difference of TLR4 expression in serum and cell levels of patients infected with hypervirulentK.pneumoniae(hvKP) and classicalK.pneumoniae(cKP) and clinical prognosis. Methods The strains and serum of patients with KP were collected, the virulence genes were detected by PCR amplification, and the drug susceptibility was detected; Strains were divided into the hvKP group and cKP group. The expression level of TLR4 in serum was detected by ELISA. A549 human alveolar epithelial cells were infected with hvKP and cKP. The expression of TLR4 was determined by quantitative PCR. Results The derection rates of kfu, rmpA1 and rmpA2 in virulence genes of hvKP group were the highest, reaching 100%, 87.8% and 80.5% respectively. The serum TLR4 level was significantly higher in the hvKP groups than that in the control and cKP groups, which was positively correlated with PSI score and CURB-65 score about clinical prognosis. The expression of TLR4 mRNA was significantly higher in the hvKP groups than that in the control and cKP groups. Conclusion The combination of rmpA1, rmpA2 and magAgenes has higher accuracy in diagnosing hvKP infection. Higher expression of TLR4 in serum and cells induced by hvKP infectiong, and TLR4 level positively correlated with the clinical severity of infected KP, suggesting that TLR4 may be involved in the pathophysiological process of hvkp infection. Serum TLR4 can be used as an detection index for the diagnosis and prognosis of patients with KPN, especially patients with hvkp infection.

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Objective To investigate the effect of vascular endothelial growth factor(VEGF) and clinical features on prognosis of diffuse large B-cell lymphoma(DLBCL). Methods The general data, laboratory and imaging examination, and immunohistochemical staining results of 203 cases of newly diagnosed DLBCL were collected.MaxStat statistics were used to calculate the optimal cut-off points of VEGF, albumin, β2-microglobulin, RBC count, hemoglobin, lactate dehydrogenase and age. Univariable and multivariable analyses were performed using Cox proportional risk model to determine the variables affecting the survival outcome of DLBCL patients.Results The 203 patients included 104 males(51. 2%) and 99 females(48. 8%), with a median age of 59 years, 97 patients(47. 8%) with Ann Arbor stage Ⅲ/Ⅳ, with a median follow-up time of 37. 6 months and a 5-year OS of 62%. There were significant differences in VEGF, age, RBC count, hemoglobin, platelets, albumin, β2-microglobulin, lactate dehydrogenase and fibrinogen between the survival group and the death group(P<0. 05). The optimal cutoff point of VEGF was 266. 8 pg/ml. Univariable and multivariable analyses showed that VEGF, RBC count, hemoglobin and albumin affected the prognosis of DLBCL. Patients with increased VEGF were associated with higher Ann Arbor stage, ECOG score, IPI score, MYC positive and BCL-2 positive expression. In addition,patients with high levels of VEGF in the low-risk IPI group had poorer survival.Conclusion High level of VEGF was associated with poor prognosis of DLBCL patients, and VEGF could perform more accurate stratification for patients with Ann Arbor stage Ⅲ/Ⅳ, ECOG<2 points, IPI low-risk group, BCL-2 negative expression group and MYC positive expression group.

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Objective To investigate the expression of LAMP5 in colorectal cancer(CRC) and the relationship between the expression of LAMP5 and prognosis of patients with colorectal cancer, reveal the role of LAMP5 in the progression of CRC. Methods GEPIA and LinkedOmics database were used to analyze the expression of LAMP5 in CRC tissues and paired adjacent tissues, and the correlation with clinical parameters and the prognosis of CRC patients followed by validation of clinical samples. Gene Set Enrichment Analysis was used to study the LAMP5 related molecular functions and related signal pathways involved in the occurrence and development in CRC. Results The results of immunohistochemistry showed that the expression of LAMP5 in CRC tissues was significantly higher than that in paired adjacent normal tissues(n=120,P<0.05). LAMP5 expression in CRC tissues was negatively correlated with the degree of differentiation(P<0.05), and positively correlated with N stage and M stage(P<0.05), but not correlated with age, gender and T stage. GEPIA and clinical tissues analysis showed that CRC patients with high expression of LAMP5 had shorter overall survival and disease-free survival than those with low expression of LAMP5(P<0.05). Cox Proportional Hazards Model analysis revealed that the High expression level of LAMP5 was an independent risk factor for OS and DFS in CRC patients(P<0.05).GO analysis showed that LAMP5 had significant correlation with extracellular matrix, extracellular matrix adhesion, extracellular matrix localization, and collagen localization in CRC(P<0.05). KEGG analysis showed that LAMP5 might be involved in multiple pathways, including TGF-β and PI3 K-AKT signaling pathways(P<0.05). Conclusion The expression of LAMP5 is up-regulated in CRC, and correlated with tumor dedifferentiation, lymph node metastasis, distant metastases, and prognosis. Therefore, LAMP5 can be used as a novel biomarker for diagnosis, treatment and prognosis of CRC.