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Objective To study the protein expression and cell localization of RB1-C-terminal phosphorylation site mutants, and to investigate the change of co-localization with Sedlin. Methods The primers of RB1-C-terminal phosphorylation site mutants were designed, and pcDNA3.1-FLAG-RB1-C was used as the template to amplify pcDNA3.1-FLAG-RB1-C-S788 A,S795 A,S807 A,T811 A,T821 A,T826 A. The above mutants were separately transfected into HEK 293 T and COS7 cells, and their protein expression and cell localization were observed by Western blot and immunofluorescence technology; the co-localization of the above mutants and Sedlin were observed by IF technology, and compared with the results of wild type RB1-C. Results The sequencing results showed that the six phosphorylation sites of RB1-C were mutated successfully and expressed normally in eukaryotic cells, which was basically consistent with the cell localization of wild type RB1-C, and they were mainly located in the nucleus. In addition, compared with the wild-type RB1-C, the co-localization of RB1-C-S788 A,S795 A,T821 A,T826 A and Sedlin did not change, that is, they were mainly co-located in the nucleus, but the co-localization of RB1-C-S807 A,T811 A and Sedlin changed significantly, in addition to co-localization and nucleus, there was also significant co-localization in the cytoplasm. Conclusion RB1-C phosphorylation site mutants were successfully constructed and expressed. The mutations of two phosphorylation sites S807 A or T811 A can significantly change the co-localization region of RB1-C and Sedlin in cells, indicating that they are one of the key binding sites of RB1-C and Sedlin, which lays a foundation for further study of the interaction between RB1-C and Sedlin.

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Objective To study the protective effect of berberine(BBR) on podocytes in high glucose environment and to discuss its mechanism. Methods The exponential growth stage of the glomerular podocyte was selected. The podocyte were divided into 5 groups, including normal control group, high-glucose model group and BBR was given in concentrations of 30, 60 and 90 μmol/L. Annexin FITC/PI double staining was used to detect the apoptosis rate of podocytes. High-intension imaging system was used to detect cell proliferation,podocyte migration was measured by transwell and cell scratch assay,Western blot detected the expression of AKT/NF-κB signaling pathway related proteins,apoptotic protein Bim,and the podocyte marker proteins WT-1,Podocin and desmin. Results Compared with the normal group,the results of Annexin FITC/PI double staining showed that the apoptosis rate of podocytes in the high glucose group increased( P<0. 01); The high-content imaging system showed that the number of podocytes in the high glucose group decreased( P<0. 01); Transwell showed that the number of podocytes migrate ability increased in the high glucose group( P<0. 01); The cell scratch showed that the scratch healing rate of the high glucose group increased( P<0. 01); Western blot showed that the expression levels of p-AKT,p-p65,p-IκBα,Bim,and desmin protein in the podocytes of the high glucose increased( P<0. 01),and the expression of WT-1 and Podocin proteins decreased( P<0. 01). Compared with the high glucose group,the apoptosis rate of podocytes decreased after add BBR( P<0. 05); the number of podocytes increased in the different concentrations of BBR( P<0. 01); the number of podocyte migration was lower( P<0. 05); Scratch healing rate decreased( P<0. 01); In addition,BBR reduced the protein expression levels of p-AKT,p-p65,p-IκBα,Bim and desmin( P<0. 05),the expression level of WT-1 and Podocin protein increased( P<0. 01). Conclusion BBR may improve the podocyte injury,inhibit the podocyte apoptosis,and weaken the podocyte migration ability induced by high glucose through regulating AKT/NF-κB signaling pathway.

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Objective To investigate the effect of neuropathic pain on myocardial ischemia-perfusion injury in rats and the regulatory mechanism of AKT/mTOR signaling pathway involved in this process. Methods Forty-two Sprague-Dawley rats weighing 250-320 g(10 weeks old) were randomly divided into 3 groups(n=14): control group(group Con), myocardial ischemia-reperfusion injury group(group I/R) and neuropathic pain with myocardial ischemia-reperfusion injury group(group NP+I/R). The neuropathic pain model was prepared by the chronic constriction injury method(CCI) of the sciatic nerve. The mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL)of each group of rats were measured before and at 3, 7, and 14 days after the operation. The left anterior descending coronary artery is ligated for 30 minutes and opened for 120 minutes 14 days after the CCI operation to establish a model of myocardial ischemia-reperfusion injury. A slice of the heart was taken to detect the infarct size(IS) and area at risk(AAR) to calculate the myocardial infarction area. Western blot was used to detect the expression of AKT, mTOR and LC3 proteins, which was related to the AKT/mTOR signal pathway and autophagy. Results Compared with group I/R, MWT and TWL of group NP+I/R decreased at 3, 7, and 14 days after CCI.Compared with group Con, IS/AAR increased in group I/R, the ratio of p-AKT/AKT and p-mTOR/mTOR decreased, the ratio of LC3-Ⅱ/Ⅰincreased, and the differences were statistically significant(P<0.05). Compared with group I/R, IS/AAR decreased in group NP+I/R, the ratio of p-AKT/AKT and p-mTOR/mTOR increased, the ratio of LC3-Ⅱ/Ⅰ decreased, and the differences were statistically significant(P<0.05). Conclusion Neuropathic pain reduces myocardial ischemia-reperfusion injury in rats. The mechanism may be related to neuropathic pain regulating AKT/mTOR signaling pathway and inhibiting excessive autophagy.

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Objective To study the effect of eupatilin on miR-188-5 p overexpression and its effect on the proliferation, invasion and apoptosis of retinoblastoma. Methods Y79 cells were selected for research in retinoblastoma lines. Different concentrations of eupatilin(1, 2.5, 5, 10, 20, 40, 100, 200, 300 μmol/L) were used to treat Y79 cells and MTT assay was used to detect cell viability. Y79 cells were cultured with 0, 10, 20, 40 μmol/L eupatilin, and the effects of eupatilin on proliferation, apoptosis and invasion of Y79 cells were tested. Control group, mimic-NC transfection group, miR-188-5 p mimic transfection group were established, then the cells were treated with concentration of 40 μmol/L eupatilin each group to detect the effect of eupatilin on the expression of miR-188-5 p, the proliferation, apoptosis and invasion of Y79 cells. RT-qPCR was used to detect the expression of miR-188-5 p, colony formation test, flow cytometry and Transwell test were used to detect cell proliferation, apoptosis and invasion.Western blot was used to detect the protein expression of E-cadherin, N-cadherin, Bax and Bcl-2. Results MiR-188-5 p overexpression promoted the Y79 cells proliferation, reduced cell apoptosis and increased cell invasion. Eupatilin could inhibit miR-188-5 p overexpression, thereby inhibiting retinoblastoma Y79 cell proliferation, inducing apoptosis. Conclusion Eupatilin inhibits the cell invasion and induces apoptosis of retinoblastoma by inhibiting the expression of miR-188-5 p.

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Objective To investigate the effect of high-fat diet on hippocampal morphological changes and synapse-related protein Synapsin-1(Syn-1) expression in rats. Methods 18 male SD rats were randomly divided into a control group and a model group, with 8 rats in the control group and 10 rats in the model group. Rats in the model group were given high-fat diet lasting for 4 weeks, and rats in the control group were given standard control diet. The changes of blood lipid in the rats of the two groups were detected, the morphological changes of the hippocampal tissues were observed by hematoxylin-eosin(HE) staining and silver staining, and the mRNA and protein expression of Syn-1 in the hippocampus was detected via real-time fluorescent quantitative PCR(qRT-PCR) and immunohistochemistry technique,respectively. Results Compared with those in the control group,the serum levels of total cholesterol( TC),low density lipoprotein cholesterol( LDL-C),and high density lipoprotein( HDL) in the model group significantly increased,and the level of leptin significantly reduced( P<0. 01). Results of HE staining showed that the neurons in the DG,CA3 and CA1 regions of model rats were irregular in shape,loosely arranged,and hyperchromatic in nuclei. Consistently,the neurons in DG,CA3 and CA1 regions of the model rats were also hyperchromatic via silver staining. Compared with that in the hippocampus of control rats,the mRNA and protein expression of Syn-1 in the model group was significantly lower. Conclusion High-fat diet can induce hyperlipidemia,hippocampal neuron damage,and decrease expression of synaptic binding protein Syn-1 in the hippocampus of rats.

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Objective To study the effect of non-small cell lung cancer(A549 cells) on the sensitivity of the chemotherapy drug pemetrexed(Pe) under hypoxia environment. Methods The A549 cell line was cultured in groups and divided into cell control group, normoxia solvent group, normoxia dosing group, hypoxia cell group, hypoxia solvent group and hypoxia dosing group. First, MTT was used to detect the effect of Pe on the proliferation of A549 cells, thereby screening out the suitable experimental concentration of Pe. Then Hoechst staining was used to observe the apoptosis of these six cell groups. Later, Western blot was applied to detect the expressions of apoptosis-related proteins Bcl-2, Bax, and p53. Results MTT results showed that compared with the normoxia control group, the cell inhibition rate after Pe treatment gradually increased(P<0.05), and the inhibition rate of A549 cells was directly proportional to the action time and concentration of Pe. The results of Hoechst staining indicated that cell apoptosis rate increased in the normoxia-dosed group compared with the normoxia control group(P<0.05). But compared with normoxia-dosed group, the cell apoptosis rate decreased under hypoxic environment(P<0.05). Western Blot experiment showed an increase in p53 protein and a decrease in Bcl-2 protein expression in the normoxia dosing group compared to the normoxia control group(P<0.05). There was no change of p53 protein expression identified in the hypoxia dosing group, but Bcl-2 protein expression decreased(P<0.05); compared with the hypoxic cell group, Bcl-2 protein expression decreased(P<0.05) in the hypoxia dosed group, while p53 protein expression was unchanged. Conclusion Hypoxia environment reduces the apoptotic effect of Pe on non-small cell lung cancer A549 cells, and thus reducing the sensitivity of A549 cells to pemetrexed.

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Objective To investigate the effect of naringenin(NAR) on mechanical ventilation-induced lung injury(VILI) and its possible mechanism. Methods Sixty C57 BL/6 mice were divided into sham operation group(sham), mechanical ventilator model group(Model), naringenin low-dose group(L-NAR, 15 mg/kg) and naringenin high-dose group(H-NAR, 60 mg/kg), with 15 mice in each group. The drug was administered orally 7 days before the modeling, once a day for 7 consecutive days. Then the VILI model was established by mechanical spring flow ventilation, the oxygenation index(OI) was determined by blood collection with internal carotid artery. Mice were sacrificed after 4 h mechanical ventilation and bronchoalveolar lavage fluid(BALF) was collected. The contents of TNF-α, IL-1β and IL-18 in BALF were determined by ELISA. Lung tissue was taken and the wet/dry ratio(W/D) of lung tissue was determined. Histopathological changes were observed by HE staining and scored. The mRNA and protein expression levels ofNLRP3、ASC and Caspase-1 were detected in macrophages by RT-PCR and Western blot, respectively. Results Compared with the sham group, the Model group had severe lung tissue injury, the lung histopathological score, W/D value, the content of TNF-α, IL-1β and IL-18 in BALF, and the mRNA and protein expression levels ofNLRP3,ASC and Caspase-1 in lung tissue increased(P<0.01), while the OI value decreased(P<0.01). Compared with the Model group, the degree of lung tissue injury improved in the H-NAR group, the lung histopathological score, W/D value, the content of TNF-α, IL-1β and IL-18 in BALF, and the mRNA and protein expression levels of NLRP3, ASC and Caspase-1 in lung tissue decreased(P<0.01), the OI value increased(P<0.01), However, there was no significant difference in relevant indicators between the L-NAR group and the Model group. Conclusion NAR pretreatment reduced VILI in mice, and the mechanism may be related to the inhibition of NLRP3 inflammasome activation.

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Objective To investigate the effect and mechanism of microRNA-223(miR-223) on the inflammatory response, oxidative stress and apoptosis of human bronchial epithelial cells(BEAS-2 B) induced by influenza A virus(IAV). Methods RT-qPCR was used to detect the expression of miR-223 andNLRP3 mRNA in sera of influenza patients and normal people, and the effect of IAV treatment at different time on the mRNA expression of miR-223 and NLRP3 was detected; CCK-8 was used to detect the cell viability of BEAS-2 B. Annexin V-FITC/PI apoptosis detection kit was used to detect the apoptosis level of BEAS-2 B cells. The expression of apoptosis related proteins Caspase-3, Caspase-9, Bax and Bcl-2 was detected by Western blot; ROS was detected by reactive oxygen species indicator DCFH-DA. Results The results showed that the expression of miR-223 in the serum of influenza patients was lower than that of normal people; IAV inhibited miR-223 and promoted the mRNA expression ofNLRP3, which was time-dependent(P<0.05). Overexpression of miR-223 reduced the apoptosis rate and inflammatory factor levels of BEAS-2 B induced by IAV(P<0.05). In addition, overexpression of miR-223 alleviated oxidative stress of BEAS-2 B induced by IAV(P<0.05). Further study showed that miR-223 inhibited the expression of TLR4, p-p65 and NLRP3(P<0.05). Conclusion miR-223 alleviated the oxidative stress, apoptosis and reduced cell damage of pulmonary epithelial cells induced by IAV by inhibiting TLR4/NF-κB signaling pathway and activating NLRP3 inflammasome.

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Objective To investigate the role and possible mechanism between phosphatidylinositol-3-kinase/protein kinase B( PI3 K/AKT) pathway and transient receptor potential cation channel protein 6( TRPC6) in the process of angiotensin Ⅱ( Ang Ⅱ)-induced glomerular podocyte injury. Methods Mouse glomerular podocytes were cultured in vitro and treated with Ang Ⅱ,Losartan,PI3 K/AKT pathway inhibitor( LY294002),TRPC6 channel blocker( SKF96365). The cells were divided into blank control group and Ang Ⅱ group,Ang Ⅱ + Losartan group,Losartan group,Ang Ⅱ + LY294002 group,LY294002 group,Ang Ⅱ + SKF96365 group,SKF96365 group.The qRT-PCR was used to detect the changes of TRPC6 mRNA in each group. Western blot was used to detect the changes of AKT,phosphorylated protein kinase B( p-AKT),and TRPC6 protein expression in each group. The apoptosis of podocytes was detected by Hoechst 33258 staining. Results Compared with the blank control group,the AKT phosphorylation level of the Ang Ⅱ group decreased,the expression of TRPC6 increased,and the apoptosis of podocytes increased( P<0. 05). Compared with the Ang Ⅱ group,the AKT phosphorylation level of the Ang Ⅱ + SKF96365 group increased,the expression of TRPC6 decreased,and the apoptosis of podocytes decreased( P<0. 05); the AKT phosphorylation level of the Ang Ⅱ + LY294002 group decreased,and the apoptosis of podocytes increased( P<0. 05); the AKT phosphorylation level of the Ang Ⅱ + Losartan group increased,the expression of TRPC6 decreased,and the apoptosis of podocytes decreased( P<0. 05). Conclusion Ang Ⅱ may induce podocyte injury by activating the TRPC6 channel and thereby inhibiting the activation of PI3 K/AKT pathway.

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Objective To investigate the effects of miR-138-5 p and TCF4 on the malignant biological behavior of MuM-2 B cells in uveal melanoma. Methods MuM-2 B cells were transfected or co-transfected with miR-138-5 p mimic and pcDNA-TCF4, respectively. A mouse model ofin vivoxenograft tumor was established. Cell transfection efficiency was detected by RT-qPCR. Bioinformatics software predicted miR-138-5 p and TCF4 target sites. Dual luciferase reporter gene assay was used to verify the targeted relationship. Clone formation assay was used to detect the ability of cell clone formation. The invasion ability of cells was detected by Transwell assay. Western blot analysis of TCF4 and epidermal mesenchymal transformation(EMT) related protein expression. Immunohistochemistry was used to detect the expression of Ki67, VEGF and Vimentin in tumor tissues. Results Compared with control group, miR-138-5 p and TCF4 were up-regulated in transfected group(P<0.05). Between miR-138-5 p and TCF4 were target relationship. Overexpression of miR-138-5 p inhibited MuM-2 B cell proliferation, invasion ability as well as up-regulating the protein expressive levels of E-cadherin and Vimentin and down-regulating the protein expressive levels of N-cadherin by targeting TCF4 in Mum-2 B cells(P<0.05). In transplanted tumorsin vivo, overexpression of miR-138-5 p significantly reduced tumor weight(P<0.05), and overexpression of miR-138-5 p down-regulated the positive cell rate of Ki67, VEGF and Vimentin in tumor tissues(P<0.05). Conclusion The overexpression of miR-138-5 p down-regulated the TCF4 levels, and further inhibited the cell proliferation, invasion and EMT of uveal melanin Mum-2 B cells.

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Objective Microinjection of NG2 cell antibodies(NG2-Ab),the effect of NG2 cells on the change of γ-aminobutyric acid( GABA) and glutamate( Glu) transmitter content in the raphe nucleus of rats were studied by microdialysis to investigate the interaction between 5-hydroxytryptamine( 5-HT) and NG2 in the raphe nucleus.Methods Seventy rats were randomly divided into normal group,control group,NG2-AB group,PCPA group,5-HT group,PCPA + NG2-Ab group and 5-HT + NG2-Ab group. Normal group did not receive any treatment. The other groups were microinjected with saline( control group),NG2-Ab、PCPA、5-HT、PCPA + NG2-Ab and 5-HT +NG2-Ab,into the middle suture nucleus. The corresponded drugs were microinjected into the raphe nucleus. Microdialysis was used to collect extracellular fluid of the raphe nucleus 20,40,60,80,100 and 120 min after injection,respectively. High performance liquid chromatography( HPLC) was used to analyze the content changes of GABA and Glu in the microdialysate in corresponding time. Results Compared with the control group,NG2-Ab group decreased the content of GABA and Glu( P<0. 05),and the difference was statistically significant; PCPA group decreased content of GABA and increased Glu( P<0. 05). 5-HT group content increased of GABA( P<0. 05) and decreased Glu( P<0. 01). Compared with the PCPA group,there was no statistical difference in GABA content after PCPA + NG2-Ab injection,while Glu content decreased( P<0. 05). Compared with the 5-HT group,there was no statistical difference in GABA content after 5-HT + NG2-Ab injection in the raphe nucleus,while Glu content decreased( P<0. 05). Conclusion The content changes of GABA and Glu in raphe nucleus is affected by the inhibition of excitability of NG2 cells. The regulation effect of NG2 on Glu content is related to the synthesis of 5-HT,but its regulation effect on GABA content may not be directly related to the synthesis of 5-HT.

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Objective To evaluate the effects of long-term exposure to high altitude hypoxia(HAH) on cardiac function in rats, and to explore whether the mechanism is related to autophagy in the process of myocardial fibrosis mediated by AMPK pathway. Methods Fifty male SD rats were housed at an altitude of 4 500 m. Ten rats were randomly selected for body weight, blood pressure, echocardiography, cardiac histology and autophagy protein examination at baseline, 1 week, 2 weeks, 4 weeks and 8 weeks. Thirty rats were randomly divided into two groups with 15 animals in each group, including control group and rapamycin group. Rapamycin group was administered by intraperitoneal injection [1 mg/(kg·d)]. The control group was intraperitoneally injected with the same volume of normal saline. The above examinations were carried out before the test and at the end of the 8 th week. Results Body weight, blood pressure and echocardiography showed that the systolic blood pressure, body weight, right ventricular end diastolic diameter(RVEDd) and left ventricular ejection fraction(LVEF) of rats exposed to HAH environment increased, while the pulmonary artery acceleration time(PAAT) decreased. Rapamycin reversed these changes. Histological analysis showed that the rats exposed to HAH environment had myocardial degeneration, necrosis and fibrosis, accompanied by monocyte infiltration, and increased myocardial fibrosis. Rapamycin reduced these changes. Western blot analysis showed that the Beclin-1, LC3-Ⅱ/Ⅰ and AMPK protein increased significantly up to 4 weeks, but were impaired after 8 weeks of HAH treatment in heart tissue. Rapamycin increased autophagy related markers Beclin-1, LC3-Ⅱ/Ⅰ and AMPK protein expression. Conclusion Long term exposure to HAH may damage cardiac function in rats, and its mechanism is related to autophagy in the process of myocardial fibrosis mediated by AMPK pathway.

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Objective To observe and analyze the effect of ORP8 protein on the proliferation, migration and invasion ability of human colorectal cancer DLD-1 cells, and to preliminarily explore the mechanism of its influence. Methods The expression levels of ORP8 in colorectal cancer were up-regulated and down-regulated by transfection of ORP8 expression plasmid or ORP8 siRNA in colorectal cancer DLD-1 cell. The effects of interference or overexpression on the proliferation,migration and invasion of colorectal cancer cell DLD-1 were detected by CCK-8 test,transwell test and wound healing test. Western blot was used to detect the total protein and phosphorylation levels of ORP8 in the extracellular regulated protein kinases( ERK1/2) and the expression of epithelial-mesenchymal transition( EMT) related molecular markers protein E-cadherin,N-cadherin and Vimentin are studied. Results In colorectal cancer cell line DLD-1,when ORP8 was silenced,the cell proliferation,migration and invasion capacity significantly increased( P<0. 01),the expression levels of epithelial marker E-cadherin decreased,the expression levels of mesenchymal marker Vimentin and N-cadherin increased,and the expression level of p-ERK1/2 also increased. On the contrary,when ORP8 was overexpressed,the cell proliferation,migration and invasion capacity significantly decreased,the expression levels of epithelial marker E-cadherin increased( P<0. 01、P<0. 01、P<0. 05),the expression levels of mesenchymal marker N-cadherin and Vimentin protein decreased,and the expression level of p-ERK1/2 also decreased. Conclusion ORP8 protein can inhibit the proliferation,migration and invasion of colorectal cancer cell DLD-1,and its molecular mechanism may be related to blocking EMT transformation and inhibition of phosphorylation of p-ERK1/2.

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Objective To compare the advantages and disadvantages between caudal vein injection and subcutaneous injection of human Jurkat cells in the establishment of xenograft acute T lymphocytic leukemia model. Methods Female BALB/c nude mice were randomly divided into normal group, tail vein transplantation group and subcutaneous transplantation group. The tail vein transplantation group and subcutaneous transplantation groups were given cyclophosphamide for 2 consecutive days before modeling. 5×106Jurkat cells were inoculatedviatail vein in tail vein transplantation group for 2 consecutive days, and 5×106Jurkat cells were subcutaneously injected into subcutaneous transplantation group for 2 consecutive days. The same volume of phosphate buffer saline was injected into the normal group by tail vein. The body weight, bow back and mental state of nude mice were observed and recorded. The percentages of Jurkat cells in peripheral blood were monitored by flow cytometry on the 7 th and 14 th day. On the 28 th day, the mice appeared signs of failure and were close to death, and they were put to death. At the same time, the body weight was recorded. Wright′s stain was used to detect the infiltration of Jurkat cells in peripheral blood and bone marrow, spleen and bone marrow of the Jurkat invasion was detected by flow cytometry. Pathology of liver and spleen was also observed by HE staining, and infiltration of Jurkat cells was observed by immunohistochemistry. The level of IL-2 secreted by Jurkat cells in serum was detected by ELISA. Results Compared with the normal group, the tail vein transplantation group and subcutaneous transplantation groups nude mice gradually showed weight loss, bow back and loss of appetite, and Jurkat cells were expressed in the liver, spleen and bone marrow of the mice in each group. The success rate of tail vein transplantation group was 73%, and the success rate of subcutaneous transplantation group was 40%. Conclusion Both tail vein injection and subcutaneous injection of human Jurkat cells can successfully establish the animal model of acute T-lymphocyte leukemia, and tail vein injection transplantation is an optimal option for experimental requirements.

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Objective To explore the potential mechanism of differentiation of human acute promyelocytic leukemia NB4 cells induced by a new retinoic acid derivative 4-amino-2-trifluoromethyl-phenyl retinoate(ATPR). Methods After the differentiation of NB4 cells induced by ATPR and alltransretinoic acid(ATRA),we analyzed the long-chain non-coding RNA(lncRNA) and mRNA expression profile of the treated cells with gene chip technology. Results The results showed that 775 lncRNAs and 904 mRNAs were screened in the ATRA group compared with the ATRA group, and the chromosomal location of some key lncRNAs was analyzed by cis regulatory genes.The results oftransanalysis showed that the related transcription factors could regulate the expression of lncRNA. Conclusion In conclusion, lncRNA plays an important role in the differentiation of NB4 cells induced by ATPR.

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Objective To study the function and molecular mechanism of the acetylation of malic enzyme 2(ME2) in the proliferation of malignant glioma U87 MG cells. Methods Based on the conserved sequence and combined with the mass spectrometry database, the acetylation modification sites of ME2 were predicted, point mutants were constructed, transfected cells were transfected, and the modification sites were determined by IP and Western blot. The wild-type and mutant of ME2 were transfected into 293 T cells and purified to detect the effect of acetylation on ME2 enzyme activity. The effects of deacetylate Sirtuins 3(SIRT3) on the activity of ME2, ROS and NADH were detected through co-transfection, purification, combined with enzyme activity experiment. After knockdown of endogenous ME2 with shRNA in U87 MG cells, the effects of ME2 acetylation on cell proliferation were detected by stable ectopic expressing wild type ME2 and mutant, which was verified at the animal level by tumor formation in nude mice. Western blot was used to detect the difference of the acetylation level of ME2 in the clinical samples of malignant glioma. Results Results showed that K156 was the main acetylation site of ME2, which inhibited the activity of ME2 and the proliferation of U87 MG cells. SIRT3 deacetylated ME2, improved its activity, thus promoting the formation of NADH and reducing the intracellular ROS level. Conclusion SIRT3-deacetylated ME2 can promote the proliferation of U87 MG cells by increasing the activity of its own enzymes, up-regulating the level of NADH and reducing the level of ROS.

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Objective To investigate the protective effect of grape seed proanthocyanidins(GSP)on heart injury and its possible mechanism in diabetic mice. Methods Diabetic mice was induced by intraperitoneal injection of alloxan. The mice were randomly divided into model group, GSP low, middle and high dose group(65, 130, 195 mg/kg) and metformin positive control group(220 mg/kg). Meanwhile, the control group was set up. The control group and model group were given normal saline. Dynamic blood glucose, glucose tolerance and water intake were recorded during the administration.At the end of the experiment, BL-420 S was used to detect the hemodynamics of mice; the heart weight and kidney weight index were calculated; pathologicalchanges were observed by pathological staining; serological indexes were detected by enzyme-linked immunosorbent assay and colorimetry; protein expression in heart tissue was observed by immunofluorescence and western blotting. Results Compared with the control group, the blood glucose, glucose tolerance, diet and drinking water were significantly higher in the model group; the cardiac function, pathomorphology, serological indexes had changed abnormally, such as the increase of endothelin-1 A receptor(ETA) and the decrease of the endothelial nitric oxide synthase(eNOS). GSP and metformin could significantly improve the above indicators. Conclusion GSP has protective effect on heart injury in diabetic mice, and its mechanism may be related to anti-oxidant stress and improvement of endothelial dysfunction.

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Objective To investigate the curative effects of a new sustained release agent of glucocorticoid on doxorubicin-induced nephropathy rats. Methods The nephrotic model was prepared by injecting doxorubicin into caudal vein in rats with a total dose of 7.5 mg/kg for two consecutive weeks. The test group was given dexamethasone implant(DexI, 2.8, 1.4, 0.7 mg/kg) one time through renal capsule injection, while the intragastric administration of dexamethasone(Dex, 0.1 mg/kg, qd) for 8 weeks was used as a positive control. Serum and urine were collected regularly before and after modeling to detect renal function and 24 h urine protein. After the experiment, kidney specimens were taken for histopathological examination, immunohistochemical analysis and the expression of nephrin protein in the ruptured septum of renal podocytes. Results DexI could improve the general condition of rats with adriamycin nephropathy to different degrees, prevent weight loss and show a better mental state; DexI could also significantly reduce the 24 h urine protein in kidney rats and improve kidney function(reduce blood creatinine and urea nitrogen levels); DexI could fight kidney pathological damage caused by doxorubicin. Conclusion Sustained-release dexamethasone implants can significantly improve doxorubicin-induced kidney injury in rats, which may be related to its enhancement of the podocyte filtration barrier barrier function.

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Objective To study the changes of red blood cell distribution width(RDW) coefficient of variation(RDW-CV) and red blood cell distribution width standard deviation(RDW-SD) in patients with cirrhosis, and to explore the clinical significance of RDW-CV and RDW-SD in the differential diagnosis of compensatory and decompensated cirrhosis. Methods A total of 120 inpatients with cirrhosis diagnosed were selected, including 60 patients at the compensatory stage and 60 patients at the decompensation stage. The differences between RDW-CV and RDW-SD in patients at different stages were analyzed, and then the correlation of RDW-CV, RDW-SD with liver function indexes total protein(TP), albumin( ALB), alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL),alkaline phosphatase(ALP), γ-glutamyl transpeptidase(γ-GT) and inflammation indexes C-reaction protein(CRP) was discussed. Receiver operating characteristic curve(ROC) curve evaluated the specificity and sensitivity of RDW-CV and RDW-SD in the diagnosis of decompensated cirrhosis. Results Both RDW-CV and RDW-SD in the compensation period were lower than that in the decompensation period, while RDW-SD was more significant(P<0.001). In the compensatory stage of cirrhosis, RDW-SD and RDW-CV were negatively correlated with TP, in addition, RDW-SD was positively correlated with γ-GT; In the decompensated stage, RDW-SD was negatively correlated with ALB and positively correlated with TP, ALT, AST and TBIL. However, RDW-CV was also positively correlated with TP, ALT and TBIL, but the correlation was not as good as that of RDW-SD. CRP at decompensation was significantly positively correlated with RDW-SD, but not with RDW-CV. ROC curve analysis showed that the area under curve(AUC) of RDW-SD was 0.788, significantly higher than that of RDW-CV(0.648). Conclusion There are differences between RDW-CV and RDW-SD in different stages of cirrhosis, and the difference between RDW-SD is more significant, which has potential clinical value in differential diagnosis of decompens-ated cirrhosis.

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Objective To provide references for the diagnosis, treatment, prevention and control of the disease by analyzing the clinical characteristics, epidemiological characteristics and prognostic factors of patients infected with novel Bunyavirus. Methods Retrospective analysis was performed on 202 patients infected with novel Bunyavirus. According to the prognosis of the patients, the patients were divided into the survival group(n=164) and the death group(n=38). The general data, clinical manifestations, and laboratory test indicators of patients in the survival group and the death group(within 24 hours) were compared. Multivariate Logistic regression analysis was conducted on the basis of univariate analysis for the two groups of data, and the independent influencing factors of mortality of patients infected with Bunyavirus were obtained. Results Among the 202 confirmed cases, 86 were male and 116 were female, aged 52～73(62.8±10.6) years. The survival group was significantly younger than the death group, and the heat course was significantly longer than that of the survival group(P<0.05). However, there was no statistically significant difference between the two groups in terms of gender, region, occupation, onset distance to visit the doctor, insect bite history, etc. There was no significant difference in clinical symptoms(fever, hyperneckiness, headache, fatigue, insufficiency, nausea, diarrhea, vomiting, cough, muscle aches, shivers, lymph node enlargement, abdominal discomfort, etc.) between the two groups. The proportion of acute pancreatitis, central nervous system damage, bleeding, myocardial damage and arrhythmia in the death group was significantly higher than that in the survival group(P<0.01).In the death group, viral load, ferritin, lactic lehydrogenase(LDH) and other indicators were significantly higher than those in the survival group, while lymphocyte count, platelet count, Th cell count and IgM positive rate were all lower than those in the survival group(P<0.01). Among them, thermal course, age, lymphocyte count and active partial thromboplastin time(APTT) were independent influencing factors of death. Conclusion The older the age, the longer the heat course, the longer the APTT and the lower the lymphocyte count, the worse the prognosis of the patients infected with the novel Bunyavirus.

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Objective To analyze the relationship between postoperative pathological types of colon polyps andHelicobacter pylori(Hp) infection and its related factors. Methods A total of 350 patients receiving colon polypectomy in the outpatient department of gastroenterology and in hospital were selected, including 192 high-risk adenomas, 158 low-risk adenomas and inflammatory or proliferative polyps(control group). Complete clinical data of the subjects were obtained by self-designed questionnaire. Results The diameter of adenomatous polyps and different sites of polyp growth in the Hp positive group were compared with that in the Hp negative group, and the differences were statistically significant(P<0.05).The risk of colon adenoma was correlated with Hp infection, age, body mass index(BMI), fruits and vegetables, meat, smoking and family history of primary cancer(P<0.05), but not with physical activity, diabetes, hypertension and alcohol consumption. Conclusion Postoperative pathological types of colon polyps are correlated with the diameter, size and location of adenomatous polyps and Hp infection. High-risk colon adenomas are correlated with Hp infection, age, BMI, fruits and vegetables, meat, smoking and first-degree relatives of tumors, but not with exercise, diabetes, hypertension and alcohol consumption.

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Objective To investigate the expression level and clinical significance of SAMHD1 protein in esophageal squamous cell carcinoma tissue. Methods Immunohistochemically staining was used to detect SAMHD1 protein expression in 204 cases of esophageal squamous cell carcinoma tissues. Then we analyzed the relationship between it and the gender, age, clinic pathological characteristics of patients with esophageal squamous cell carcinoma and the five-year survival prognosis of patients. Results SAMHD1 was mainly expressed in the cytoplasm or nucleus of esophageal squamous cell carcinoma cells. The positive rate of SAMHD1 expression was not correlated with the patient′s age, gender, tumor T, N, TNM stage, and upper mediastinal lymph node metastasis. However, in large tumors, the low expression of SAMHD1 was more significant(P=0.013). The poorer the degree of tumor differentiation, the lower the expression of SAMHD1(P=0.001). The expression of SAMHD1 was lower in cases of esophageal cancer with abdominal lymph node metastasis(P=0.044). Compared with the high expression SAMHD1 group, the low expression SAMHD1 group had lower cumulative survival(rate) and disease-free survival(rate)(P<0.05). Conclusion SAMHD1 is lowly expressed in esophageal squamous cell carcinoma, the lower the expression level, the worse the prognosis survival.

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Objective To screen the diagnostic indicators of obese patients with obstructive sleep apnea(OSA) and to evaluate the screening effect of these indicators in obese patients with OSA. Methods Retrospective analysis was performed in 32 patients with simple obesity and 87 patients with obesity combined with OSA. The general data were improved and polysomnography( PSG) was performed on the night after admission,and in the morning after PSG monitoring,oral glucose tolerance test( OGTT),serum glycosylated hemoglobin( Hb A1 c),fasting plasma glucose( FPG) and postprandial 2 hours plasma glucose( 2 hPG) were measured to analyze the clinical characteristics and influencing factors of obesity complicated with OSA. Results Multivariate Logistic regression analysis showed that the independent influencing factors of OSA included neck circumference and HbA1 c. The analysis of the working characteristic curve of the subjects( ROC) showed that the area under the curve( AUC) of HbA1 c and neck circumference were 0. 813 and 0. 909 respectively,which were better than the Epworth sleepiness scale and STOP-Bang rating scale( AUC: 0. 638、0. 739). The combined use of Hb A1 c and neck circumference could improve AUC to 0. 934,with a sensitivity of 85. 1% and specificity of 96. 9% in the diagnosis of obese patients with OSA. Conclusion For obese patients,the combined detection of neck circumference and Hb A1 c has good sensitivity and specificity,which can be used for screening obese patients with OSA.

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Objective To explore the clinical characteristics and prognostic factors of the primary gastrointestinal mantle cell lymphoma(PGIMCL). Methods With the SEER database, clinical treatment information of PGIMCL patients from 1983 to 2015 was collected. Kaplan-Meier function was used to draw the overall survival(OS) and cause-specific survival(CSS) curves, then univariate and multivariate Cox regression analysis was used to evaluate the influence of various clinical factors on the prognosis. Results A total of 420 patients were subsumed into this case, most of them were elderly men, and the primary site was mostly in the intestine. Old age(OS:HR=2.210, 95%CI1.646-2.968,P<0.001; CSS:HR=2.024, 95%CI1.437-2.852,P<0.001), Ⅲ/Ⅳ stage(OS:HR=1.397, 95%CI1.065-1.833,P=0.016; CSS:HR=1.935, 95%CI1.391-2.692,P<0.001), the primary site was in the stomach(OS:HR=1.599, 95%CI1.205-2.121,P=0.001; CSS:HR=1.516, 95%CI1.079-2.130,P=0.017) patients with PGIMCL had poor OS and CSS. Female OS was superior to male(HR=1.390, 95%CI1.013-1.908,P=0.041). Conclusion Age, primary site and clinical stage are independent risk factors for prognosis of PGIMCL patients.

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Objective To investigate the expression of miR-23 a in the peripheral blood of patients with acute gouty arthritis(AGA) and its correlation with clinical parameters. Methods Peripheral blood samples were collected from 20 cases of AGA group and 20 cases of healthy controls, the expression of miR-23 a in peripheral blood was detected by quantitative PCR, and the correlation between miR-23 a expression level and clinical parameters was analyzed. Results (1) The relative expression of miR-23 a in the AGA group was higher than that in the normal group, and the difference in relative expression was statistically significant(P=0.005).(2) Correlation analysis showed that the peripheral blood miR-23 a expression level was positively correlated with low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), Triglyceride(TG)(P<0.05).(3) Correlation analysis showed that the expression level of miR-23 a in peripheral blood was negatively correlated with emotional function(P<0.05). Conclusion The expression of miR-23 a may reflect the disease progression of AGA and the prognostic process of patients, and miR-23 a may be related to metabolic indicators and emotional functions, and its mechanism of action needs to be further explored.

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To explore the therapeutic effect and clinical value of classification and treatment of children′s trigger thumb. The clinical data of 128 cases(164 thumbs) of children with trigger thumb were retrospectively analyzed. The author had proposed a new subtype classification based on the Jung′s. The different treatment methods had been applied in specific classification based on Lee′s criteria to evaluate the clinical outcome, and the overall effect could be divided into cure, improvement and ineffective. Then the therapeutic effects were compared respectively. In type I, 18 thumbs were treated with simple manual massage, and 13 thumbs(72.22%) were cured. The remaining 24 thumbs were treated with low-temperature thermoplastic splint external fixation, and 18 thumbs were cured ( 75%). There was no statistically significant difference in the cure rate between the two treatment methods; In type IIa,36 thumbs were treated with low-temperature thermoplastic splint external fixation and 26 thumbs were cured( 72. 22%),13 thumbs were under general anesthesia had undergone tendon sheath release operation,12 thumbs were cured( 92. 31%). There was no statistically significant difference between the two groups in the cure rate; 21 thumbs in type Ⅱb were treated with low-temperature thermoplastic splint external fixation,and 10 thumbs were cured( 47. 62%),the remaining 20 thumbs had undergone tendon sheath release operation,and 18 thumbs were cured( 90%). The cure rate of brace treatment was significantly lower than that of surgical treatment; All 32 thumbs of type Ⅲ had undergone tendon sheath release operation,and 28 thumbs( 87. 5%) were cured after follow-up. For children with trigger thumb,they should be diagnosed and treated correctly according to different types,with strong pertinence and good recovery of thumb function.

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The clinical date of 146 patients with wake-up strokes(WUS) were retrospectively analyzed. Reperfusion therapy in 62 patients with WUS was performed according to the mismatch of cerebral MRI DWI/FLAIR or PWI/Core Infarct Volume. The scores of 24 h NIHSS and 90 d mRS in reperfusion were lower than those of the 84 patients in WUS non-reperfusion. The incidence symptomatic intracranial hemorrhage and mortality did not increase. Reperfusion therapy for WUS patients under the guidance of multimodal magnetic resonance imaging is safe and effective.

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To investigate the effect of tibial intramedullary nailingviasuprapatellar and infrapatellar approaches on knee function after tibial shaft fracture. 85 cases of tibial shaft fractures were treated with tibial intramedullary nailing, including 40 cases of suprapatellar approach(suprapatellar approach group) and 45 cases of infrapatellar approach(infrapatellar approach group). Range of motion(ROM) and Hospital of Special Surgery(HSS)coring system of knee joint were evaluated 12 months after operation. There was no significant difference in operation time and intraoperative blood loss between the two groups. 12 months after operation, the total HSS score, pain score and functional activity score were(97.78±4.14),(28.44±2.78) and(21.47±2.50) for suprapatellar approach group respectively while they were(97.11±5.90),(29.13±1.92) and(21.73±1.26) for infrapatellar approach group respectively. There was no significant difference between the two groups. In the ROM score of knee joint, the range of motion of the infrapatellar approach group(17.47±1.06) was larger than that of the suprapatellar approach group(16.85±1.49)(P=0.023). Tibial intramedullary nailingviasuprapatellar approach and infrapatellar approach can achieve good results in the treatment of tibial shaft fractures, but the range of knee flexion and extension in the infrapatellar approach group is larger than that in the suprapatellar approach group, so the suprapatellar approach needs further study to reduce the damage to quadriceps femoris.

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Objective To establish a rapid detection method of fluorescent RT-RAA for novel coronavirus(SARS-CoV-2). Methods Using SARS-CoV-2 RNA transcribedin vitroas template,orf1 abgene andSgene were amplified rapidly by single strand DNA binding protein, recombinant enzyme and DNA polymerase at constant temperature of 40.5 ℃. Pharyngeal swabs from patients with known COVID-19 nucleic acid positive and other respiratory virus infections were used for preliminary evaluation. Results The method established in this research took less than 20 minutes to detect the two SARS-CoV-2 genes separately. The sensitivity of the two genes was 2 copies per reaction tube, and the specificity was 100%. The lowest detection limit of two primers was 100%. The peak time of the amplification reaction of the three repeated experiments was the same, and the shape of the curve was similar. Conclusion The method established in this study has high sensitivity, specificity and good reproducibility. The test results of clinical specimens have a high coincidence rate and do not require expensive equipment, so this method is suitable for rapid detection on site.

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Objective In this study, we detected angiotensin-converting enzyme 2(ACE2) and transmembrane protease, serine 2(TMPRSS2) expression of different ages(newborn, juvenile stage, adulthood and aged mice) and different tissues(lung, heart and small intestine) in mice to reveal the potential age and target organ that were susceptible to corona virus disease 2019(COVID-19) infection. Methods The conservatism of TMPRSS2 and ACE2 among different species was analyzed by online bioinformatics analysis website(Uniprot). Real-time Quantitative PCR was used to analyze relative expression of ACE2 and TMPRSS2 mRNA at different ages and different tissues in mice. Western blot was used to analyze relative expression of ACE2 and TMPRSS2 protein at different ages and different tissues in mice. Results (1)The homology of ACE2 between human and mice was 82.1%, and the homology of TMPRSS2 was 78.4%;(2)Compared with 1-3 day old(newborn group) mice, the expression levels of ACE2 and TMPRSS2 mRNA in lung and small intestine tissues of 1-month old(young group), 3-month old(adult group) and 18-month old(old group) mice increased(P<0.05); compared with newborn mice, ACE2 and TMPRSS2 mRNA expression in heart tissues of young and adult mice increased(P<0.05), and TMPRSS2 and ACE2 mRNA expression in elderly group decreased(P<0.05);(3)Differences in ACE2 and TMPRSS2 mRNA expression levels in different tissues of mice of the same age group were statistically significant, and the highest expression levels were in lung tissue(P<0.05);(4)The tendency of ACE2 and TMPRSS2 protein and gene expression was consistent. Conclusion With the increase of age, the expression of ACE2 and TMPRSS2 in lung and small intestine of mice also increased, which may mean that advanced age is one of the independent risk factors for infection of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2) in mice and even humans. In addition, the high expression of ACE2 and TMPRSS2 in the lung tissue of the same age mice group may suggest that the lung is the main target organ for SARS-CoV-2 invasion.

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Objective To investigate the clinical improvement of glucocorticoid in the treatment of mild, severe, and critical cases of coronavirus disease 2019(COVID-19). Methods A retrospective study included 248 patients with confirmed COVID-19. The severity of the patient was assessed as mild, severe, and critical. Patients were divided into non-hormonal group and hormonal group according to whether they received glucocorticoid therapy during their hospitalization. The non-hormone group was treated with oxygen support and antiviral therapy, and the hormone group was treated with methylprednisolone for a short term. The discharge mortality rate, hospitalization time, and time from symptom onset to discharge were analyzed. The dynamic changes of S-IgG, S-IgM, S-RBD-IgG, N-IgG, and N-IgM antibodies were detected by ELISA. Results There was no difference in discharge mortality between mild, severe patients in the glucocorticoid therapy group and non-glucocorticoid treatment group, while the mortality was higher in critical patients treated with glucocorticoid(68%vs40%,P<0.05). The hospitalization time in mild patients receiving glucocorticoid therapy was higher than that in the non-glucocorticoid treatment group [22(17-28)dvs16(12-22)d], and the difference was statistically significant(P<0.05). The S-IgG, S-RBD and N-IgG antibodies showed a gradually increasing trend, while the S-IgM and N-IgM antibodies showed a gradually decreasing trend, and there was no statistical significance between the two groups. Conclusion Short-term glucocorticoid therapy can not improve the mortality rate and clinical outcome, and has no significant effect on the immune function of patients with COVID-19.