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  • Table of Content
      28 November 2021, Volume 37 Issue 6 Previous Issue   
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    ORIGINAL ARTICLES
    Effects of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition of type II alveolar epithelial cell and its mechanism
    TANG Juan, KE Jie, FU Yu, HE Xiao-wei, LI Xian-wei
    CJAP. 2021, 37 (6): 577-583.   DOI: 10.12047/j.cjap.6124.2021.048
    Abstract   PDF (2038KB) ( 4 )
    Objective: To study the role of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition (EMT) of type II alveolar epithelial cells in pulmonary fibrosis (PF) and hope to provide a new theoretical basis for the pathogenesis of PF. Methods: Thirty rats were randomly divided into control group and bleomycin (BLM) group, 15 rats in each group. The PF rat model was induced by intratracheal injection of BLM (7 500 U/kg). Excised inferior lobe of left lung was fixed in 10% formalin for HE staining, Masson staining and transforming growth factor-beta 1 (TGF-β1) immunohistochemistry staining after BLM injection for 28 days. The cultured type II alveolar epithelial cells (RLE-6TN) were divided into 4 groups (Control group, transforming growth factor-beta 1 (TGF-β1) group, Notch-1 negative control siRNA (NC siRNA, 100 pmol/L) group and Notch-1 siRNA (100 pmol/L) group), each group was established nine holes. The cells were treated with TGF-β1 (5.0 ng/ml) for 24 h following NC siRNA or Notch-1 siRNA for 48 h. The mRNA and (or) proteins levels of TGF-β1, collagen I, collagen III, E-Cadherin, zonula occludens-1 (ZO-1), Vimentin, E-Cadherin, Notch-1, Notch-1 intracellular domain (NICD), Hes-1 and Twist-1 were detected in lung tissue and type II alveolar epithelial cells. Results: In vivo, compared with the control group, the alveolar atrophy, collapse and fusion occurred, alveolar septum widened significantly, and a large number of inflammatory cells infiltrated in the pulmonary interstitial of the rats in the BLM group. And compared with control group, BLM obviously increased collagen deposition and collagen I and collagen III expressions, while the expressions of ZO-1 and E-cadherin were decreased, and the expressions of Vimentin and N-cadherin were increased, and concomitantly with increasing Notch-1, NICD, Hes-1 and Twist-1 expression in lung tissues of rats (P<0.01). In vitro, compared with control group, TGF-β1 treatment obviously induced collagen I, collagen III, Notch-1, NICD, Hes-1 and Twist-1 expressions, and the expressions of E-cadherin and ZO-1 were decreased and the expressions of Vimentin and N-cadherin were increased(P<0.01). Compared with TGF-β1 group, Notch-1 siRNA treatment significantly inhibited the expressions of Notch-1, NICD, Hes-1 and Twist-1, and the expressions of E-cadherin and ZO-1 were increased and the expressions of Vimentin and N-cadherin were decreased, and also obviously reduced the expressions of collagen I and collagen III induced by TGF-β1 (P<0.05 or P<0.01). Conclusion: Notch-1/Twist-1 axis is involved in the EMT process of type II alveolar epithelial cells, suggesting that Notch-1/Twist-1 signaling may be involved in the development of pulmonary fibrosis.
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    Interventional effect of IL-17A on chronic obstructive pulmonary disease and its mechanism
    BAI Lin-lin, WANG Zhi-hua, NING Xue-cong, GONG Cui-ke, LI Ai-min, DUAN Yu-ling, JIAO Yu-jiao
    CJAP. 2021, 37 (6): 584-588.   DOI: 10.12047/j.cjap.6135.2021.093
    Abstract   PDF (1127KB) ( 3 )
    Objective: To investigate the intervention effects and mechanism of interleukin-17A (IL-17A) on chronic obstructive pulmonary disease (COPD). Methods: C57BL/6 mice were randomly divided into wild type blank control group, wild type COPD group and IL-7A knockout COPD group. Mice in wild type blank control group received no treatment, and mice in the other two groups were exposed to cigarette smoke to induce COPD (Cigarette: 1 cigarette / time, 4 times/day, 45 minutes/time; interval time: 1 hour; total intervention time: 90 days). Lung function of mice was assessed using animal pulmonary function machine. Bronchoalveolar lavage fluid (BALF) of mice was collected and BALF cell count and classification were determined. The lung tissue of mice was collected, the expression level of IL-17A in airway epithelium was determined by flow cytometry, and the levels of inflammatory factors in lung tissue were determined by enzyme-linked immunosorbent assay. The expression level of JNK/AP1 signaling pathway protein in mouse lung tissue was determined by Western blot. Results: Compared with the wild type blank control group mice, the wild type COPD group mice had significantly higher expression level of IL-17A, significantly lower peak inspiratory flow rate (PIF) and peak expiratory flow rate (PEF), significantly higher number of BALF neutrophils, eosinophils, lymphocytes and macrophage, significantly higher expression levels of CXC chemokine 1(CXCL1), CXC chemokine 2 (CXCL2), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and significantly higher phosphorylation level of JNK, cJun and cFos and AP1 expression levels (P<0.05). Compared with COPD mice, IL-17A expression level in airway epithelium of mice in IL-7A knockout COPD group was significantly lower, PIF and PEF were higher, the number of BALF neutrophils, eosinophils, lymphocytes and macrophage was significantly lower, the expression levels of CXCL1, CXCL2, IL-1β and IL-6 in lung tissue were lower, and the phosphorylation levels of JNK, cJun and cFos and AP1 expression levels were significantly lower (P<0.05). Conclusion: Cigarette smoke can induce the production of IL-17A and reduce (or inhibit) the production (or expression or secretion) of IL-17A in mouse airway epithelium, thus inhibiting the JNK/AP1 signaling pathway to reduce the airway inflammation and improve the lung function of COPD mice.
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    Effects of L-carnitine on autophagy and apoptosis of mouse pulmonary microvascular endothelial cells induced by lipopolysaccharide
    XUE Feng, WANG Hui, XU Si-duo, LIU Shou-ting, GONG Yong-sheng, FAN Xiao-fang
    CJAP. 2021, 37 (6): 589-593.   DOI: 10.12047/j.cjap.6077.2021.086
    Abstract   PDF (1603KB) ( 1 )
    Objective: To investigate the protective effects of L-carnitine (LC) on lipopolysaccharide (LPS) - injured mouse pulmonary microvascular endothelial cells (PMVECs) and its effects on autophagy and apoptosis. Methods: Cultured mouse PMVECs were divided into three groups: ① Control group, ② LPS group (10 μg/ml, 3, 6, 12, 24 h), ③ LPS (10 μg/ml, 24 h)+LC (2.5, 5.0, 10 μg/ml) (LPS+LC) group. PMVECs apoptosis was examined by Annexin V-FITC/PI double labeling method. Autophagosome was detected by immunofluorescence staining. Levels of autophagy-related protein LC3 and apoptosis-related protein caspase-3 were detected by Western blot. PMVECs viability was measured by CCK-8. Results: ① Compared with the control group, LPS treatment inhibited the PMVECs viability significantly, whereas the apoptosis rate and the expression of autophagy protein LC3 II were markedly increased after LPS treatment for 6 h, 12 h and 24 h. ② Compared with LPS group (10 μg/ml, 24 h), the PMVECs viability, levels of autophagy protein LC3 II and caspase-3 protein expression as well as apoptosis rate in LPS+LC group were increased significantly. Conclusion: LC can increase the activity of PMVECs injuried by LPS, promote autophagy and inhibit apoptosis of PMVECs.
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    Effects of miR-133b on oxLDL-induced vascular endothelial cell injury by targeting SGTB
    CHEN Gang, CHEN Jiu-lin, WU Jun
    CJAP. 2021, 37 (6): 594-600.   DOI: 10.12047/j.cjap.6111.2021.064
    Abstract   PDF (1874KB) ( 4 )
    Objective: To investigate the effect of microRNA-133b (miR-133b) on oxidized low density lipoprotein (oxLDL) induced vascular endothelial cell injury by targeting small protein molecules rich in glutamine 34-tetrapeptide repeats (SGTB). Methods: Human umbilical vein endothelial cells (EVC-304) were induced by 100 μg/ml oxLDL for 24 h to construct a vascular endothelial cell injury model. EVC-304 cells were divided into control group, oxLDL group (oxLDL treatment), oxLDL+miR-NC group (transfectted with 20 nmol/L miR-NC+oxLDL treatment), oxLDL+miR-133b group (transfectted with 20 nmol/L miR-133b mimics +oxLDL treatment), oxLDL+si-NC group (transfectted with 20 nmol/L si-NC+oxLDL treatment), oxLDL+si-SGTB group (transfected with 20 nmol/L si-SGTB+oxLDL treatment), oxLDL+miR-133b+ pcDNA group (transfected with 20 nmol/L si-SGTB and pcDNA+oxLDL), oxLDL+miR-133b+pcDNA-SGTB group (transfected with 20 nmol/L si-SGTB and pcDNA-SGTB), 9 wells in each group. Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-133b and SGTB; flow cytometry was used to detect cell apoptosis; kits were used to detect malondialdehyde (MDA) content and the activities of superoxide disproportionation enzyme (SOD) and glutathione peroxidase (GSH-Px). The expression levels of Bcl-2 and Bax protein were detected by Western blot. The dual luciferase reporter gene assay and Western blot were used to verify the targeted and regulatory between miR-133b and SGTB. Results: Compared with the control group, the expressions of miR-133b and Bcl-2 in EVC-304 cells were decreased significantly after oxLDL induction, while the expression levels of SGTB and Bax were sincreased ignificantly (P<0.05), the MDA content and apoptosis rate were increased significantly (P<0.05), and the activities of SOD and GSH-Px were decreased significantly (P<0.05). Over-expression of miR-133b or interfering with SGTB inhibited oxLDL-induced apoptosis and oxidative stress in EVC-304 cells (P< 0.05). miR-133b directly bound to SGTB, miR-133b overexpression significantly down-regulated SGTB expression (P<0.05), miR-133b inhibition significantly up-regulated SGTB expression (P<0.05) Over-expression of SGTB reversed the effect of over-expressing miR-133b on oxLDL-induced vascular endothelial cell injury (P<0.05). Conclusion: miR-133b could attenuate oxidative stress damage and apoptosis induced by oxLDL in vascular endothelial cells by targeting and inhibiting SGTB expression.
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    Effects of miR-155-3p on the degradation rate of EAF1 mRNA and malignant proliferation in HANK1 cells
    LIU Li, XU Kai, WANG Lu-qiang, XU Mei-li
    CJAP. 2021, 37 (6): 601-605.   DOI: 10.12047/j.cjap.6159.2021.095
    Abstract   PDF (1448KB) ( 0 )
    Objective: To investigate the effects and mechanisms of miR-155-3p on the malignant behavior of human NK/T cell lymphoma cell line HANK1. Methods: Targetscan database was used to predict the target gene of miR-155-3p. HANK1 cells in logarithmic growth period were cultured, and the cells were divided into blank group, over-expressed group, control group and interference group, which were transfected with pENTER-puro vector, pENTER-miR-155-3p vector, GV248 control vector and GV248-miR-155-3p siRNA interference vector, respectively. Meanwhile, actinomycin D (ActD) was used to treat each group of cells, and the expressions of miR-155-3p, EAf1, β-catenin and c-Myc in each group were detected by real-time fluorescence quantitative PCR (n=5). The degradation rate of EAF1 mRNA, the expressions of EAF1, β-catenin and c-Myc protein were detected by Western blot (n=3), and the malignant proliferation abilities of cells were detected by CCK-8 (n=5). Results: Compared with the blank group, the expression levels of miR-155-3p, β-catenin and c-Myc in the over-expressed group were significantly higher, the expression level of EAF1 was lower, the half-life of EAF1 mRNA was shortened, and the malignant proliferation ability of the cells was strengthened (P<0.05). Compared with the control group, the expression levels of miR-155-3p, β-catenin and c-Myc in the interference group were significantly lower, and the expression level of EAF1 was increased, the half-life of mRNA was prolonged and the ability of cell proliferation was decreased (P<0.05). Conclusion: miR-155-3p can promote EAF1 mRNA degradation and proliferation in HANK1 cells.
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    Effects of PD-L1 on immunosuppression of bacterial sepsis and its relevant mechanism
    WANG Fang, YANG Mu-yu, WANG Bin, QU Zhen-li, HU Qing-ru
    CJAP. 2021, 37 (6): 606-610.   DOI: 10.12047/j.cjap.6112.2021.091
    Abstract   PDF (1358KB) ( 1 )
    Objective: To investigate the expression of programmed death ligand-1 (PD-L1) in dendritic cells (DCS) and its related signaling pathway in lipopolysaccharide (LPS)-induced immunosuppression of bacterial sepsis.Methods: Stimulating with bacterial LPS, bone marrow-derived dendritic cells could induce T lymphocyte immunosuppression imitating bacterial sepsis model. The experiments were divided into 5 groups: control group, LPS group, 2-(4-morpholinyl)-8-phenyl-4H-1- benzopyran-4-one (LY294002)+LPS group, pyrrolidinedithiocarbamate(PDTC)+LPS group and LPS+anti-PD-L1 group with 6 multiple wells in each group. After mice bone marrow source monocytes were cultured with rmGM-CSF (10 ng/ml) and rmIL-4 (1 ng/ml) in 10% fetal bovine serum 1640 for 4 days, DCs cells were treated with with 10 ng/ml LPS for 12 h to obtain immunosuppressive cells with high expression of PD-L1. Pathway-inhibitors LY294002 (10 μmol/L) and PDTC (20 μmol/L) were used to block PI3K and NF-κB signals. Flow cytometry and confocal laser scanning microscopy were used to detect the PD-L1 expression and phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signal activation on DCs. BrdU cell proliferation assay and γ-interferon enzyme-linked immunospot assay were used to detect ovalbumin specific T lymphocyte proliferation response and cytotoxic T cell response, respectively. Results: Compared with the control group, the percentage of PD-L1 positive cells and PD-L1 red fluorescence intensity of DCs were all increased(P<0.01), while DCs- mediated T cell proliferation and γ-interferon spot-forming cell number were decreased (P<0.01).PI3K inhibitor LY294002, NF-κB inhibitor PDTC and PD-L1 blocking antibody could significantly reverse the inhibition of DCs mediated T lymphocytes immunosuppression above (P<0.01). Conclusion: PD-L1 was a key molecule that mediates immunosuppression in lipopolysaccharide induced bacterial sepsis. PI3K Signal and NF- κB signal were also involved in this immunosuppressive process.
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    Effects of curcumol on liver function and fibrosis in rats of nonalcoholic fatty liver disease and its mechanism
    QI Shu-yan, HUANG Hua, LI Yong-kun, PEI Lin-guo, ZHANG Wen-juan
    CJAP. 2021, 37 (6): 611-615.   DOI: 10.12047/j.cjap.6058.2021.082
    Abstract   PDF (1578KB) ( 2 )
    Objective: To investigate the effects and mechanism of curcumol (CC) on liver function and fibrosis in rats of nonalcoholic fatty liver disease (NAFLD). Methods: The rat models of nonalcoholic steatohepatitis (NASH) combined with liver fibrosis were constructed by high-fat diet. Sixty SD rats were randomly divided into blank control group, model group (NASH), NASH + Compound Biejiarangan Troche (CBT) group (positive control group), and NASH + CC groups (25, 50, 100 mg/kg) , 10 rats in each group. The percentage of liver to body weight, and the levels of high density lipoprotein (HDL), triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. The liver fibrosis was observed by HE staining. The expressions of α-smooth muscle actin (α-SMA) and positive staining of nuclear factor κB p65 (NF-κB p65) were detected by immunohistochemistry. The expression levels of α-SMA, matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and toll-like receptor-4 (TLR4), transforming growth factor-activated kinase-1 (TAK1), NF-κB p65 and vascular cell adhesion molecule-1 (VCAM-1) were detected by Western blot. The expression levels of interleukin (IL-6, IL-10, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). Results: Compared with blank control group, the contents of HDL and IL-10 and the expression level of MMP-1 protein were decreased in model group significantly (P<0.05), while the levels of TG, ALT and AST, the positive rate of P65, α-SMA, TIMP-1, TLR4, TAK1, NF-κB p65, VCAM-1, IL-6, TNF-α and IL-1β were increased significantly (P<0.05). Compared with model group, the levels of HDL, IL-10 and MMP-1 protein were significantly increased after treatment with CBT and CC (P<0.05), while the levels of TG, ALT, and AST, the positive rate of P65, α-SMA, TIMP-1, TLR4, TAK1, NF-κB p65, VCAM-1, IL-6, TNF-α and IL-1β were decreased significantly (P<0.05). The improvement in model+high- concentration CC group was the most significant, and which in all concentration groups was lower than that in model+CBT group (P<0.05). Conclusion: CC can reduce inflammation response and improve liver function by regulating TLR4, TAK1 and NF-κB/p65 signaling pathway, and thus alleviating liver fibrosis, showing concentration-dependence within certain range.
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    Effects of TUG1 on hepatic fibrosis and its mechanism
    ZHU Hai-dong, LYU Chang-kun, MA Fei-fei
    CJAP. 2021, 37 (6): 616-621.   DOI: 10.12047/j.cjap.6139.2021.094
    Abstract   PDF (1389KB) ( 1 )
    Objective: To investigate the effects and mechanisms of taurine up-regulated gene 1 (TUG1) in hepatic fibrosis. Methods: According to the literature, the classic hepatic fibrosis model of rats induced by 1%DMN(1ml/kg/d) was established. The rats with hepatic fibrosis and activated hepatic stellate cells (HSC) were divided into model control group, negative control group (transfected with siRNA negative control), siRNA interference group (transfected with TUG1). At the end of the experiment, hematoxylin eosin (HE) staining was used to detect the pathological changes of liver tissue; reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to determine the expression levels of α-smooth muscle actin (α-SMA), TUG1, collagen I, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), Smad2 and Smad3 in rat liver tissue and activated hepatic stellate cells. Results: Compared with the model control group, the protein and gene levels of TUG1 and α-SMA in the negative control group were increased significantly(P<0.05). The protein and gene levels of TUG1, α-SMA, collagen I, MMP-2, TIMP-1, Smad2 and Smad3 in the liver tissue and activated hepatic stellate cells in the siRNA interference group were decreased (P<0.05) while compared with the blank control group and the negative control group. There were no significant differences in the levels of TUG1, α-SMA, collagen I, MMP-2, TIMP-1, Smad2 and Smad3 in the liver tissue and activated hepatic stellate cells between the control group and the negative control group (P>0.05). Conclusion: TUG1 level is elevated in hepatic fibrosis tissue and activated hepatic stellate cells. Silencing TUG1 may improve the pathological damage of hepatic fibrosis induced by 1% DMN by inhibiting the transforming growth factor(TGF-β1)/ Smad signaling pathway.
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    Effects of alcohol extract of radish seed on blood lipid, glucose and liver steatosis in ApoE-/- mice
    LI Li, HUANG Han-cheng
    CJAP. 2021, 37 (6): 622-627.   DOI: 10.12047/j.cjap.6143.2021.078
    Abstract   PDF (1515KB) ( 2 )
    Objective: To investigate the effects and mechanisms of alcohol extract of Raphani seed (AERS) on blood lipid, blood glucose and hepatic steatosis in ApoE-/- mice. Methods: Sixty ApoE-/- mice were randomly divided into control group (CG), normal saline group (NG), rosiglitazone group (RG) and AERS treatment groups (AERS-L / M / H). Except CG, other groups were fed with high-fat, high-sugar and high-salt diet for 9 weeks. The mice in RG were treated with rosiglitazone (1.33 mg·kg-1, 0.2 ml ·10 g-1) by gavage daily. The mice in CG and NG were treated with equal volume of NS by intragastric administration daily. The mice in AERS groups were treated with AERS at the doses of 100, 300 and 900 mg·kg-1 for 9 weeks, respectively. FPG and Fins were detected. Insulin resistance index (IRI) and liver coefficient (LC) were calculated. The levels of ALT, AST, Leptin (LEP), TNF - α and blood lipid (TC, FFA, etc.) were tested. The expressions of proteins related to liver lipid metabolism (HMG-R、LDL-R、LEP-R) were detected by Western blot. Results: Compared with NG or RG, CG showed significant changes in liver appearance (color, swelling, etc.) and pathology (steatosis, hepatocyte necrosis, etc.), while AERS-M/H groups were similar to CG. Compared with CG, the serum levels of FPG, Fins, IRI, ALT, AST, TNF-α, LC, TG, LDL-C, FFA and LEP were increased significantly (P<0.05) . Compared with NG, AERS could decrease the above mentioned indicators in a dose-dependent manner (P<0.05). Compared with RG, the levels of FPG and Fins of AERS-H were increased significantly, while the level of IRI was decreased (P<0.05). Compared with NG and RG, the protein expressions of HMG-R and LEP-R in AERS groups were decreased, while the protein expression of LDL-R was increased in a dose-dependent manner (P<0.05). Conclusion: AERS can prevent the increase of blood lipid, glucose and hepatic steatosis induced by high-fat and high-sugar diet in ApoE-/- mice. The mechanism is related to the decrease of FFA and LEP, the inhibition of TNF-α, HMG-R, LEP-R protein expressions and the promotion of LDL-R protein expression.
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    BRIEF REPORTS
    Effects of simulated high-altitude hypobaric hypoxia on iron metabolism in mice spleen
    HU Jia-nan, JIANG Hui, LI Mei-qi, ZHU Li, LUO Qian-qian
    CJAP. 2021, 37 (6): 628-631.   DOI: 10.12047/j.cjap.6142.2021.077
    Abstract   PDF (1608KB) ( 0 )
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    ORIGINAL ARTICLES
    Effects of hypoxia combined with LPS on the expression of pro- inflammatory cytokines and BNIP3 in primary cultured astrocyte
    DING Li-ping, HAN Ying, CHENG Xiang, ZHAO Tong, ZHU Ling-ling , LIAO Hong
    CJAP. 2021, 37 (6): 632-637.   DOI: 10.12047/j.cjap.6177.2021.096
    Abstract   PDF (1105KB) ( 0 )
    Objective: To investigate the expression of Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and inflammation in astrocytes under lipopolysaccharide ( LPS ) combined with hypoxia. Methods: Primary cultured astrocytes and neurons in vitro were divided into four groups: normoxia group; hypoxia group; LPS group; LPS plus hypoxia group (each group is provided with 3 duplicate holes). After treated with LPS(100 ng/ml), hypoxia group and LPS plus hypoxia group were placed in hypoxia cell incubator with 0.3% O2, and normoxia group and LPS group were placed in normal cell incubator for 24 h. Primary astrocytes were divided four groups as above for 6 h,12 h and 24 h. The expression of BNIP3 in astrocytes was detected by Western blot. The expressions of tumor necrosis factor-α(TNF-ɑ), interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA in astrocytes were detected by RT-PCR. The levels of TNF-ɑ, IL-1β and IL-6 in cultured medium were detected by ELISA. Results: Compared with the normoxia group, the expressions of inflammatory cytokines TNF-ɑ, IL-1β and IL-6 mRNA had no change in hypoxia group and were increased in LPS group and LPS plus hypoxia group (P<0.01). Compared with the LPS group, the expressions of inflammatory cytokines IL-1β and IL-6 mRNA were increased in LPS plus hypoxia group (P<0.05, P<0.01). Compared with the normoxia group, the levels of inflammatory cytokines had no change in hypoxia group and the levels of TNF-ɑ and IL-6 were increased in LPS group and LPS plus hypoxia group (P<0.01), the level of IL-1β had no change in LPS group and LPS plus hypoxia group. Compared with the LPS group, the levels of TNF-ɑ and IL-6 had no more change in LPS plus hypoxia group. BNIP3 was expressed in primary neurons and astrocytes in vitro. Compared with astrocytes in the normoxia group, the expression of BNIP3 in LPS group had no change and was increased markedly in hypoxia group and LPS plus hypoxia group (P<0.01). Compared with neurons in the normoxia group, the expression of BNIP3 in LPS group had no change and was increased in hypoxia group and LPS plus hypoxia group (P<0.05, P<0.01). Compared with neurons in the hypoxia group, the expression of BNIP3 in astrocytes of hypoxia group was increased (P<0.01). Compared with the normoxia group at the same time point, the expression of BNIP3 in LPS group had no change and was increased in hypoxia group and LPS plus hypoxia group (P<0.05, P<0.01). Compared with the hypoxia group at the same time point, the expression of BNIP3 was increased markedly in LPS plus hypoxia group at 6 h and 12 h (P<0.01). Conclusion: The combination of hypoxia with LPS augmented inflammation in astrocyte and LPS enhanced the expression of BNIP3 in astrocyte under hypoxia, suggesting BNIP3 might be involved in regulating astrocyte inflammation.
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    Effects of spicy leaves on cognitive function and apoptosis of hippocampal neurons in diabetic rats
    SUN Yang, ZHAO Can, LIU Yuan-yuan, SUI Yue-lin, ZHU Jian-zhong
    CJAP. 2021, 37 (6): 638-643.   DOI: 10.12047/j.cjap.6123.2021.070
    Abstract   PDF (1173KB) ( 0 )
    Objective: To investigate the effects of Moringa leaves on the cognitive dysfunction and apoptosis of hippocampal neurons in diabetic rats induced by streptozotocin (STZ). Methods: Fifty male SD rats were selected, and 10 rats were randomly selected as the control group. The other 40 rats were treated with STZ at the dose of 25 mg/kg by intraperitoneal injection. The 40 diabetic rats were randomly divided into model group, Moringa oleifera low-dose, medium-dose and high-dose group. The rats in Moringa oleifera groups were treated with Moringa oleifera at the doses of 2.0, 4.0 and 8.0 g/kg by gavage, the control group and model group were treated with the same amount of normal saline once a day, for 8 weeks. Morris water maze test was used to evaluate the learning and memory ability of rats. Pathological changes of hippocampal neurons and expressions of Bax, caspase-3 and bcl-2 protein in each group were observed by the sections were stained with HE staining and immunohistochemistry. Enzyme linked immunosorbent assay (ELISA) was used to detect tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in rat. Results: compared with the control group, the blood glucose of the model group was increased significantly (P<0.01), and the blood insulin level was decreased significantly (P<0.05); compared with the model group, the blood glucose values of Moringa oleifera groups were decreased significantly (P<0.05, P<0.01), and the blood insulin levels of middle and high dose Moringa oleifera group were increased significantly (P<0.05, P<0.01). There was no significant difference in FBG and INS among the three groups (P>0.05). In Morris water maze test, compared with the model group, the latency of Moringa oleifera groups was significantly shorter (P<0.05); the residence time in target quadrant of Moringa oleifera groups with different doses was significantly prolonged (P<0.05). Compared with the model group, the contents of TNF - α, IL-6 and protein expression in low, medium and high dose groups of Moringa oleifera were decreased significantly (P<0.05). HE staining and immunohistochemical staining results showed that Moringa oleifera medium dose group was positive, brown yellow, fine granular, compared with the model group. The number of neuronal apoptosis was significantly reduced in the middle dose group (53.21±7.19,P<0.01); the protein expressions of Bax, caspase-3 and the ratio of Bax/Bcl-2 in hippocampus were significantly decreased in the middle dose group (P<0.05). Conclusion: The mechanisms of Moringa leaves on the cognitive dysfunction and apoptosis of hippocampal neurons may be related to regulating the protein expressions of Bax, Bcl-2 and Caspase-3, reducing the contents of inflammatory factors TNF-α and IL-6.
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    Effects of Metformin and Sitagliptin on islet cell function in insulin-resistant prediabetic KKay mice
    LI Ping, LIANG Lin-lang, WANG Yu , HOU Da, YANG Xin, YANG Bo
    CJAP. 2021, 37 (6): 644-649.   DOI: 10.12047/j.cjap.6062.2021.084
    Abstract   PDF (1756KB) ( 0 )
    Objective: To investigate the effects of metformin and sitagliptin on the function of islet β cells in insulin resistance pre-diabetic KKay mice. Methods: Thirty 6-week-old KKAy mice were randomly divided into two groups: normal diet fed group (NC group, n=10) and high-fat diet fed group (n=20). At 8 weeks, KKAy mice were randomly divided into two groups: metformin intervention group (met group, n=10) and sitagliptin intervention group (SP group, n=10), which were treated by gavage for 8 weeks. Glucose tolerance was measured by oral glucose tolerance test (OGTT), serum insulin level and plasma lipid level were measured by tail blood sampling, and HOMA-β and HOMA-IR were calculated. The mice were killed after blood collection, and the pancreas of KKAy mice was taken. The β cell volume of each group was compared by immunofluorescence staining of insulin and glucagon, respectively. The proliferation and apoptosis of β cell were analyzed by Ki67/INS. The expressions of pancreatic transcription factors PDX-1 and MafA were detected by Western blot. Results: ① The OGTT results indicated that blood glucose of KKAy mice at fast, 30, 60 and 120 min after oral administration of glucose in the Met and SP groups were decreased significantly compared with the NC group, and the area under the blood glucose time curve (AUC) was significantly reduced (P<0.01, P<0.01). There was no significant difference between the Met group and the SP group in blood glucose level at 30 and 60 min after oral administration of glucose. Compared with the SP group, the blood glucose of Met group at 120 min was decreased significantly (P<0.05). There was no significant difference in AUC between the two groups. ② The results of the insulin tolerance test (ITT) indicated that, compared with NC, the fasting blood glucose and the blood glucose at 30, 60 and 90 min after insulin injection in KKAy mice in the Met and SP groups were decreased significantly, and the area under the ITT blood glucose curve (AUC) was increased significantly (P<0.01), while there was no significant difference between the Met and SP groups. ③ In the NC group, the brightness of the areas of the islet β cells was low and the edges were scattered. After treated with metformin, the areas and brightness of the β cells were increased. After treatment with sitagliptin, the area and brightness of the β cells were increased significantly. In the NC group, the α cells were disordered in the islet distribution and the brightness was large. After the administration of metformin, the α cell area and the brightness were decreased, and distributed to the edge of the islet to a certain extent. After the administration of sitagliptin, there was a significant decrease in the area of the α cells, with a significant decrease in the brightness and distribution at the edge of the islet. ④ Compared with the NC group, the expression levels of pancreatic MafA in the Met group and SP group were increased significantly, which were 1.63 times and 1.58 times, respectively (P<0.01, P<0.01). There was no significant difference in the expression of pancreatic PDX-1 between the groups. Conclusion: In pre-diabetes mellitus KKAy mice with insulin resistance, metformin can maintain the function and morphology of pancreatic islets, and sitagliptin may promote the proliferation of islet βcells, improve the expression level of insulin transcription factor MafA, and prevent the occurrence and development of diabetes.
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    BRIEF REPORTS
    Effects of repeated transcranial magnetic stimulation combined with low-dose fluoxetine on CUMS depression mice
    YUAN Qian-fa, HE Jue, XU Zhi-zhong, LIN Duo-duo, ZHU Jing-jing, WEN Chun-yan, DUAN Hai-zhen, WANG Wen-qiang
    CJAP. 2021, 37 (6): 650-653.   DOI: 10.12047/j.cjap.6070.2021.035
    Abstract   PDF (1109KB) ( 0 )
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    ORIGINAL ARTICLES
    Effects of ropivacaine hydrochloride on the proliferation, invasion and apoptosis of osteosarcoma cells and its mechanism
    YU Peng, YIN Tian-xiang, LUO Cai-yun, ZENG Ping
    CJAP. 2021, 37 (6): 654-659.   DOI: 10.12047/j.cjap.6082.2021.088
    Abstract   PDF (1636KB) ( 0 )
    Objective: To investigate the effects and molecular mechanism of ropivacaine hydrochloride on osteosarcoma cell proliferation, invasion, and apoptosis. Methods: The osteosarcoma doxorubicin-resistant cell line (U2OS/DOX) was established by gradually increasing the drug doses. U2OS/DOX cells were treated with ropivacaine hydrochloride at the concentrations of 0, 20, 50 and 100 μg/ml, respectively; as different concentrations treatment groups of ropivacaine hydrochloride. pcDNA3.1 and pcDNA3.1-Livin were transfected into U2OS/DOX cells and then treated with 100 μg/ml ropivacaine hydrochloride, which were defined as ropivacaine hydrochloride 100 μg/ml+pcDNA3.1 group, ropivacaine hydrochloride 100 μg/ml+pcDNA3.1-Livin group. MTT was used to detect the cell proliferation inhibition rate and inhibitory concentration (IC50). Western blot was used to detect the expressions of cyclin-dependent kinase inhibitor 1A (P21) and activated cysteine aspartic protease-3 (Cleaved Caspase-3), E-cadherin, matrix metalloproteinase 2 (MMP-2) and Livin; clone formation experiments were used to detect the number of cell clones formed; flow cytometry was used to detect apoptosis; Transwell was used to detect cell migration and invasion; real-time fluorescent quantitative PCR (RT-qPCR) was used to detect Livin mRNA expression. Results: When the concentration of doxorubicin was more than 1 μg/ml, the proliferation inhibition rate of osteosarcoma cells U2OS was significantly increased in a concentration-dependent manner (P<0.05); when the concentration of doxorubicin was more than 10 μg/ml, the proliferation inhibition rate of osteosarcoma resistant cell U2OS/DOX was significantly increased, and it was dose-dependent (P<0.05). In U2OS/DOX cells treated with ropivacaine hydrochloride, the expressions of P21, Cleaved Caspase-3, and E-cadherin were increased significantly, the expression of MMP-2 was decreased significantly, the cell proliferation inhibition rate was increased significantly, the number of colony formation was decreased significantly, and the cells apoptosis rate was increased significantly, the number of cell migration and invasion was decreased significantly, and the expression of Livin was significantly reduced, in a concentration-dependent manner (P<0.05). Overexpression of Livin partially reversed the inhibitory effect of ropivacaine hydrochloride on proliferation, migration, invasion, and promotion effect on apoptosis of cell U2OS/DOX. Conclusion: Ropivacaine hydrochloride can significantly inhibit the proliferation, migration and invasion of doxorubicin-resistant osteosarcoma cells, and significantly promote osteoma cell apoptosis. The mechanism may be related to Livin.
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    Effects of Hedyotis diffusa polysaccharide extract on autophagy of endoplasmic reticulum in laryngeal cancer Hep-2 cells
    ZHANG Yan-yan, LIU Hua, SONG Yang, GUO Hai-yan
    CJAP. 2021, 37 (6): 660-664.   DOI: 10.12047/j.cjap.6096.2021.089
    Abstract   PDF (1166KB) ( 1 )
    Objective: To investigate the effects of Hedyotis diffusa polysaccharide extract (HDPE) on endoplasmic reticulum autophagy in laryngeal cancer Hep-2 cells. Methods: The cells were divided into control group, HDPE 100, 200, 400 mg/L group and 3-MA(autophagy inhibitor) group. MTT assay was used to detect the inhibition rate of cell proliferation after 24, 48 and 72 h of culture, and TUNEL was used to detect the apoptosis of cells in each group. The changes of autophages and autophagic lysosomes were observed by MDC staining, and the formation of autophagic vesicles around endoplasmic reticulum was observed by transmission electron microscopy. Western blot was used to detect the expressions of Beclin-1, microtubule-associated light chain protein 3I (LC3I), microtubule-associated light chain protein 3II (LC3II), glucose regulatory protein 78 (GRP78), activated transcription factor 6 (ATF6) and CCAAT enhancer binding protein homologous protein (CHOP) in each group. Results: Compared with the control group, the inhibition rate of cell proliferation and apoptotic index AI in HDPE 100, 200, 400 mg/L group and blocker group were increased, the positive rate of MDC was decreased, and the autophagic vesicles around endoplasmic reticulum were reduced. The expression levels of GRP78, ATF6 and CHOP and the ratio of LC3I/LC3II were increased, and the expression of Beclin-1 was decreased (P<0.05). Compared with 3-MA group, the inhibition rate of cell proliferation and apoptotic index AI in HDPE 400 mg/L group were increased, the positive rate of MDC was decreased, the expressions of GRP78, ATF6 and CHOP and LC3I/LC3II ratio were increased, and the expression of Beclin-1 was decreased (P<0.05). Conclusion: HDPE may inhibit the proliferation of Hep-2 cells by inhibiting endoplasmic reticulum autophagy and promoting the apoptosis of endoplasmic reticulum stress in laryngeal cancer Hep-2 cells.
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    The effects of aerobic exercise plus spirulina polysaccharide supplement on learning and memory function of diabetes rats and its mechanism
    ZHU Hong-zhu, ZHU Mei-ju, ZHANG Ying
    CJAP. 2021, 37 (6): 665-672.   DOI: 10.12047/j.cjap.6065.2021.058
    Abstract   PDF (1588KB) ( 0 )
    Objective: To investigate the effects of aerobic exercise plus spirulina polysaccharide(SP) supplement on the related protein expressions of p75NTR signal in hippocampal and the improvement of learning and memory of type 2 diabetes rats. Methods: The model of type 2 diabetic rats was established by fed high-fat diet for four weeks together with intraperitoneal injecting a low dose of STZ. The model rats were randomly divided into diabetic model group(B),diabetic exercise group(C),diabetic+SP group(D)and diabetic exercise+SP group(E), another normal control group(A)without any intervention was set up,12 rats in each group. The rats in Group C and E were treated with intervention of swimming training for six weeks. The rats in Group D and E were treated with SP intragastrically for 6 weeks. Learning and memory abilities were observed by Morris water maze test. The hippocampus cell apoptosis was observed by Tunnel staining, and BDNF content and the expressions of p75NTR, cleaved caspase-3 of hippocampus were tested by ELISA, Western blot and immunohistochemistry, respectively. At the same time, the changes of blood glucose and levels of serum insulin were examined. Results: ①Compared with Group A at different time points, the body weight of Group B was decreased significantly(P<0.01). Compared with Group B at different time points, the body weight of Group C,D and E had no difference (P>0.05). Compared with Group A, levels of the blood glucose and serum insulin Group B were increased significantly(P<0.01).Compared with Group B, the levels in the intervention groups were decreased significantly (P<0.01); ②Compared with Group A, the escape latencies of Group B were prolonged significantly(P<0.01), platform quadrant residence duration and the times of crossing platform were decreased (P<0.01). Compared with Group B, the escape latencies of the intervention groups were shortened (P<0.05 or P<0.01), and the times of crossing platform were increased (P<0.05 or P<0.01). ③Compared with Group B, the neural cells apoptosis of the intervention rats was decreased, and the protein expressions of p75NTR and cleaved caspase-3 were decreased (P<0.05 or P<0.01), however the expression of BDNF was increased significantly (P<0.05 or P<0.01). Conclusion: Aerobic exercise and SP supplement can improve the learning-memory ability of type 2 diabetes rats, and the improvement effect of exercise combined with SP is markedly better than that of exercise and SP alone, the mechanism might be related to better regulating p75NTR signal related protein expressions and then inhibiting apoptosis in hippocampus of rats with type 2 diabetes.
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    Effects of long-term moderate-small intensity aerobic exercise on differential expression of proteome in left ventricular muscle of rats
    GUO Yuan-pan, SHI Shao-rong, XU Zhe, WANG Juan, HUANG Si-min, WAN Li-li, LEI Xiong, PENG Yong
    CJAP. 2021, 37 (6): 673-677.   DOI: 10.12047/j.cjap.6081.2021.091
    Abstract   PDF (1298KB) ( 0 )
    Objective: To investigate the effects of long-term moderate-small intensity aerobic exercise on the differential expression of proteome in left ventricular muscle of rats, and to screen the target proteins sensitive to moderate-small intensity aerobic exercise stimulation. This study will enrich the basic theory of exercise and fitness and provide new ideas and experimental basis for the rehabilitation treatment of chronic cardiovascular disease. Methods: Twenty male SD rats were randomly divided into exercise group and control group (n=10). The treadmill training model of long-term moderate-small intensity aerobic exercise was established, and the whole protein samples of left ventricular muscle were extracted and separated by two-dimensional gel electrophoresis (2-DE). The two-dimensional gel electrophoresis map was analyzed by Bio PD quest image analysis software. The protein spots with differential expression more than 5 times or down-regulated over 80% after exercise were identified by tandem time-of-flight mass spectrometry (ULGRAFL-FLEX-TOF/TOF). Results: Compared with the group C, the heart weight index of the group E was increased by 32.0%, and the difference was significant (P<0.05). Compared with the group C, there were 71 protein spots expression were up-regulated≥2 times or down-regulated≥50% in the group E. 4 protein spots expression were up-regulated≥5 times or down-regulated≥80% were identified by mass spectrometry, 3 proteins and 1 unknown protein were identified. Conclusion: After long-term moderate-small intensity aerobic exercise, the rats heart had a good adaptive change, and the proteome of left ventricular muscle changed significantly. Long-term moderate-small intensity aerobic exercise can effective enhance the ability of myocardial antioxidation.
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    Effects of the combination of tangeretin and whey protein on testosterone and cortisol in sprinters at winter training season
    LIU Meng, MO Shi-wei, QIN Chun-li, LYU Bing-qiang, ZHOU Zhi-qin, GAO Bing-hong
    CJAP. 2021, 37 (6): 678-682.   DOI: 10.12047/j.cjap.6100.2021.062
    Abstract   PDF (1011KB) ( 2 )
    Objective: To evaluate the regulation efficacy of oral tangeretin on testosterone and cortisol in sprinters at winter training season. Methods: Twenty-four sprinters were paired and randomly divided into experimental group (EG) and control group (CG). During winter training season, EG were treated with 200 mg tangeretin by oral, and CG were treated with placebo for 4 weeks. Blood samples were collected on the first day of each week (T1, T2, T3, T4) and after the intervention (T5) to detect serum levels of testosterone, cortisol, superoxide dismutase (SOD), and adrenocorticotropic hormone (ACTH). The body composition was tested at T1 and T5. Results: After 4 weeks, ①the serum cortisol level of CG was increased, and the serum levels of testosterone and SOD were decreased significantly (P<0.05). ②In EG, the serum levels of cortisoland ACTH were decreased significantly (P<0.05, P< 0.01), while the serum testosterone level was remained stable, and the level of SOD was increased slightly. ③The muscle mass of EG and CG were increases, but that of EG was increased higher than that of CG. Conclusion: Tangeretin reduces the oxidative stress response that caused by high-intensity exercise during winter training, which maintain the serum testosterone level and inhibit cortisol excessive secretion and promote muscle synthesis.
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    Effects of 12-week Tai Chi exercise on the microvascular reactivity of the middle-aged and elderly patients with mild hypertension and its mechanism
    HU Qing-hua, QIAN You-ling, LIU Xiao-li, ZHU Huan, XIAO Jie-wen
    CJAP. 2021, 37 (6): 683-687.   DOI: 10.12047/j.cjap.6164.2021.080
    Abstract   PDF (951KB) ( 0 )
    Objective: To study the effects of 12-week Taijiquan exercise on the microvascular reactivity of middle-aged and elderly patients with mild hypertension and to explore the mechanisms of microvascular reactivity. Methods: Thirty patients with mild hypertension were divided into exercise group (53.8±6.3 years old) and control group (52.6±7.5 years old). The number and gender ratio of the two groups were the same. The exercise group performed Tai Chi exercise for 12 weeks, and the control group maintained the original lifestyle and did not do other regular sports. The two groups of subjects were tested for microvascular reactivity, blood pressure, serum nitric oxide content, and nitric oxide synthase activity before exercise intervention, 6th week and 12th week, respectively. Results: There was no significant difference in the basic values of each index between the two groups of subjects before the test (P> 0.05). In the 6th week, the microvascular reactivity (blood flow increase rate), systolic blood pressure, diastolic blood pressure, nitric oxide content, nitric oxide synthase activity of the exercise group did not significantly change from the basic value (P>0.05). At the 12th week, the microvascular reactivity, nitric oxide content, c nitric oxide synthase activity were significantly higher than those of the base values and the control group (P<0.05), but the systolic blood pressure and diastolic blood pressure were significantly lower than those of the base values and control group (P<0.05). In the control group, there were no significant changes in the 6th and 12th week values of each index from the basic value (P>0.05). Conclusion: Twelve weeks of Taijiquan exercise can improve the microvascular reactivity of middle-aged hypertensive patients, reduce blood pressure, and increase the nitric oxide content and c nitric oxide synthase of patients. The increase of endogenous nitric oxide production is one of the biological mechanisms of Tai Chi exercise to improve the microvascular responsiveness of hypertension patients.
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    Effect of swimming training on the expression of PKC δ/p66Shc protein in mouse myocardium
    XIE Wen-jie, ZHOU Gang, LI Peng-fei, YANG Fan, AN Jing-fang, LI Hang
    CJAP. 2021, 37 (6): 688-693.   DOI: 10.12047/j.cjap.6160.2021.079
    Abstract   PDF (1333KB) ( 0 )
    Objective: To investigate the effects of different intensity of swimming training on p66Shc protein in mouse myocardium. Methods: Fifty Kunming mice were randomly divided into control group (Group C), weight-bearing swimming group (Group E), weight-bearing swimming + drug group (Group ER), non weight-bearing swimming group (Group P), non weight-bearing swimming + drug group (Group PR), with 10 mice in each group. Group C did not exercise. Groups E, ER, P, and PR received swimming training for 4 weeks. Groups E and ER performed weight-bearing swimming with a 3% body weight, and Group P and Group PR were swimming without weight-bearing, 60 min/d, 6 times/w. Mice in ER and PR groups were injected intraperitoneally with Rottlerin (0.3 mg/kg), a PKCδ inhibitor, before the last two exercises. Groups C, E, and P were injected with the same dose of normal saline. Samples were collected after training finished for 24 hours. The protein expressions of PKCδ, P-PKCδ, P66Shc, P-P66shc and NOX2 were detected by Western blot; PKCδ and P66Shc were detected by immunoprecipitation; malondialdehyde (MDA), reactive oxygen species (ROS) and superoxide dismutase (SOD) in myocardium and serum were analyzed by biochemistry. Results: Compared with Group C, the protein expressions of PKCδ, P-PKCδ, P66Shc, P-P66shc and NOX2 in Group E were increased significantly (P< 0.01), the serum and myocardial MDA levels, myocardial ROS were increased significantly (P<0.05 or P<0.01), and the myocardial SOD activity was decreased (P<0.01), the PKCδ, P-PKCδ, P-P66shc and NOX2 in Group P were increased significantly (P<0.05 or P<0.01), and the myocardial SOD activity was enhanced (P<0.05). Compared with Group E, the protein expressionS of PKCδ (P<0.01), P-PKCδ (P<0.01), P66Shc (P<0.05), P-P66shc (P<0.01), NOX2 (P<0.05) in Group ER was decreased significantly, the protein expression of P66Shc in Group P was decreased significantly (P<0.05), the myocardial MDA (P<0.01) and ROS (P<0.05) were decreased, and the activity of SOD was enhanced (P<0.01). Compared with Group P, the protein expressions of PKCδ, P-PKCδ and P-P66shc in Group PR were decreased significantly (P<0.01), while the expression of NOX2 was increased (P<0.05). Conclusion: Both swimming training of two intensities promoted the increase of PKCδ protein and its phosphorylation in mouse cardiomyocytes. High-intensity swimming training could significantly enhance the expression and phosphorylation level of p66Shc protein, resulting in the production of ROS and the decrease of antioxidant enzyme activity. Low-intensity swimming training enhanced the phosphorylation of p66Shc, but did not promote its protein expression, resulting in the enhancement of myocardial antioxidant capacity and exercise adaptation.
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    Effects of Gui pi tang on myocardial ischemia in rats
    WU Jiang-li, WANG Xiao-ying, DAI Cheng, LI Rui-jia, ZHANG Yue, SUN Jia-huan, LI Ai-ying
    CJAP. 2021, 37 (6): 694-698.   DOI: 10.12047/j.cjap.6099.2021.090
    Abstract   PDF (1223KB) ( 2 )
    Objective: To investigate the effects of Guipitang (GPT) on myocardial ischemic (MI) injury of rats. Methods: Forty male SD rats were randomly divided into five groups as control, model, GPT low-dose and high-dose groups (7.52, 15.04 g/kg), and positive-drug trimetazidine group (2 mg/kg). Rat myocardial ischemia model was induced by feeding high fat forage and intraperitoneal injection of isoprenaline (ISO). After 15 days intragastric administration, rats were injected with ISO once a day for 3 days again. Subsequently, Electrocardiograph (ECG) was examined, serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and glucose (GLU) were detected using an automatic biochemical analyzer. The histopathological alterations of heart were assessed using HE and Masson staining. The protein expressions of Collagen I and Collagen III in heart were evaluated by Western blot. Results: Compared with control group, the electrocardiogram S-T segment of model rats moved down, the serum levels of TC, AST, CK, LDH and GLU in model group were increased significantly (P<0.05), the expressions of collagen I and collagen III in heart were increased (P<0.05), and the hearts were damaged severely. However, no significant changes of TG, HDL-C, LDL-C and ALT were observed (P>0.05). Compared with the model group, the high and low dose groups of GPT and trimetazidine could inhibit the descent of S-T segment, reduced serum TC, AST, CK, LDH and GLU levels (P<0.05), and decreased collagen III expression in heart (P<0.05), and alleviated myocardial pathological damage as well. The high dose group of GPT could decrease the protein expression of collagen I. Conclusion: GPT could improve heart function and alleviate the injury of myocardial ischemia, especially the high lose.
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    TECHNICAL NOTES
    An improved method for isolation and culture of primary cardiomyocytes from SD neonatal rats
    MENG Xiang-hong, ZENG Bin, CHEN Hui-ling, LI Wei-dong, LI Na, XU Xiao-yong
    CJAP. 2021, 37 (6): 699-704.   DOI: 10.12047/j.cjap.6063.2021.085
    Abstract   PDF (1351KB) ( 0 )
    Objective: To establish a stable, rapid and improved method for isolation and culture of primary cardiomyocytes from neonatal rats. Methods: Ventricular tissues from neonatal SD rats were digested with 0.12% collagenase Ⅱ, and then subjected to Percoll density gradient centrifugation. The original cardiomyocytes were cultured in modified DMEM/F12 containing 5% horse serum and 5-bromodeoxyuracil(5-BrdU) in vitro for further purification, and medium was changed to normal high glucose DMEM with 10% FBS the next day. The difference between the improved method and traditional differential attachment one used for isolation and culture of primary cardiomyocytes was compared. Results: Cardiacmyocytes obtained through the improved method grew well. 24 hours after plating, most cells adhered to the dishes, with shapes looked triangular, fusiform or irregular, and some of them showed spontaneously contract at a frequency varying from 10~30 times/min. After 48 h culture, the cardiomyocytes became longer and stretched out pseudopodia. Some cells showed synchronous beats with the frequency close to 50~80 times/min. 72 hours later, cardiomyocytes were interwoven into a network in chrysanthemum patterns, and spontaneous beats tended to be more synchronous, with a frequency of 80-100 times/min. After 96 h, cells gathered into clusters as islands, with synchronous beat at a frequency of around 100~120 times/min. All cardiomyocytes were in good condition within one week. Yields((1.17±0.15)×106 vs (1.21±0.22)×106,P>0.05)and survival rate of primary cardiomyocytes obtained by the improved method was comparable to that gained using traditional differential attachment way (93.3%±1.4% vs 92.2%±0.7%, P>0.05), but the purity of primary cardiomyocytes obtained through the improved method was much higher (94.7%±2.1% vs 89.5%±1.3%, P<0.05), while with less time consuming ((3.1±0.4)h vs (4.3±0.3)h, P<0.01). Conclusion: This improved method is an ideal and simple method for the isolation and culture of primary cardiomyocytes with shorter time-consuming, high purity, intact structure and function, and with great repeatability and stability.
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  2019-03-15

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Chinese Association for Physiological Sciences Academy of Military Medical Sciences Institute of Health and Environmental Medicine
Associate Sponsor
Institute of Basic Medical Sciences
Editor in Chief
WANG Hai
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Editorial Board,Chinese Journal of Applide Physiology;Dali Dao,Tinanjin 300050,China



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