Objective: To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro. Methods: The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments. (1) The cells were divided into 6 groups according to the random number table (the same grouping method below), i. e., normal control 3, 6, and 9 h groups and ischemia-hypoxia 3, 6, and 9 h groups, with 4 wells in each group. After being regularly cultured for 48 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), the cells in normal control 3, 6, and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3, 6, and 9 h, respectively, and the cells in ischemia-hypoxia 3, 6, and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3, 6, and 9 h, respectively. Cell viability was measured by the cell counting kit 8 (CCK-8) method. (2) The cells were grouped and treated the same as those in experiment (1), with 1 well in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3Ⅱ, p62, and hexokinase Ⅱ. (3) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, and ischemia-hypoxia 9 h+ 2-deoxyglucose (2-DG) group, with 4 wells in each group. After a regular culture for 48 h, the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h; the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium, and the cells in ischemia-hypoxia 9 h+ 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 μmol), and then they were cultured with hypoxia for 9 h. Cell viability was measured by CCK-8 method. (4) The cells were grouped and treated the same as those in experiment (3), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, and p62. (5) The cells were grouped and treated the same as those in experiment (3), with 2 wells in each group. Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes. (6) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ hexosinase Ⅱ small interfering RNA1 (HK-ⅡsiRNA1) group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group, with 4 wells in each group. The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h, and the cells in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then also cultured for 48 h. The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h, and the cells in simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h. Cell viability was measured by CCK-8 method. (7) The cells were grouped and treated the same as those in experiment (6), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, p62, and hexokinase Ⅱ. Except for experiment (5), each experiment was repeated 3 times. Data were processed with one-way analysis of variance and lest significant difference t test, and Bonferroni correction. Results: (1) The viabilities of cardiomyocytes in ischemia-hypoxia 3, 6, and 9 h groups were 0.450±0.022, 0.385±0.010, and 0.335±0.015, respectively, which were significantly lower than 0.662±0.026, 0.656±0.028, and 0.661±0.021 of the corresponding normal control 3, 6, and 9 h groups, respectively (t=6.21, 9.12, 12.48, P<0.01). (2) Compared with those of corresponding normal control 3, 6, and 9 h groups, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3, 6, and 9 h groups were significantly increased (t_{3 h}=16.15, 10.99, 5.30, t_{6 h}=6.79, 10.42, 9.42, t_{9 h}=15.76, 16.51, 7.20, P<0.05 or P<0.01). (3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353±0.022, which was significantly lower than 0.673±0.027 of normal control group (t=9.29, P<0.01). The viability of cardiomyocytes in ischemia-hypoxia 9 h+ 2-DG group was 0.472±0.025, which was significantly higher than that of simple ischemia-hypoxia 9 h group (t=3.60, P<0.05). (4) Compared with those of normal control group, the LC3Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=9.45, 8.40, P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group were significantly decreased (t=4.39, 4.74, P<0.05). (5) In cardiomyocytes of normal control group, only single autophagosome/autolysosome with bilayer membrane structure was observed. Compared with that of normal control group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly. Compared with that of simple ischemia-hypoxia 9 h group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group was significantly decreased. (6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358±0.023, which was significantly lower than 0.673±0.026 in normal control group (t=9.12, P<0.01). The viabilities of cardiomyocytes in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were 0.487±0.027 and 0.493±0.022, respectively, which were significantly higher than the viability in simple ischemia-hypoxia 9 h group (t=3.63, 4.28, P<0.05). (7) Compared with those of normal control group, the LC3Ⅱ/Ⅰratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=6.08, 6.31, 4.83, P<0.05 or P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were significantly decreased (t=5.10, 7.76, 15.33, 4.17, 8.42, 12.11, P<0.05 or P<0.01). Conclusions Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro, which decreases the viability of cardiomyocytes by impairing autophagic flow. To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.

Objective: To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro. Methods: The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased (t=12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P<0.05 or P<0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased (t=6.88, 10.56, 5.76, 9.91, P<0.05 or P<0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased (t=6.01, P<0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups (F=1.09, P>0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups (t=43.05, 11.07, 5.39, P<0.05 or P<0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups (t=11.18, 12.71, P<0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased (t=4.82, 4.44, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased (t=4.39, 6.27, P<0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group (t=10.13, P<0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=3.28, P<0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group (t=4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P<0.05 or P<0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased (t=5.51, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=5.97, P<0.05). Conclusions After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.

Objective: To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro. Methods: The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco′ s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t_{3 h}=4.891, 5.890, 4.928; t_{6 h}=9.790, 6.750, 10.590; t_{9 h}=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01). Conclusions TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.

Objective: To explore the effects of cardiac support on delayed resuscitation in extensively burned patients with shock. Methods: Clinical data of 62 extensively burned patients with shock on admission, admitted to the 159th Hospital of PLA (hereinafter referred to as our hospital) from January 2012 to January 2017, were retrospectively analyzed. They were divided into cardiac support group (n=35) and control group (n=27) according to the use of deslanoside and ulinastatin. All patients were treated with routine fluid resuscitation based on the formula of the Third Military Medical University till post injury hour (PIH) 48. Patients in cardiac support group were given slow intravenous injection of deslanoside which was added in 20 mL 100 g/L glucose injection with first dose of 0.4 to 0.6 mg, 0.2 to 0.4 mg per 6 to 8 h, no more than 1.6 mg daily, and slow intravenous injection of 1×10⁵U ulinastatin which was added in 100 mL 50 g/L glucose injection, once per 12 h. Other treatments of patients in the two groups followed the same conventional procedures of our hospital. The following data of the two groups of patients were collected. (1) The data of urine volume per hour within PIH 48, heart rate, mean arterial pressure (MAP), central venous pressure (CVP), blood lactic acid, base excess, hematocrit, and albumin at PIH 48 were recorded. (2) The input volumes of electrolyte, colloid within the first and second 24 hours post burn and the total fluid input volumes within PIH 48 were recorded. (3) The data of creatine kinase, creatine kinase isoenzyme-MB, lactate dehydrogenase, total bile acid, alanine aminotransferase, aspartate aminotransferase, β₂-microglobulin, urea nitrogen, and creatinine at PIH 48 were recorded. (4) The complications including cardiac failure, pulmonary edema, pleural effusion, seroperitoneum, renal failure, sepsis, and death were also recorded. Data were processed with independent sample ttest, Fisher′s exact test, Pearson chi-square test, or continuous correction chi-square test. Results: (1) There were no statistically significant differences in urine volume within PIH 48, heart rate, MAP, CVP, hematocrit, or albumin at PIH 48 between the patients of two groups (t=0.150, 0.488, 0.805, 0.562, 1.742, 0.696, P>0.05). While the levels of blood lactic acid and base excess were respectively (4.2±2.2) and (-4.3±2.0) mmol/L in patients of cardiac support group, which were significantly better than (5.9±1.7) and (-6.0±3.1) mmol/L in patients of control group (t=3.249, 2.480, P<0.05 or P<0.01). (2) There was no statistically significant difference in input volume of colloid within the first 24 hours post burn between the patients of two groups (t=0.642, P>0.05). The input volume of electrolyte within the first 24 hours post burn, the input volumes of electrolyte and colloid within the second 24 hours post burn, and the total fluid input volume within PIH 48 of patients in cardiac support group were significantly less than those in control group (t=2.703, 4.223, 3.437, 2.515, P<0.05 or P<0.01). (3) The levels of creatine kinase, creatine kinase isoenzyme-MB, lactate dehydrogenase, total bile acid, alanine aminotransferase, aspartate aminotransferase, β₂-microglobulin, urea nitrogen, and creatinine of patients in cardiac support group at PIH 48 were significantly lower than those in control group (t=3.066, 3.963, 3.225, 2.943, 2.431, 3.084, 4.052, 2.915, 3.353, P<0.05 or P<0.01). (4) The occurrences of pleural effusion and seroperitoneum and mortality of patients in cardiac support group were significantly lower than those in control group (χ²=5.514, 6.984, 4.798, P<0.05 or P<0.01). There were no statistically significant differences in cardiac failure, pulmonary edema, renal failure, and sepsis between the patients of two groups [χ²=1.314 (sepsis), P>0.05]. Conclusions The cardiotonic and cardiac protection treatments in delayed resuscitation of extensively burned patients with shock contribute to improving the cellular anonic metabolism, reducing the volume of fluid resuscitation, and mitigating the ischemic and hypoxic damage to organs, so as to lay foundation for decreasing further complication incidences and mortality.

Objective: To explore the effects of decline of pH value on cardiomyocyte viability of rats, and to analyze the possible mechanism. Methods: Hearts of five newborn Sprague-Dawley rats were isolated, and then primary cardiomyocytes were cultured and used in the following experiments. (1) The primary cardiomyocytes were divided into pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups according to the random number table, with 4 wells in each group. After being routinely cultured for 48 h (similarly hereinafter), cells in pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, and pH 6.0+ 6 h groups were cultured with pH 7.4, pH 7.0, pH 6.5, and pH 6.0 DMEM-F12 medium (similarly hereinafter), respectively, and then they were cultured for 6 h. Cells in pH 6.5+ 1 h and pH 6.5+ 3 h groups were cultured with pH 6.5 medium, and then they were cultured for 1 h and 3 h, respectively. Viability of cells was detected by methyl-thiazolyl-tetrazolium (MTT) method. (2) The primary cardiomyocytes were divided into pH 7.4, pH 6.5, and pH 6.5+ taxol groups according to the random number table, with 2 wells in each group. Cells in pH 7.4 group were cultured with pH 7.4 medium, while cells in pH 6.5 and pH 6.5+ taxol groups were cultured with pH 6.5 medium. Cells in pH 6.5+ taxol group were added with taxol of a final molarity of 0.2 μmol/L in addition, and then they were cultured for 6 h. Morphology and density of microtubule of cells was detected by immunofluorescence assay. (3) The primary cardiomyocytes were grouped and treated as in experiment (2), with 2 wells in each group. The expressions of polymerized microtubulin and free microtubulin were determined with Western blotting. (4) The primary cardiomyocytes were grouped and treated as in experiment (2), with 4 wells in each group. Viability of cells after treated with taxol was detected by MTT method. Data were processed with one-way analysis of variance and LSD-t test. Results: (1) The viability of cells in pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups were 1.00±0.08, 0.90±0.08, 0.85±0.06, 0.83±0.04, 0.91±0.10, and 0.89±0.10, respectively. Compared with that in pH 7.4+ 6 h group, viability of cells in pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups were all decreased in different degrees (t=2.476, 4.002, 4.996, 2.168, 2.400, P<0.05). (2) Microtubules of cells in pH 7.4 group were radially distributed around the nucleus with clear tubular structure. Compared with that in pH 7.4 group, the skeleton of microtubules of cells in pH 6.5 group was obviously damaged, with broken structure of microtubule and reduced density. Compared with that in pH 6.5 group, the damage degree of microtubules of cells in pH 6.5+ taxol group was obviously alleviated, and the structure of microtubules basically returned to normal. (3) Compared with that in pH 7.4 group, the expression of free microtubulin of cells in pH 6.5 group was significantly increased (t=3.030, P<0.05), while the expression of polymerized microtubulin of cells was significantly decreased (t=8.604, P<0.05). Compared with that in pH 6.5 group, the expression of free microtubulin of cells in pH 6.5+ taxol group was significantly decreased (t=4.559, P<0.05), while the expression of polymerized microtubulin of cells was significantly increased (t=5.472, P<0.05). (4) Viability of cells in pH 7.4, pH 6.5, and pH 6.5+ taxol groups were 1.00±0.10, 0.83±0.04, and 0.93±0.10, respectively. Compared with that in pH 7.4 group, the viability of cells in pH 6.5 group was obviously declined (t=4.412, P<0.05). Compared with that in pH 6.5 group, the viability of cells in pH 6.5+ taxol group was obviously increased (t=2.461, P<0.05). Conclusions The decline of pH value reduces the viability of cardiomyocytes of rats through destroying the skeleton of microtubule. Stabilizing microtubule skeleton can significantly reduce acidic treatment-induced damage and ameliorate cardiomyocyte viability.

Objective To investigate influence of nicotinic acid adenine dinucleotide phosphate (NAADP) on autophagy in hypoxic cardiomyocytes of rats and its mechanism.Methods Five neonatal Sprague-Dawley rats were collected and sacrificed to harvest the hearts,and primary cardiomyocytes were separated for the following experiments.(1) Primary cardiomyocytes were collected and divided into normoxia group,hypoxia 9 h group,and hypoxia 9 h + NAADP group according to random number table,with 5 wells in each group.Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours.Cells in hypoxia 9 h group and hypoxia 9 h + NAADP group were cultured in hypoxic incubator with volume fraction 94％ nitrogen,5％ carbon dioxide,and 1％ oxygen for 9 hours.Before hypoxia,cells in hypoxia 9 h + NAADP group were dealt with final amount-of-substance concentration 10 μmol/L NAADP.Cell counting kit 8 was used to measure cell viability.(2) Primary cardiomyocytes were collected and divided into normoxia group,hypoxia 9 h group,hypoxia 9 h + NAADP group,hypoxia 9 h + tran-Ned-19 group,and hypoxia 9 h + trans-Ned-19 + NAADP group according to the random number table,with 2 wells in each group.Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours.And cells in the other 4 groups were cultured in hypoxic incubator as that in experiment (1) Before hypoxia,cells in hypoxia 9 h + NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP,cells in hypoxia 9 h + tran-Ned-19 group were dealt with amount-of-substance concentration 1 μmol/L trans-Ned-19,and cells in hypoxia 9 h + trans-Ned-19 + NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP and 1 μmol/L trans-Ned-19.Protein expressions of microtubule associated protein 1 light chain 3-Ⅱ and P62 were detected by Western blotting.(3) Primary cardiomyocytes were collected and grouped as those in experiment (1).The lysosomal acidity was determined by immunofluorescence method.Data were processed with one-way analysis of variance and least-significant difference test.Results (1) The cell viability in normoxia group was 1.114 ± 0.024,which was significantly higher than 0.685 ± 0.079 of cells in hypoxia 9 h group (P ＜ 0.01).The cell viability of hypoxia 9 h + NAADP group was 0.886 ± 0.061,which was obviously higher than that of cells in hypoxia 9 h group (P ＜0.05).(2) Expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 of cells in hypoxia 9 h group were significantly higher than those of cells in normoxia group (P ＜ 0.0l).Compared with those in hypoxia 9 h group,expression of P62 in hypoxia 9 h + NAADP group was significantly decreased (P ＜ 0.01),while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (P ＞ 0.05).There were no significantly statistical difference in expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 between hypoxia 9 h group and hypoxia 9 h + trans-Ned-19 group (P ＞ 0.05).Compared with those of cells in hypoxia 9 h + NAADP group,expression of P62 of cells in hypoxia 9 h + trans-Ned-19 + NAADP group was obviously increased (P ＜ 0.01),while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (P ＞ 0.05).(3) The intensity of green fluorescence of cells in normoxia group was strong and co-localized well with red fluorescence,and internal environment of lysosome was with stronger acidity.The intensity of green fluorescence in cells of hypoxia 9 h group was significantly lower than that of cells in normoxia group,and acidity of internal environment of lysosome was weakened.The intensity of green fluorescence and acidity of internal environment of lysosome in hypoxia 9 h + NAADP were significantly stronger than those of cells in hypoxia 9 h group,but significantly lower than those of cells in normoxia group.Conclusions NAADP can improve myocardial cell viability through acidifying internal environment of lysosome of cardiomyocyte after hypoxia,promoting degradation of autophagosomes,reducing autophagic lysosomal accumulation,and repairing damaged autophagie flow.

Objective: To survey the prevalence of drug resistant HIV-1 in Shandong province in 2013-2015. Methods: WHO truncated sequential sampling technique was adopted by using 77 and 53 samples of newly diagnosed as HIV-1 positive and aged 16-25 years in Shandong province in 2013 and 2015. RNA was prepared and HIV-1 pol region was amplified by RT-PCR and nested PCR. Pol genetic mutation associated with drug resistance was analyzed. Results: The success rates for sequence acquisition of the survey were 100% (77/77) and 94% (50/53) in 2013 and 2015, and the main subtype was CRF01_AE. A total of 2 surveillance drug-resistance mutation(SDRMs) and 3 SDRMs were found by analyzing the 47 sequences each year, sampled in 2013 and 2015, indicating that the prevalence of drug resistant HIV-1 stains was low in 2013, and moderate in 2015. A total of 5 individuals with drug resistant HIV-1 stains found in this study were mainly infected by homosexual transmission (3 cases), and the other two samples were different: one was infected by heterosexual transmission, the other was infected by IDU. The subtype was CRF01_AE (2 cases) , CRF07_BC (2 cases) and B (1 case) . SDRMs for protease inhibitor (PIs), nucleotide HIV-reverse transcriptase inhibitor (NRTIs) and non-NRTI (NNRTIs) were all found in the individuals with drug resistant HIV-1 stains. Conclusion CRF01_AE were the main HIV-1 subtypes of recently reported HIV-infected individuals in Shandong province, and the HIV-1 drug resistant strains transmission was catalogued as at low and moderate prevalence level in 2013 and 2015.

OBJECTIVETo retrospectively analyze the risk factors and clinical manifestations of myocardial damage of patients with severe burn in order to provide evidence for its prevention and treatment.

METHODSTwo hundred and fifty-two patients with severe burn admitted to 5 burn centers from January 2010 to June 2015, conforming to the study criteria, were treated in accordance with the fluid resuscitation formula of the Third Military Medical University. According to the creatine kinase isoenzyme-MB (CK-MB) level before treatment on admission, patients were divided into non-myocardial damage group (n=118, CK-MB level less than 75 U/mL) and myocardial damage group (n=134, CK-MB level higher than or equal to 75 U/mL). Data of patients in two groups were collected and evaluated such as gender, age, body mass, number of patients with chemical burn, admission time after injury, total burn area, full-thickness burn area, number of patients with inhalation injury, levels of haemoglobin, hematocrit, and blood lactate on admission and at post injury hour (PIH) 24 and 48, volumes of urine output and fluid input at PIH 24 and 48, levels of creatinine, urea nitrogen, total bile acid, diamine oxidase on admission and at PIH 24 and 48, and mortality. Furthermore, patients were divided into three groups, i. e. less than 50% total body surface area (TBSA) group (n=110), larger than or equal to 50% TBSA and less than 80% TBSA group (n=83), and larger than or equal to 80% TBSA group (n=59) according to the total burn area, and the incidence rates of myocardial damage in patients of three groups were recorded. Data were processed with chi-square test, t test, Wilcoxon test, analysis of variance for repeated measurement, and the values of P were adjusted by Bonferroni. Basic data of 252 patients were processed with binary logistic regression analysis. Receiver operating characteristic curve of total burn area of 252 patients was drawn to predict myocardial damage.

RESULTS(1) There were no statistically significant differences in age, body mass, number of patients with chemical burn, number of patients with inhalation injury, and full-thickness burn area between two groups (with t values respectively 0.20 and 0.31, χ(2) values respectively 0.49 and 4.10, Z=1.42, P values above 0.05). There were statistically significant differences in gender, admission time after injury, and total burn area of patients between two groups (χ(2)=5.00, with t values respectively 2.44 and 3.13, P<0.05 or P<0.01). (2) Gender, admission time after injury, and total burn area were independent risk factors related to myocardial damage in the patients (with odds ratios respectively 2.608, 3.620, and 1.030; 95% confidence intervals respectively 1.315-5.175, 1.916-6.839, and 1.011-1.049; P values below 0.01). (3) The incidence rates of myocardial damage of patients in less than 50% TBSA group, larger than or equal to 50% TBSA and less than 80% TBSA group, and larger than or equal to 80% TBSA group were 38.2% (42/110), 54.2% (45/83), and 61.0% (36/59) respectively, and there was statistically significant difference among them (χ(2)=9.46, P<0.05). (4) The total area under receiver operating characteristic curve of total burn area to predict myocardial damage of 252 patients was 0.706 (with 95% confidence interval 0.641-0.772, P<0.01), and 51.5% TBSA was chosen as the optimal threshold value, with sensitivity of 62.6% and specificity of 65.3%. (5) Compared with those in non-myocardial damage group, except the levels of haemoglobin and hematocrit at PIH 48 (with t values respectively -0.76 and -0.61, P values above 0.05), the levels of haemoglobin, hematocrit, and blood lactate of patients in myocardial damage group were significantly increased at each time point (with t values from -2.80 to -2.06, P<0.05 or P<0.01). Compared with those in non-myocardial damage group, the volume of urine output of patients was significantly declined (with t values respectively 2.05 and 3.68, P<0.05 or P<0.01), while the volume of fluid input of patients was not obviously changed in myocardial damage group at PIH 24 and 48 (with t values respectively 1.01 and 1.08, P values above 0.05). (6) Compared with those in non-myocardial damage group, the level of creatinine of patients was significantly increased on admission and at PIH 24 and 48 (with Z values from -2.91 to -1.99, P<0.05 or P<0.01), the level of urea nitrogen of patients was only significantly increased at PIH 24 and 48 (with t values respectively -4.75 and -5.24, P values below 0.01), the level of total bile acid of patients was not obviously changed on admission and at PIH 24 and 48 (with t values from -0.81 to -0.20, P values above 0.05), and the level of diamine oxidase of patients was only significantly increased on admission and PIH 24 in myocardial damage group (with t values respectively -3.97 and -2.02, P<0.05 or P<0.01). (7) Compared with that in myocardial damage group, the mortality of patients in non-myocardial damage group was significantly declined (χ(2)=5.81, P<0.05).

CONCLUSIONSPatients with severe burn have high incidence of myocardial damage, which may be predicted by total burn area. Severely burned patients with myocardial damage are more likely to suffer from decline of effective circulating volume, tissue oxygenation disorders, and damage in other organs in shock stage.

Body Surface Area ; Burn Units ; Burns ; pathology ; Fluid Therapy ; Hematocrit ; Hemoglobins ; analysis ; Humans ; Lactic Acid ; blood ; Myocardium ; pathology ; Retrospective Studies ; Shock

Objective To study the dynamic change of polysaccharide from Asarum insigne in Guizhou during different harvest period and its hemostatic effect. Methods Asarum insigne polysaccharide was extracted by water isolation and alcohol precipitation. We measured the polysaccharide content by UV spectrophotometry after impurity and purification and detected the bleeding and clotting time by tail cutting and slide methods in mice. Results There was significant variation in polysaccharide content of Asarum insigne at different harvest time, which was at a higher level in June(1. 78%-1. 82%). The bleeding time in mice of normal control group was (6. 73±1. 21) min,and that in mice treated with refined polysaccharide at high dose was (4. 91±1. 58) min,the difference between two groups was statistically significant(P<0. 01). The clotting time in mice of normal control group was (7. 27±2. 09) min,and that in the refined polysaccharide at middle and high dose groups was (3. 96±1. 78) min and (3. 27±1. 61) min,respectively. The latter two groups were obviously different from the normal control group(P<0. 05 or P<0. 01). Conclusion The polysaccharide is an active hemostatic substance in Asarum insigne and the optimum harvest time of it is in June for the clinical use.

OBJECTIVETo analyze survival time of AIDS death cases receiving Antiretroviral Therapy and related factors.

METHODSA retrospective cohort study was carried out to collect the data on death cases receiving Antiretroviral Therapy by the National HIV/AIDS Comprehensive Response Information Management System. Kaplan-Meier was used to calculate the median survival time, and compare survival time among different groups of age, sex, marriage status, infectious routes, WHO clinical stage, baseline CD4(+)T cell counts, and interval time from the start of ART to HIV confirmation. Life table and survival curve were applied to describe survival distribution. Cox proportional hazard model was used to determine the factors associated with the survival time.

RESULTSAmong 142 AIDS death cases, 125 (88.03%) were related with AIDS and 17(11.97%) were not. The total median survival time was 3.100 months (95%CI: 2.279-3.921). The cumulative survival rate was (52 ± 4)%, (33 ± 4)%, (26 ± 4)% in the first 3 months, 3-6 months, and 6-12 months. The median survival time of married or cohabitation group was 2.670 months (95%CI:1.470-3.870), and single (unmarried, divorced, separation, widowed) group was 5.870 months (95%CI: 2.617-9.123). The median survival time of WHO clinical stage I or II group was 5.870 months (95%CI: 3.989-7.751), and WHO clinical stage III or IV group was 1.700 months (95%CI: 0.885-2.515). The median survival time of baseline CD4(+)T cell counts ≤ 50 /µl group was 1.670 months (95%CI: 0.759-2.581), and 51-199 /µl group was 4.400 months (95%CI: 2.735-6.065), and ≥ 200/µl group was 7.100 months (95%CI: 0.000-14.542). The survival time was significantly different among different baseline marital status groups, different WHO clinical stage groups, and different CD4(+)T cell counts groups. The mortality risk of Single (unmarried, divorced, separation, widowed) group was 0.641 times of the risk in married or cohabitation group. The mortality risk of WHO clinical stage III or IV was 1.856 times of the risk in stage I or II. The mortality risk of baseline CD4(+)T cell counts 51-199 /µl group was 0.582 times of the risk in ≤ 50 /µl group, and ≥ 200 /µl group was 0.551 times of the risk in ≤ 50 /µl group.

CONCLUSIONThe total median survival time was relatively short. Most AIDS deaths happened in the first 3 months or 3-6 months after they received Antiretroviral Therapy, and the mortality trend slowed down in the following months. Married or cohabitation, low-baseline CD4(+)T cell counts, or WHO clinical stage III or IV were found to be the risk factors associated with AIDS death cases receiving Antiretroviral Therapy.

Acquired Immunodeficiency Syndrome ; Antiretroviral Therapy, Highly Active ; CD4 Lymphocyte Count ; Cohort Studies ; Disease Progression ; HIV Infections ; Humans ; Marital Status ; Proportional Hazards Models ; Retrospective Studies ; Risk Factors ; Survival Rate