BACKGROUND:As a common complication of diabetes mellitus,male reproductive disorders have received increasing attention in recent years.Ganoderma spore have hypoglycemic,antioxidant and anti-inflammatory effects,but the regulatory mechanism for diabetic testicular tissue has not been fully elucidated. OBJECTIVE:To investigate the effect of ganoderma spore on the PTEN-induced kinase 1/E3 ubiquitin ligase pathway and cell apoptosis in testicular tissue of diabetic rats. METHODS:Forty male Sprague-Dawley rats were randomly divided into normal group,high fat and high sugar group,diabetic group and ganoderma spore group,with 10 rats in each group.The latter three groups were given high fat/high sugar diet until the end of the experiment.After 1 month of high fat/high sugar diet,the diabetic and ganoderma spore groups were given intraperitoneal injection of streptozotocin(30 mg/kg per day)to establish type 2 diabetic rat models.After successful modeling,the ganoderma spore group was intragastrically given ganoderma spore(300 mg/kg per day),and the other groups were given the same amount of normal saline for continuous 12 weeks.The sperm number and morphology were detected.The histopathological changes of the testicle were observed.Serum testosterone and oxidative stress levels in testicular tissue were measured.The levels of PTEN-induced kinase 1,E3 ubiquitin ligase,and anti-nucleoporin p62 were analyzed by immunohistochemistry and the expression of PTEN-induced kinase 1,E3 ubiquitin ligase,anti-nucleoporin p62,programmed cell death-1,microtubule-associated protein light chain 3 Ⅱ/Ⅰ,caspase 3,cleaved-caspase 3 were detected by western blot assay. RESULTS AND CONCLUSION:Compared with the normal group and the high fat and high sugar group,the diabetic group had decreased sperm number(P＜0.01),increased sperm malformation rate(P＜0.01),and decreased serum testosterone level(P＜0.01).Compared with the diabetic group,ganoderma spore intervention could increase the sperm number(P＜0.05),decrease the malformation rate(P＜0.01),and increase the serum testosterone level(P＜0.01).Compared with the normal group and the high fat and high sugar group,the malondialdehyde level in testis tissue was increased in the diabetic group(P＜0.01),while the levels of glutathione deoxidase and superoxide dismutase decreased(P＜0.01).Compared with the diabetic group,the malondialdehyde level in testis tissue was decreased in the ganoderma spore group(P＜0.01),and the levels of glutathione deoxidase and superoxide dismutase increased(P＜0.01).Immunohistochemical staining showed that compared with the normal group and the high fat and high sugar group,the positive expressions of PTEN-induced kinase 1 and E3 ubiquitin ligase in testicular tissue were decreased in the diabetic group,while the positive expressions of anti-nucleoporin p62 were increased.Compared with the diabetic group,the positive expressions of PTEN-induced kinase 1 and E3 ubiquitin ligase in testicular tissue e were increased in the ganoderma spore group,while the positive expression of anti-nucleoporin p62 was decreased.Western blot assay results indicated that compared to the normal group and the high fat and high sugar group,the expression of PTEN-induced kinase 1 and E3 ubiquitin ligase,programmed cell death-1 and the ratio of microtubule-associated protein light chain 3 Ⅱ/Ⅰ protein were decreased in the diabetic group(P＜0.05 or P＜0.01),while the expressions of anti-nucleoporin p62,caspase3 and cleaved-caspase3 were increased(P＜0.01).Compared with the diabetic group,ganoderma spore intervention could elevate the expression of PTEN-induced kinase 1 and E3 ubiquitin ligase,programmed cell death-1 and the ratio of microtubule-associated protein light chain 3 Ⅱ/Ⅰ protein(P＜0.05 or P＜0.01)as well as reduce the expressions of anti-nucleoporin p62,caspase3 and cleaved-caspase3(P＜0.05 or P＜0.01).Overall,ganoderma spores may activate the PTEN-induced kinase 1/E3 ubiquitin ligase pathway to enhance autophagy in testicular tissue and reduce apoptosis in tissue cells,so as to protect testicular tissue.

Objective To construct models of viral transfection and hypoxia/reoxygenation induced cellular injury in adult mouse cardiomyocytes(AMCMs)isolated using a non-Langendorff method.Methods AMCMs were isolated,extracted,sedimented,and plated using a non-Langendorff method.The morphology and survival rate of the isolated cells were evaluated 2,24,48 and 72 h after plating,and their integrity was observed by immunofluorescence staining for α-actinin.The isolated AMCMs were infected with adenoviruses carrying an RFP-expressing vector and fluorescence images were obtained at 36 and 48 h post-infection and used to calculate transfection efficiency.The cells were cultured under hypoxic conditions for 45 min,reoxygenated for 24 h,and then stained with propidium iodide(PI)to verify establishment of the hypoxia/reoxygenation injury model.Results The survival rates of AMCMs at 2,24 and 48 h after plating were comparable,but survival was significantly reduced at 72 h.The integrity of the AMCMs was good and＞80%of the cells were transfected with adenovirus at 48 h.After hypoxia/reoxygenation treatment,42%of cells were stained by PI,suggesting successful establishment of the AMCM injury model.Conclusions In this study,we developed a non-Langendorff method for the fast and easy isolation of AMCMs with high cell viability.The isolated cells can be efficiently infected with adenovirus and respond to hypoxia/reoxygenation injury.These findings provide a systematic method for isolating AMCMs and for applying gene modification and hypoxia/reoxygenation injury in these cells.

Objective:The aims of the study were to investigate the relationship among atherogenic index of plasma (AIP) and inflammatory adipocytokines with the severity of coronary artery calcification (CAC) score in coronary artery disease (CAD). And then we analyzed the diagnostic value of the new markers on CAC.Methods:A total of 241 patients with CAD diagnosed by coronary CT angiography (CTA) and coronary angiography in Baoding First Central Hospital from June 2019 to June 2020 were retrospectively enrolled. According to the presence of calcification in coronary CTA, they were divided into CAC group ( n=63) and non-CAC group ( n=178). The clinical data of the patients were collected, and the levels of serum inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). The correlation between CAC score and AIP and inflammatory cytokines was analyzed. The diagnostic value of AIP and inflammatory factors in the formation of CAC in patients with CAD. Results:The levels of AIP, serum osteoprotegerin (OPG) and oligomeric matrix protein (COMP) in CAC group were higher than those in non-CAC group, while the levels of serum fibroblast growth factor 21 (FGF21) were lower than those in non-CAC group, with statistically significant difference (all P<0.01). Correlation analysis showed that CAC score of CAD patients was positively correlated with AIP, OPG and COMP ( r=0.581, 0.451, 0.326, P<0.05), and negatively correlated with FGF21 ( r=-0.294, P<0.05). Receiver operating characteristic (ROC) curve analysis showed that AIP, OPG, COMP and FGF21 had diagnostic value for CAC in CAD patients (all P<0.05). AIP>0.387, OPG>5.150 ng/ml, FGF21>136.35 pg/ml, COMP>733.16 ng/ml were independent factors affecting the formation of CAC (all P<0.05). Conclusions:The increase of AIP and the change of inflammatory factors can be used as markers for the diagnosis of CAC formation in CAD patients.

Objective:To explore a new training mode for nursing professionals suitable for the 1+X certificate system, and realize the training goal of "one specialty and multi-ability" compound technical nursing talents.Methods:To take the "1+ X" certificate standard as the basis for the construction of nursing specialty, to reconstruct the talent training program of integration of graduation certificate and certificate. To take the content of "1+X" certificate as the basis for the construction of professional courses, construct the core curriculum system of integration of curriculum and certificate. To build a "new double-qualified" teaching staff and constructing of new double-qualified teachers and accelerate the development of quality resources.Results:The talent training mode of integration of graduation certificate and certificate under the 1+X certificate system was constructed. The curriculum structure has been optimized. The teachers′ability of teaching, training and examination was improved.Conclusions:The new mode of training nursing professionals under the 1+X certificate system meets 1 degree education and X vocational training of nursing students and achieve a "1" and "X" seamless convergence. It provides innovative ideas for the promotion and implementation of 1+X certificate system pilot work in the field of nursing education nationwide.

Objective:To explore the application effect of traditional Chinese medicine comprehensive nursing on elderly patients with coronary heart disease(qi stagnation and blood stasis type) constipation.Methods:A total of 126 elderly patients with coronary heart disease (qi stagnation and blood stasis type) constipation were divided into the control group and the experimental group by random digits table method, 63 cases each; the control group received routine nursing, and the experimental group was given traditional Chinese medicine comprehensive nursing; comparing the two groups of patients defecation, mental state, recurrence rate, and nursing satisfaction.Results:There was no significant difference in defecation frequency, each defecation time, defecation smoothness score, fecal trait score, fall inflated score, and self-Rating Anxiety Scale (SAS) score( P >0.05). After one month of nursing, defecation frequency, each defecation time, defecation smoothness score, fecal trait score, fall inflated score, and SAS score were (4.09±0.86) times/week, (5.12±1.24) min/time, (2.57±0.39) points, (1.53±0.31) points, (0.97±0.21) points, (49.27±4.15)points in the experimental group, (3.12±0.61) times/week, (6.87±1.52) min/time, (2.59±0.36) points, (2.02±0.44) points, (1.29±0.27) points, (54.42±6.01) points in the control group, the differences were significant ( t values were -6.638-7.081, all P<0.05). Within 1 month after nursing, the recurrence rate of constipation in the experimental group (14.3%, 9/63) was lower than that in the control group (74.6%, 47/63) , and the satisfaction rate of the experimental group (98.4%, 62/63) was higher than that in the control group (69.8%, 44/63) , the differences were significant(× 2 values were 46.414, 19.257, P<0.05). Conclusions:Traditional Chinese medicine comprehensive care for elderly patients with coronary heart disease (qi stagnation and blood stasis type) constipation can significantly improve their defecation and psychological state, and can reduce the recurrence rate and improve the patient's satisfaction with nursing, which deserves further clinical application and research.

Objective:To explore the calvarial critical size defect (CSD)in rats with type 2 diabetes mellitus(T2DM).Methods:T2DM model of SD rats(weighted 300-320 g)was induced by high fat and high sugar diet and low dose intraperitoneal streptozotocin (STZ)injection.The rats with T2DMand the normal controls were divided into 4 groups(n=3)respectively.Defects with the diame-ter(mm)of 2,3,4 and 5 were made on the central calvaria of each rat.General observation,X-ray examination and histological study were performed 8 weeks postoperatively.Results:In the T2DM group,only the defects of 2 mm diameter were healed completely,X-ray resistance and new bone formation were observed;the defects of 3,4 and 5 mm diameter were unhealed,X-ray transmission was observed and newly formed bone was insufficient.In the control group,the defects of 2,3 and 4 mm diameter were healed completely, X-ray resistance and new bone formation were observed;the defects of 5 mm diameter were unhealed,X-ray transmission was ob-served,newly formed bone was insufficient.Conclusion:The calvarial CSD of T2DM rat model can be defined as the defect with the diameter of 3 mm.

AIM: To study the mechanism of diabetic cardiomyopathy and abnormality of oxygen free radicals. METHODS: The contents of myocardial cytosolic cytochrome C, mitochondria cytochrome C, mitochondrial calcium, NO, MDA and the activity of SOD and NOS were determined in diabetic rats induced by STZ. The pathological changes were observed under transmission electron microscope. RESULTS: Compared to the normal and ganoderma group, the levels of mitochondrial NO, iNOS, MDA, calcium and plasma Cyt-C in rat myocardium were higher (P<0.05), while mitochondrial Cyt-C and SOD were lowered in model group (P<0.05). The bouncary indistinct, disorganization, a focal loss of muscular fibril, myocardium mitochondria swelling, pulmonary vascular endothelial cellular swelling and obstructed lumen of the capillary were also observed under transmission electronic microscope. CONCLUSION: The findings indicate that oxyradical and lipid peroxidation might be associated with the damage of myocardial mitochondria in NIDDM rats. Cyt-C and mitochondrial calcium is also involved in the process.

BACKGROUND: Varicocele (VC) can induce the infertility in males, so the investigation on its mechanism is important for the treatment of male infertility. OBJECTIVE: To analyze the effects of VC induced by surgical operation on the contents of mitochondria calcium, cytochrome C and cell apoptosis as well as the changes of microstructure and ultrastructure in epididymis. DESIGN: Randomized control experiment. MATERIALS: The experiment was conducted in Jiamusi University between June 2003 and May 2004. Forty male adolescent Wistar rats with the average body mass of (220±20) g were selected, which were provided by the Animal Laboratory of Jiamusi University. Rats were randomly divided into sham-operation group and deligation group with 20 rats in each group at one week after feeding at room temperature. METHODS: Rats in the sham-operation group were made into sham-op eration models by exposing the left renal vein. Rats in the deligation group were deligated of partial left renal veins so as to establish VC models. Bilateral epididymides were removed at ten weeks after operation. The levels of mitochondria calcium in head and body of epididymis as well as the contents of cytochrome C and cytoplasm cytochrome C were detected. The cell apoptosis was detected by in situ terminal deoxynucleotityl transferase mediated dTUP nick end labeling (TUNEL) technique. The specimens of corpus epididymis were routinely made for observation under optimal microscope and electron microscope. The changes of microstructure and ultrastructure of epididymis were studied.MAIN OUTCOME MEASURES: The contents of mitochondria calcium, cytochrome C and cytoplasm cytochrome C. Cell apoptosis. Changes of cellular morphous in epididymis. RESULTS: A total of 40 rats were involved in the analysis of results.① The contents of mitochondria calcium in bilateral epididymis were obviously decreased in the deligation group than the sham-operation group [(4.72±1.45), (5.90±1.97), (10.13±2.34) mg/g, (P ＜ 0.01)].②The content of mitochondria cytochrome C in right epididymis obviously increased more in the deligation group than the sham-operation group [(0.36±0.20), (0.19±0.14), (0.15±0.07) μmol/L (P ＜ 0.05)]. ③The contents of cytoplasm cytochrome C in bilateral epididymis greatly increased more in the deligation group than the sham-operation group [(8.17±1.49), (7.48 ± 1.60), (5.93±1.60) mol/L, (P ＜ 0.05)].④The apoptotic rate of bilateral cells in the deligation group was significantly increased than the sham-op eration group [( 13.3±1.9)%, ( 12.6±1.5)%, (6.2±0.3)%,(P ＜ 0.01 )]. However, there were no significant differences in mitochondria calcium, cytochrome C, cytoplasm cytochrome C and apoptotic rate between the left and right mitochondria of the deligation group (P ＞ 0.05).⑤Main changes under light microscope: cuctus epididymis shrinked, the blebbing appeared in epithelial cells, and the light cells as well as halo cells in epithelia were significantly increased. ⑥Main representation under electron microscope: the cytolysosome inside the chief cells were increased and enlarged with increased residual bodies, and the endoplasmic reticulum expanded, the mitochondria cristae was dim, the Golgi complex was vacuolated. Besides, nuclear chromatin were dense and in lump at different size, which located mainly in the nuclear membrane. The microvilli of columnar epithelial cells were sparse and local defects could be seen. CONCLUSION: The cytochrome C is released to kytoplasm via mitochondrial outer membrane, which activates the caspase 3 and leads to the apoptosis, and accordingly causes excessive apoptosis of epididymal tissues and as well as the changes of microstructure and ultrastructure. All these changes may be one of the important reasons of infertility resulting from VC.

OBJECTIVETo investigate the effects of experimental varicocele on mitochondria calcium and cytochrome C of the epididymal cells in adolescent rats.

METHODSForty male adult Wistar rats were divided into two groups randomly: varicocele group (VG) and sham operation group (SOG) by partial ligation or exposure of the left renal vein. Bilateral epididymides were removed after ten

RESULTSThe content of mitochondria weeks. Mitochondria calcium and cytochrome C levels of the epididymal cells were detected. calcium decreased (P < 0.001 ) while that of cytochrome C increased (P < 0.05) markedly in the experimental group compared with SOG.

CONCLUSIONCalcium dyshomeostasis and mitochondrial damage of the epididymal cells caused by varicocele may play an important role in leading to subfertility.

Animals ; Calcium ; metabolism ; Cytochromes c ; analysis ; Epididymis ; metabolism ; Infertility, Male ; etiology ; Male ; Mitochondria ; metabolism ; Rats ; Rats, Wistar ; Varicocele ; metabolism

AIM: To study the cell apoptosis and the change of microstructure and ultrastructure in epididymis with experimental varicocele (EVC) in rats. METHODS: Experimental varicocele model was induced by partial ligation of the left renal vein in adolescent Sprague-Dawley Wistar rats. Apoptotic cells were detected by in situ terminal deoxynucleotityl transferase-mediated dTUP nick end labeling (TUNEL) technique. The corpus epididymis of the rats was prepared for light and electron microscopic observation. The microstructure and ultrastructure of the epididymis were studied. RESULTS: There was certain proportion of apoptosis cells in epididymis cells in control rats. The incidence of apoptosis increased remarkably in experimental group than that in control group (P