Objective To investigate the different distribution and expression of mammalian target of rapamycin complex (mTORC) in the kidney of diabetic nephropathy (DN) mice.Methods Fourteen eight-week-old male C57BL/6 mice were assigned to 2 groups: the control group ( n=7 ) and the streptozotocin ( STZ )-induced DN group ( n=7 ) . Blood and urinary variables including glucose , albumin, creatinine and albumin/creatinine ratio were assessed 2 weeks after STZ injection.Hematoxylin-eosin staining was performed for renal pathological analyses .The distributions of mTOR , phosph-ser2448-mTOR(p-mTOR), mTORC1(Raptor), mTORC2(Rictor) and phosph-ser240/244-S6K1 (p-S6K1) were determined by immunofluorescence.The expression levels of mTOR, p-mTOR, mTORC1(Raptor), mTORC2(Rictor), S6K1 and p-S6K1 were detected by Western blotting .Results Two weeks after STZ injection , the diabetic mice developed albuminuria (P<0.01) and renal hypertrophy (P<0.05).The immunofluorescence positive staining for mTOR , Raptor, and Rictor was distributed in the epithelial cells of proximal tubules , glomerular mesangium and capillary loops as well as the medullary collecting ducts of the control mouse kidney .These positive signals increased in the DN mouse kidney ( P<0.05).However, pS6K1 was not detected in the inner medulla of control mouse and p-mTOR was not found in the glomeruli of both control and DN mice .Conclusion mTORC is widely expessed in the mouse kidney and participates in the development of DN , whereas the 2448 serine phosphorylation of mTOR may be not implicated in the hyperglycemia mediated glomerular injury .

Objective To study the effect of hypoxial endothelia cell conditional medium(HECCM)on the proliferation of pulmonary arterial muscle cell(PASMC).Methods MTT assay was used to test the proliferation,immuno-cytochemistry was used to identify the expression of ?-SM-actin.Results(1)HECCM could promote the proliferation of PASMC,down-regulate their expression of ?-SM-actin.(2)DDPH could up-regulate the expression of ?-SM-actin in PASMC which was time-dependant.Conclusions DDPH could reverse the phenotype transformation of PASMC exposed to HECCM,the action was time-dependant.After some time DDPH could reversly transform PASMC to the normal contractile phenotype.

Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury. Although melatonin is known as an effective antioxidant, the mechanism of the protection cannot be explained merely by antioxidation. This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron survival. To find it out, an ischemia/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti-proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow cytometry. The cell cycle of the tumor cells was also observed by flow cytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatocarcinoma cells. Incubated with melatonin, chromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G0/S increased but that of G2/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatocarcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Flow Cytometry ; Humans ; Liver Neoplasms ; pathology ; Melatonin ; pharmacology ; Tumor Cells, Cultured

To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj14-Sj26 that contains fatty binding protein (Sj14), GST (Sj26) and paramyocin (Sj97) that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was trans-fected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and pIRES-Sj97-Sj14-Sj26 plasmid DNA, pIRES-Sj14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA,plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh.Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the pIRES-Sj97-Sj14-Sj26 group as compared with the plRES blank vector, normal saline and pIRES-Sj26 groups (P<0.01) and the pIRES-Sj14-Sj26(P<0.05). Single splenocyte suspension was prepared to detected the level of IFN-γ by ELISA and the lymphocyte stimulating index (SI) by MTT SI was significantly higher of in the pIRES-Sj97-Sj14-Sj26 group than in other groups (P<0.01), while the IFN-γ, level was significantly higher the pIRES-Sj97-Sj14-Sj26 group than in pIRES blank vector and normal saline groups (P<0.01), but no significant differences were found when compared with pIRES-Sj14-Sj26 and pIRES-Sj26 groups. Flow cytometery showed that the percent-ages of CD4+ and CD8+ T cells were much higher in the pIRES-Sj97-Sj14-Sj26 group (P<0.01,P<0.05). It was concluded that pIRES-Sj97-Sj14-Sj26 vaccine may induce stronger immune response in BALB/c mice.

Objective To observe the ehanges in plasma OX40L level in patients with stable angina pectoris (SAP) after percutaneous coronary intervention (PCI) and to investigate the relationship between OX40L level and prognosis in patients with SAP.Methods We collected 200 cases of SAP after PCI.Plasma OX40L levels were examined at different time points,including before and after stent placement,in all patients withSAP.The patients with SAP were followed up for 18 months,and the end point was adverse cardiovascular events.Results The plasma OX40L level increased more apparently after PCI than before PCI (P ＜ 0.05) and peaked at 24 hours.We followed up 185 patients successfully,including 39 (21.08％) patients with and 146 (78.92％) without adverse cardiovascular events.The mean maximum OX40L level was higher in patients with than in those without adverse events (P ＜ 0.05).Multivariable logistic regression analysis revealed that the mean maximum OX40L level was related to adverse cardiovascular events (P ＜ 0.05).Conclusion PCI may cause upregulation of plasma OX40L expression.Mean maximum OX40L level showed good clinical predictive value of occurrence of cardiovascular events 18 months after PCI.

To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media. DNA fragmentation, cell viability, cytochrome C releasing, caspase3 activity and mitochondrial transmembrane potential were observed after N2a cells suffered the insults. The results showed that N2a cells in ischemic territory exhibited survival damage, classical cell apoptosis change, DNA ladder and activation of caspase3. Apoptosis-related alterations in mitochondrial functions, including release of cytochrome C and depression of mitochondrial transmembrane potential (deltapsim) were testified in N2a cells after mimic ischemia-reperfusion. Moreover, activation of caspase3 occurred following the release of cytochrome C. However, the inhibitor of caspase3, Ac-DEVD-CHO, couldn't completely rescue N2a cells from apoptosis. Administration of cyclosporine A, an inhibitor of mitochondria permeability transition pore only partly inhibited caspase3 activity and reduced DNA damage. Interestingly, treatment of Z-IETD-FMK, an inhibitor of caspase8 could completely reverse DNA fragmentation, but can't completely inhibit caspase3 activity. It was concluded that there were caspase3 dependent and independent cellular apoptosis pathways in N2a cells suffering ischemia-reperfusion insults. Mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.
Animals ; Apoptosis ; physiology ; Caspase 3 ; Caspases ; biosynthesis ; Cytochromes c ; biosynthesis ; Mice ; Mitochondria ; physiology ; Neuroblastoma ; pathology ; Neurons ; pathology ; Reperfusion Injury ; metabolism ; pathology ; Tumor Cells, Cultured

Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury.Although melatonin is known as an effective antioxidant,the mechanism of the protection cannot be explained merely by antioxidation.This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy,since autophagy has been frequently suggested to play a crucial role in neuron survival.To find it out,an ischemia/reperfusion model in N2a cells was established for examinations.The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reper-fusion onset following ischemia and greatly promoted cell survival,while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT,transmission electron microscopy,and laser confocal scanning microscopy.Besides,the protein levels of LC311 and Beclinl were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin,whereas the expression of p-PKB,key kinase in PI3K/PKB signaling pathway,showed a decrease when compared with untreated subjects as accessed by immunoblotting.Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

Objective:To compare the clinical characteristics and analyze the prognostic factors between human immunodeficiency virus (HIV)-infected patients and non-HIV-infected immunocompromised patients with pneumocystis pneumonia (PCP) complicated with acute respiratory failure (ARF) in intensive care unit (ICU).Methods:The clinical data of patients with PCP complicated with ARF admitted in ICU of The First Affiliated Hospital of Zhengzhou University and The Sixth People′s Hospital of Zhengzhou City between May 2018 and October 2020 were retrospectively reviewed. All subjects were divided into HIV-infected group and non-HIV-infected immunocompromised group. General characteristics and underlying diseases of patients in the two groups were analyzed. Laboratory parameters, treatment and outcomes between two groups were compared. Independent sample t test, Mann-Whitney U test and chi-square test were used for statistical analysis, and univariate and multivariate logistic regression models were used to identify the risk factors for the clinical outcome. Results:A total of 129 PCP complicated with ARF patients were enrolled, including 75 HIV-infected patients and 54 non-HIV-infected immunocompromised patients. Only 10.7%(8/75) patients of HIV-infected group received anti-retroviral therapy (ART), but none of the patients in either groups had previously received trimethoprim-sulfamethoxazole (TMP-SMX) for PCP prophylaxis. Acute physiology and chronic health evaluation (APACHE) Ⅱ score of HIV-infected group was 18.7±6.0, which was higher than that in non-HIV-infected immunocompromised group (13.1±4.4) when admitted in ICU ( t=-5.45, P<0.001). Hypoproteinemia was common in both groups. Ninety-six percent (72/75) of HIV-infected patients had CD4 + T lymphocyte counts lower than 200/μL and 84.0%(63/75) of patients had CD4 + T lymphocyte counts even lower than 50/μL, while 5.74%(31/54) of patients in non-HIV-infected immunocompromised group had CD4 + T lymphocyte counts lower than 200/μL. The CD4 + /CD8 + T lymphocyte counts ratio was 0.05(0.02, 0.12) in HIV-infected group, which was lower than that in non-HIV-infected immunocompromised group (0.96(0.64, 1.44)), and the difference was statistically significant ( Z=-9.16, P<0.001). The length of ICU stay and hospital stay of non-HIV-infected immunocompromised patients were 10.0(7.0, 14.0) days and 18.0(11.8, 32.5) days, respectively, which were both longer than those in HIV-infected patients (7.0(4.0, 9.0) days and 13.0(7.0, 23.0) days, respectively), and the differences were both statistically significant ( Z=-3.58 and -2.73, respectively, both P<0.050). The hospital mortality of HIV-infected patients was 57.3%(43/75), which was significantly higher than that in non-HIV-infected immunocompromised patients (38.9%, 21/54) ( χ2=4.27, P=0.039). Multivariable logistic regression identified that lactic dehydrogenase (LDH), C-reactive protein (CRP) and APACHE Ⅱ score were the risk factors for the clinical outcome of HIV-infected patients (odds ratio ( OR)= 1.006, 1.015 and 1.736, respectively, all P<0.050). The partial pressure of oxygen in arterial blood/fractional concentration of inspiratory oxygen (PaO 2/FiO 2), LDH and CD4 + T lymphocyte counts were the risk factors for the clinical outcome of non-HIV infected immunocompromised patients ( OR=0.970, 1.008 and 0.989, respectively, all P<0.050). Conclusions:PCP patients with ARF are critically ill with high mortality rate. LDH, CRP and APACHEⅡscore are predictors for prognosis of HIV-infected patients with PCP, while PaO 2/FiO 2, LDH and CD4 + T lymphocyte counts are predictors for prognosis of non-HIV infected immunocompromised patients with PCP.