Protective Effect of Salidroside on Mitochondrial Disturbances via Reducing Mitophagy and Preserving Mitochondrial Morphology in OGD-induced Neuronal Injury.

Salidroside is the active ingredient extracted from Rhodiola rosea, and has been reported to show protective effects in cerebral ischemia, but the exact mechanisms of neuronal protective effects are still unrevealed. In this study, the protective effects of salidroside (1 µmol/L) in ameliorating neuronal injuries induced by oxygen-glucose deprivation (OGD), which is a classical model of cerebral ischemia, were clarified. The results showed that after 8 h of OGD, the mouse hippocampal neuronal cell line HT22 cells showed increased cell death, accompanied with mitochondrial fragmentation and augmented mitophagy. However, the cell viability of HT22 cells showed significant restoration after salidroside treatment. Mitochondrial morphology and mitochondrial function were effectively preserved by salidroside treatment. The protective effects of salidroside were further related to the prevention of mitochondrial over-fission. The results showed that mTOR could be recruited to the mitochondria after salidroside treatment, which might be responsible for inhibiting excessive mitophagy caused by OGD. Thus, salidroside was shown to play a protective role in reducing neuronal death under OGD by safeguarding mitochondrial function, which may provide evidence for further translational studies of salidroside in ischemic diseases.

In-situ fabrication of zeolite imidazole framework@hydroxyapatite composite for dispersive solid-phase extraction of benzodiazepines and their determination with high-performance liquid chromatography-VWD detection.

A novel zeolite imidazole framework@hydroxyapatite composite (ZIF-8@HAP) was constructed via in-situ growth and developed for efficient dispersive solid-phase extraction (DSPE) of three benzodiazepines from urine samples. The prepared composite was characterized by scanning electron microscopy, energy-dispersive spectrometer, Fourier-transform infrared spectrometry, X-ray diffractometry, zeta potential analyzer, and nitrogen adsorption-desorption experiment. Characterization results showed typical dodecahedron ZIF-8 crystals that were uniformly located on the surface of rod-like HAP. The combination of ZIF-8 and HAP made the surface area significantly enhanced from 4.68 to 205.44 m2 g-1. Compared with a commercial C18 adsorbent, ZIF-8@HAP exhibited superior removal performance for interfering components from urine and offered better extraction properties for the analytes. The prepared ZIF-8@HAP was applied as an adsorbent in DSPE, and the main experimental parameters, including pH and ionic strength of solution, adsorbent amount, adsorption time, elution solvent, and volume, were investigated. Under optimal conditions, the adsorption for 250 ng mL-1 of each analyte in 4 mL of urine was accomplished within 2 min using 60 mg of adsorbent. The method of ZIF-8@HAP-based DSPE followed by high-performance liquid chromatography gave enhancement factors of 13.3-15.3, linear ranges of 2.5-500 ng mL-1, and limits of detection (S/N = 3) of 0.7-1.4 ng mL-1. The relative recoveries at three spiked levels ranged from 88.7 - 102% with intra-day and inter-day precisions from 3.0 - 10.3% and 2.3 - 12.3%, respectively. These results indicated that the proposed strategy had promising applicability for convenient, rapid, and efficient determination of benzodiazepines in urine samples.Graphical abstract In-situ fabrication of ZIF-8@HAP composite for dispersive solid-phase extraction of benzodiazepines in urine samples.

The Association between Epidermal Growth Factor Receptor Single Nucleotide Polymorphisms and Radiochemotherapy Response in Cervical Cancer.

Emerging data reveal that epidermal growth factor receptor (EGFR) single nucleotide polymorphisms (SNPs) can act as efficacy indicators for tumor treatment. Here, the association between EGFR R497K (rs11543848) and -216G/T (rs712829) SNPs and radiochemotherapy response in cervical cancer was investigated. EGFR R497K and -216G/T genotypes were analyzed by polymerase chain reaction-ligation detection reaction in 196 cervical cancer patients receiving radiotherapy alone, or in combination with chemotherapy. Compared with the 497G/G genotype, the A/A genotype significantly increased sensitivity to radiochemotherapy treatment (adjusted OR = 0.244, 95% CI = 0.087-0.680). Sensitivity to radiochemotherapy was not significantly different in carriers of the 'T' allele than that measured for the -216G/G genotype (adjusted OR = 2.412, 95% CI = 0.856-6.979). Additionally, the 497A/A genotype conferred a reduced risk of recurrence or metastasis than did the G/G genotype (adjusted OR = 0.248, 95% CI = 0.078-0.786, P < 0.05). Moreover, carriers of the 'T' allele did not have significantly modified risk of recurrence or metastasis compared with those with the -216G/G genotype (adjusted OR = 1.027, 95% CI = 0.324-3.253). Multivariate analysis revealed an association between clinical stage and treatment response (adjusted OR = 3.575, 95% CI = 1.662-7.692) and between age and the risk of recurrence or metastasis (adjusted OR = 0.319, 95% CI = 0.148-0.691). Our results show that, in patients with cervical cancer, the R497K polymorphism is correlated with treatment response and the risk of recurrence or metastasis. The R497K SNP might be a genetic marker for prediction of radiochemotherapy response and the risk of recurrence and/or metastasis in patients with cervical cancer.

Analysis of cardiovascular disease in Chinese inpatients with chronic kidney disease.

OBJECTIVE: To investigate the prevalence of cardiovascular disease (CVD) in a Chinese patient population with different stages of chronic kidney disease (CKD). METHODS: Six hundred and two CKD patients who were hospitalized in Ruijin Hospital between Jan. 2004 and Jan. 2006 were selected. Patients' medical histories and the results of laboratory tests were reviewed. RESULTS: The prevalence of CVD in 602 patients with CKD stages 1 to 5 was 1.28%, 17.24%, 22.86%, 33.33%, 56.2% respectively. The prevalence of CVD in CKD stage 5 patients with dialysis was 78.51%. In all the patients, the prevalence of coronary artery disease (CAD), left ventricular hypertrophy (LVH), and congestive heart failure (CHF) was 8.64% (52/602), 26% (154/602), and 13% (78/602), respectively. Regarding co-morbidities of CVD, 34.52% of patients had 2 or more of the above abnormalities. The prevalence of CAD in patients with CKD stages 1 to 5 respectively was 1.28%, 5.75%, 7.86%, 10.26%, 12.33%;LVH was 0%, 11.49%, 16.43%, 29.49%, 44.75%; and CHF was 0%, 3.45%, 3.57%, 8.97%, 28.77%. CONCLUSION: The occurrence of CVD started from CKD stage 1 and increased with the progression of CKD. The screening and prevention of CVD should begin at CKD stage 1.

[Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study].

OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) targeting the polymerase (P) gene sequence of hepatitis B virus (HBV) on the replication and antigen secretion of HBV. METHODS: From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 cells. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfection so as to observe the inhibitory effects. Untransfected cells and cells transfected with blank pGE-1 vector were used as controls. RESULTS: Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVP1-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2.15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88%, 32.28%, 29.10%, and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50%, 33.41%, and 12.60% respectively. In view of the transfection efficiency of 30%-40%, the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2.15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 (82%). CONCLUSION: siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.

[Expression of SARS-CoV in various types of cells in lung tissues].

OBJECTIVE: To investigate the cell types infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in lung tissues and explore the mechanism of lung injury in SARS. METHODS: In-situ hybridization(ISH) and immunohistochemistry(IHC) double staining was applied to study the lung tissues from 7 SARS cases of Beijing and one of Anhui province. According to SARS-CoV genome sequence, the cDNA probe was synthesized and labelled by digoxin. Immunohistochemically, antibodies of cytokeratin(CK), CD34, CD68, Vimentin and CD3 were applied to demonstrate bronchial epithelial cells, type II pneumocytes, endothelial cells, macrophages, fibroblasts and T cells respectively. RESULTS: The positive results of in-situ hybridization showed that the lung tissues of all cases expressed SARS-CoV RNA, and positive signals displayed in cytoplasms (purple-blue, NBP-BCIP. ISH-IHC double staining showed that positive signals of both ISH (purple-blue NBT-BCIP and IHC (red-brown, AEC expressed in the cytoplasms (purple and red). The positive results of double staining indicated that bronchial epithelial cells, type II pneumocytes, endothelial cells, macrophages, fibroblasts and T lymphocytes were diffusely infected by SARS-CoV. CONCLUSION: This study of ISH-IHC double staining in lung tissues of SARS patients showed that bronchial epithelial cells, type II pneumocytes, endothelial cells, macrophages, T lymphocytes and fibroblasts were attacked diffusely in SARS lungs. Various types of cells damaged by SARS-CoV and inflammatory mediators released by those cells play an important role in the pathogenesis of lung injury in SARS.

[Pathologic study of circulating blood leukocytes in severe acute respiratory syndrome].

OBJECTIVE: To study the pathologic characteristics and pathogenesis of circulating blood leucocytes infected by severe acute respiratory syndrome associated coronavirus (SARS CoV or SCV) in SARS patients. METHODS: Blood samples of 22 SARS patients were studied, and 4 healthy blood samples were observed as negative controls. The white blood cells were collected from whole blood. The ultrastructural characteristics were observed by transmission electron microscopy. CD45RO antibody was used for pre-embedding immunoelectron microscopy. The SARS viral sequence was detected with real-time polymerase chain reaction (RT-PCR). RESULTS: Coronavirus-like particles were founded in the leukocytes in 6 of the 22 blood samples. Five of them gave positive results in the real-time PCR. The number of granulocytes was increased (P < 0.05) and that of lymphocytes was decreased (P < 0.05) respectively. Immunoelectron microscopy showed that CD45RO positive T lymphocyte decreased to 6% - 7%. Circulating lymphocytes had the highest percentage of infection. The morphologic characteristics of coronavirus-like particles were spherical or oval in shape, about 80 - 120 nm in diameter, with a dense round core and a clear halo around the core. A distinct membrane and club-shaped surface projections were seen in the periphery. The particles were located in the cytoplasm, the cisternae of endoplasmic reticulum, Golgi apparatus and vesicles. Virus entered cells by endocytosis or membrane fusion and was released through a budding process. CONCLUSION: Our data suggested that lymphocytes, particularly T cells, were probably the target cells of SARS CoV. The viruses may actively infected the immune cells during SARS CoV acute infection phase and the destruction of target cells may be one of the important reasons for the death of the circulating leukocytes in SARS.