Effects of Hypoxia on the Growth and Development of the Fetal Ovine Hepatocytes in Primary Culture.

OBJECTIVE: To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism. METHODS: Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia (21% O2) or hypoxia (2% O2). The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry (FCM). The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope. RESULTS: The cell purity of hepatocytes was over 95%. Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability. Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm. CONCLUSION: Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics. Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.

Biological Function and Mechanism of Long Noncoding RNAs Nuclear-Enriched Abundant Transcript 1 in Development of Cervical Cancer.

BACKGROUND: Accumulating documents have demonstrated that long noncoding RNAs (lncRNAs) play critical roles in tumorigenesis. As an lncRNA, nuclear-enriched abundant transcript 1 (NEAT1) has been identified to be involved in the progression of many types of cancers. However, the biological function of NEAT1 in cervical cancer is not fully investigated. The aim of this study was to disclose the specific biological function of lncRNA NEAT1 in cervical cancer progression. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to identify the expression of lncRNA NEAT1 in the cervical cancer tissues and cell lines. All cervical cancer samples used in this study were collected from the Affiliated Suzhou Hospital of Nanjing Medical University between September 2012 and September 2017. The correlation between NEAT1 expression and the overall survival rate of cervical cancer patients was analyzed by Kaplan-Meier analysis. The effects of NEAT1 knockdown or overexpression on cell proliferation were tested by performing MTT assays and colony formation assays. Transwell assays were conducted to detect the migratory ability of cervical cancer cells, in which NEAT1 was silenced or overexpressed. Western blotting was utilized to validate whether NEAT1 promotes cervical cancer progression through activating PI3K-Akt signaling pathway. RESULTS: High expression of NEAT1 predicted poor prognosis of cervical cancer patients (χ2 = 0.735, P = 0.005). Knockdown of NEAT1 decreased the number of colonies in CaSki cell from 136.667 ± 13.503 to 71.667 ± 7.506 (t = -18.76, P = 0.003) and decreased the number of colonies in HeLa cell from 128.667 ± 13.317 to 65.667 ± 7.024 (t = -5.54, P = 0.031). However, overexpression of NEAT1 increased the number of colonies in SiHa cell from 84.667 ± 12.014 to 150.667 ± 18.037 (t = 7.27, P = 0.018). Knockdown of NEAT1 decreased the migratory number of CaSki cell from 100.333 ± 9.866 to 58.333 ± 5.859 (t = -8.08, P = 0.015) and reduced the migratory number in HeLa cell from 123.667 ± 12.097 to 67.667 ± 7.095 (t = -6.03, P = 0.026). Overexpression of NEAT1 increased the migratory number of SiHa cell from 127.333 ± 16.042 to 231.333 ± 31.786 (t = 4.92, P = 0.039). CONCLUSION: NEAT1 may exert oncogenic function in cervical cancer and serve as a novel therapeutic target for cervical cancer.

Seasonal Variations in Birth Weight in Suzhou Industrial Park.

Many environmental factors have been shown to adversely influence birth weight, and new insight has been gained into 'seasonal programming'. We studied a total of 23,064 infants. The mean birth weight varied across seasons. Logistic regression analysis was used to obtain the crude and adjusted odds ratios (ORs) for dichotomous outcomes (e.g., macrosomia, low birth weight). There were significant differences in the risks for macrosomia in infants born in different seasons. Compared with those for infants born in spring, the ORs for macrosomia were 0.85 [95% confidence interval (CI): 0.75-0.98] and 0.87 (95% CI: 0.77-0.99) for infants born in summer and autumn, respectively. These findings suggest that environmental factors may have public health implications and should be considered when primary prevention programs are developed for macrosomia or low birth weight.

[The role of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance of hepatocellular carcinoma].

OBJECTIVE: To elucidate intracellular signal pathway in formation of multidrug resistance (MDR) of hepatocellular carcinoma (HCC) induced by its microenvironment, and to explore the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process. METHODS: Activity of ERK/MAPK was examined by Western blot technique through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein in HepG2 cells exposed to hypoxia, low glucose or transfected by plasmid pcDNA3/HBX. After being treated by the specific ERK/MAPK pathway inhibitor U0126, Western blot technique was used to analyze the alterations of the expression of P-gp, MRP1, LRP and HIF-1alpha at protein level. RT-PCR was used to analyze the alterations of the expression of HIF-1alpha mRNA. Cellular location of HIF-1alpha protein was determined by immunocytochemistry after being treated by U0126. RESULTS: The activations of ERK/MAPK determined by the ratio of phosphorylated ERK/MAPK to the total ERK/MAPK were increased in varying degrees in HepG2 cells respectively exposed to different microenvironment. After being treated by U0126 for 12 h, the expressions of mdr1, MRP1, LRP genes and protein in those cells were decreased to some extent. However, the gene expression of HIF-1alpha was not influenced and only its protein was decreased. HIF-1alpha protein was reversely translocated into cytoplasm from nucleus after being treated by U0126. CONCLUSIONS: ERK/MAPK pathway is involved in the course of the formation of MDR of HCC induced by microenvironment.

Chromium(III) complexes of D-glucosaminic acid and their effect on decreasing blood sugar in vivo.

Two chromium(III) complexes of glucosaminic acid were synthesized by neutralization and exchange reaction. The formation of 1 : 1 and 2 : 3 (Cr : glucosaminate) complexes was confirmed by elemental analyses and spectroscopic studies. The effect of the complexes on decreasing blood sugar was investigated on type-2 diabetes model rats induced by tetraoxypyrimidine. The results indicated that the effect on decreasing blood sugar was comparable to that of picolinate chromium complex (Cr(pic)(3)) currently used world wide.

Puerarin reduces increased c-fos, c-jun, and type IV collagen expression caused by high glucose in glomerular mesangial cells.

AIM: Increased expression of c-fos, c-jun and type IV collagen (CoIV) in glomerular mesangial cells (GMC) are important characteristics of diabetic nephropathy. Both c-fos and c-jun regulate the gene expression of extracellular matrix components, and CoIV is the main component of the extracellular matrix. It has been reported that puerarin inhibits aggregation of the extracellular matrix in diabetic rats by an as yet unknown mechanism. The aim of this study is to investigate the effect of puerarin on c-fos, c-jun and CoIV expression in GMC cultured in medium containing 5.6 or 27.8 mmol/L glucose. METHODS: The expressions of c-fos and c-jun were measured at the protein level using flow cytometry. CoIV content was detected using radioimmunoassay. Protein kinase C (PKC) activity was measured using liquid scintillation counting. RESULTS: Puerarin (10(-5) mmol/L) significantly ameliorated the high-glucose effect on c-fos, c-jun and CoIV expression. This effect is accompanied by a reduced PKC activity in these cells. CONCLUSION: Our results suggest that reduced PKC activity and expression of c-fos and c-jun in GMC might participate in the mechanisms underlying the therapeutic effect of puerarin on diabetic nephropathy.

Effects of konjac extract on insulin sensitivity in high fat diet rats.

AIM: To evaluate the effects of konjac extract (KE) on insulin sensitivity in insulin resistance (IR) rats induced by high fat diet (HFD). METHODS: Wistar rats were fed on HFD for 4 weeks, then treated with KE 1.5, 3.0 g/kg/d and metformin (Met) 0.1 g/kg/d for 4 weeks, respectively. The effects of KE on intake of food and drink, body weight, and excretion were investigated. Serum insulin was measured by double-radioimmunoassay. Blood glucose, total cholesterol (TC), triglycerides (TG), and high-density lipoprotein-cholesterol (HDL-C) were measured by enzyme methods, respectively. Low-density lipoprotein-cholesterol (LDL-C) was calculated. Tissue glycogen was determined by modified anthracene ketone method and tissue TG by glycerin phosphor sour oxidation enzyme method. Insulin sensitivity was measured by modified glucose-insulin tolerance test (K value). RESULTS: HFD caused IR after 4 weeks (K value: 5.2+/-0.9 vs 8.3+/-0.7, P<0.01), the levels of blood insulin, TG, and LDL-C increased, while HDL-C, glycogen in liver and skeletal muscle decreased. The storage of TG in liver and skeletal muscle increased. After HFD rats were treated with KE 1.5 and 3.0 g/kg/d for 4 weeks, respectively, the fasting blood glucose (FBG) was decreased from 6.4+/-0.4 to 6.05+/-0.26, 6.0+/-0.3 (P<0.01). Serum TC, TG, LDL-C were decreased, while HDL-C/TC was increased as compared with HFD rats. There was no significant effect on insulin level. KE 1.5, 3.0 g/kg/d, and Met 0.1 g/kg/d could improve insulin sensitivity (K values were 6.1+/-0.5, 5.9+/-0.6, and 6.5+/-0.8 vs 5.2+/-0.9, P<0.05), elevate glycogen, and decrease TG in liver and skeletal muscle. CONCLUSION: KE could promote glycogen syntheses and adjust blood lipid metabolism so as to improve IR in HFD rats.

Effects of xiaoyu tablet on endothelin-1, nitric oxide, and apoptotic cells of atherosclerotic vessel wall in rabbits.

AIM: To investigate the mechanism of xiaoyu tablet on reduction of smooth muscle cells (SMC) in atherosclerotic vessel wall. METHODS: The atherosclerotic model was performed in male New Zealand rabbits that were given high fat diet and abrasion of the abdominal aorta endothelial cells. The rabbits were then administered with xiaoyu tablet 0.16-0.32 g x kg(-1) x d(-1) for 16 weeks. Changes in morphology, endothelin (ET)-1, nitric oxide (NO), and apoptotic cells of atherosclerotic vessel wall were determined by the microscopy, radioimmunoassay, colorimetric method, the techniques of DNA in situ end labeling, and image pattern analysis, respectively. RESULTS: After 16 weeks of xiaoyu tablet treatment, intimal thickness and SMC in atherosclerotic vessel wall were diminished, ET-1 was decreased by 8.2 %-42.6 %, NO was increased by 7.5 %-54.2 %, and labeled apoptotic nuclei were markedly decreased, the area and integral optical density of positive granule were (846+/-308) microm2 and 3425+/-1374 in atherosclerotic group and (225+/-60) microm2 and 1445+/-606 in xiaoyu tablet 0.32 g/kg group, respectively. CONCLUSION: Xiaoyu tablet not only inhibited proliferation of SMC through reducing ET-1 in atherosclerotic vessel wall, but also induced apoptosis of SMC by increasing NO in vessel wall.