Downregulation of miR­16 protects H9c2(2­1) cells against hypoxia/reoxygenation damage by targeting CIAPIN1 and regulating the NF­κB pathway.

The aim of the present study was to determine the function of microRNA­16 (miR­16) in myocardial hypoxia/reoxygenation (H/R)­induced cardiomyocyte injury and the possible mechanism underlying its involvement. An H/R model was constructed using H9c2(2­1) cells in vitro. The results of reverse transcription­quantitative PCR demonstrated that the expression levels of miR­16 were significantly upregulated in H9c2(2­1) cells in the H/R group compared with the sham group (1.53±0.09 vs. 1.0±0.08; P=0.0019). Cell Counting Kit­8 assays revealed that the relative proliferative ability of H9c2(2­1) cells was significantly decreased in the H/R + negative control (NC) group compared with the sham group (0.53±0.05 vs. 1.0±0.08; P=0.00005). Upregulation of miR­16 using miR­16 mimics further decreased the proliferative ability of cells (0.31±0.03 vs. 0.53±0.05; P=0.0097), whereas downregulation of miR­16 using an miR­16 inhibitor increased the proliferative ability of cells compared with the H/R+NC group (0.89±0.08 vs. 0.53±0.05; P=0.000385). Flow cytometric analysis found that the apoptotic rate of H9c2(2­1) cells was increased significantly following H/R compared with the sham group (25.86±2.62% vs. 9.29±0.82%, P=0.000014). Upregulation of miR­16 further increased the apoptotic rate (38.62±2.04% vs. 25.86±2.62%; P=0.000099), whereas downregulation of miR­16 decreased the apoptotic rate compared with the H/R+NC group (15.14±0.92% vs. 25.86±2.62%; P=0.000343). miR­16 directly bound to the 3'­untranslated region of cytokine­induced apoptosis inhibitor 1 (CIAPIN1) and negatively modulated CIAPIN1 expression. Overexpression of CIAPIN1 reversed the changes in the expression of apoptosis­associated proteins caused by H/R. Western blot analysis revealed that the levels of phospho­(p­)nuclear factor­κB (NF­κB) and p­NF­κB inhibitor α (IκBα) were upregulated following H/R (1.82±0.11 vs. 1.0±0.08; P=0.000152; and 1.77±0.07 vs. 1.0±0.00; P=0.000024, respectively), and these changes were further enhanced when miR­16 expression levels were increased (3.10±0.14 vs. 1.82±0.11; P=0.000006; and 2.19±0.10 vs. 1.77±0.07; P=0.0017, respectively). Downregulation of miR­16 exhibited the opposite effect on p­NF­κB and p­IκBα expression levels. The present study illustrates that downregulation of miR­16 may protect against H/R­induced injury partially by targeting CIAPIN1 and the NF­κB signaling pathway.

Effect of di-(2-ethylhexyl) phthalate exposure on placental development in pregnant mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) exposure on the growth and development of placenta, uterine natural killer (uNK) cell number and angiogenesis at the maternal-fetal interface in pregnant mice.</p><p><b>METHODS</b>From day 1 of pregnancy, pregnant mice were exposed daily to DEHP by oral gavage at 125, 250, or 500 mg/kg for 13 consecutive days. The uterine and placental tissues were then harvested for HE staining and immunohistochemistry to examine the effect of DEHP exposure on the growth and development of the placenta and angiogenesis and uNK cell number at the maternal-fetal interface.</p><p><b>RESULTS</b>Compared with the control group, the mice exposed to 500 mg/kg DEHP, but not those exposed to 125 and 250 mg/kg, showed significantly reduced number of embryo implantation (P<0.05). DEHP exposure significantly increased the rate of abortion. DEHP exposure at 125, 250, and 500 mg/kg significantly and dose-dependently lowered the placental weight compared with that in the control group (0.0637±0.0133, 0.0587±0.0176, 0.0524±0.0183 g vs 0.0786±0.0143 g, respectively; P<0.01), and significantly reduced the total area of the placenta and area of spongiotrophoblasts. DEHP exposure resulted in a significant reduction in the number of fetal vascular branches, and collapse and atresia of blood vessels. The mice exposed to DEHP at 125, 250, and 500 mg/kg had significantly lowered numbers of uNK cells (83.2±10.3, 60.7±12.4, and 50.4±14.5/HP, respectively) as compared with the control group (105.1±14.2/HP) at the maternal-fetal interface (P<0.01).</p><p><b>CONCLUSION</b>DEHP exposure significantly affects the growth and development of the placenta in mice possibly by suppressing angiogenesis and reducing uNK cell number at the maternal-fetal interface during pregnancy.</p>

Whole blood viscosity is negatively associated with bone mineral density in postmenopausal women with osteoporosis.

Osteoporosis (OP) is associated with cardiovascular disease. Moreover, osteoporosis has been shown to be an independent predictor of cardiovascular mortality. Recent studies revealed that altered blood rheology plays a critical role in atherosclerosis. A study confirmed that whole blood viscosity (WBV) is a predictor of cardiovascular events. However, little research has been conducted to investigate the relationship between blood viscosity and osteoporosis. In this cross-sectional study, we investigated the relationship between the rheological parameters and bone mineral density (BMD) in 481 subjects in the International Physical Examination and Healthy Center of the Second Affiliated Hospital, Harbin, China. Different biochemical stress and physical activity are correlated to lumbar spine BMD. Stepwise multivariate linear regression analysis revealed that WBV was a significant factor for decreased BMD (ß=-0.513; P<0.001 for lumbar spine L2-4 BMD; ß=-0.157; P=0.003 for femoral neck BMD). In conclusion, The findings show that WBV is elevated in osteoporosis and negatively correlated with BMD. Further studies are warranted to investigate whether antiosteoporosis medication could normalize whole blood viscosity in postmenopausal women with osteoporosis.

[Study of the correlation about IGF-I receptor and ß-amyloid in Alzheimer's disease].

OBJECTIVE: To detect the expression of IGF-I receptor in the hippocampus neuron of rat treated by Aß(1-42), and thus from the receptor level explore the disorder of central nervous insulin signaling and the possible molecular mechanism of Alzheimer disease. METHODS: Cultured primary hippocampus neurons were treated with different concentrations of Aß(1-42), apoptosis rate was detected by flow cytometry, real-time quantitative PCR and Western blot were used to detect IGF-I receptor expression. RESULTS: Primary cultured cells mature in 7(th) days; after detected by flow cytometry, early apoptosis rate in Aß(1-42) 0, 30, 60, 100 µmol/L groups showed a concentration-dependent increase. PCR results showed that, in 30 (1.72 ± 0.33) and 60 µmol/L (1.86 ± 0.36) treatment groups levels of the IGF-I receptor gene were significantly higher than the control group (regarded as 1) (P < 0.01), 100 µmol/L group (0.70 ± 0.15) was significantly lower than the control group (P < 0.05). Results of Western blot showed 30 and 60 µmol/L protein level of the treatment groups are 1.08 ± 0.04, 1.74 ± 0.08 (P < 0.01) and 100 µmol/L group was 0.79 ± 0.11(P < 0.05), which had same trend with PCR. CONCLUSIONS: Aß(1-42) induced altered expression of IGF-I receptors in rat hippocampus cells, maybe one of the molecule mechanisms of Alzheimer disease.

[Abnormal expression of insulin receptor induced by Aß(1-42) in rat hippocampus neuron].

OBJECTIVE: To detect the expression change of insulin receptor under the induction of Aß(1-42) in rat hippocampus neuron and thus from the receptor level explore the disorder of central nervous insulin signaling and molecular mechanism of Alzheimer's disease. METHODS: Cultured primary hippocampus neuron was treated with different concentrations of Aß(1-42). Apoptosis was detected by flow cytometry. And real-time quantitative PCR (polymerase chain reaction) and Western blot were used to detect the expression of insulin receptor. RESULTS: Primary cultured cells, mature at Day 7, were identified as hippocampal cells. After the treatment with different concentrations of Aß(1-42) (0 - 150 µmol/L), the ≥ 30 µmol/L treatment groups had greater early apoptosis rates (32.4%, 36.1%, 51.0%, 53.6%) than that in the control group (13.4%) in a concentration-dependent fashion. The PCR results showed that the levels of insulin receptor gene were significantly higher in 30 (2.56 ± 0.19) and 60 µmol/L (3.44 ± 0.23) treatment groups than that the control group (regarded as 1) (P < 0.01) while the 100µmol/L group (0.74 ± 0.15) was significantly lower than the control group (P < 0.01). And the results of Western blot had the same trend with those of PCR. The 30 and 60 µmol/L protein level of the treatment groups were 1.27 ± 0.13, 1.82 ± 0.10 (P < 0.01) and 100µmol/L group was 0.82 ± 0.08 (P < 0.05). CONCLUSIONS: Aß(1-42) induces an altered expression of insulin receptors in rat hippocampus cells and results in its functional defects. It may cause insulin resistance in center nervous system.

Human thioredoxin exerts cardioprotective effect and attenuates reperfusion injury in rats partially via inhibiting apoptosis.

BACKGROUND: Thioredoxin is one of the most important redox regulating proteins. Although thioredoxin has been shown to protect cells against different kinds of oxidative stress, the role of thioredoxin in myocardial ischemia and reperfusion injury has not been fully understood. This study was conducted to explore the protective role of human thioredoxin on myocardial ischemia and reperfusion injury and its potential mechanisms. METHODS: Purified human thioredoxin was injected into adult Wistar rats, which were subjected to 30 minutes of myocardial ischemia followed by 2 or 24 hours of reperfusion. We detected 1) the infarct size; 2) the level of malondisldehyde (MDA) in serum; 3) the expression of caspase-9, and cytochrome c in/out of mitochondria by Western blotting; 4) apoptosis by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay and caspase-3 and its protein by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting; 5) the expression of bcl-2 and bax in cardium by immunohistochemical (IHC) assay. RESULTS: Human thioredoxin reduced myocardial ischemia/reperfusion injury as evidenced by significant decrease of myocardial infarct size (P < 0.01), notable reduction of myocyte apoptosis (P < 0.01), lower systemic oxidative stress level (P < 0.01) after reperfusion for 2 hours, and few inflammatory cell infiltration after reperfusion for 24 hours in rats. Furthermore, treatment with human thioredoxin significantly reduced the release of mitochondrial cytochrome C (P < 0.05), and inhibited the activity of caspase-9 (P < 0.05) and caspase-3 (P < 0.01 in mRNA and P < 0.05 at protein level). Meanwhile, human thioredoxin markedly increased bcl-2 expression (P < 0.05). CONCLUSIONS: These results strongly suggest that human thioredoxin has cardioprotective effects on myocardial ischemia/reperfusion and its anti-apoptotic role may be mediated by modulating bcl-2 and the mitochondria-dependent apoptotic signaling pathway.

[Phosphorylation of PKCdelta participates in the toxicity of 6-hydroxydopamine on dopaminergic neuroblastoma cell].

OBJECTIVE: To investigate the role of phosphorylation of protein kinase C (PKC) delta in the toxicity of 6-hydroxydopamine (6-OHDA) to the death of dopaminergic neurons. METHODS: Human neuroblastoma cells of the line SH-SY5Y were cultured 6-OHDA of the concentrations of 0, 50, 100, 200, and 400 micromol/L was added to observe its toxicity. Rottlerin (PKCdelta inhibitor, 2 micromol/L), bisindolylmaleimide (Bis, general PKC inhibitor, 10 nmol/L), Gö6976 (calcium-dependent PKC inhibitor, 5 nmol/L), and phobol-12-myristate-13-acetate (PMA, PKC activator, 100 nmol/L) were added into the culture fluid of another SH-SY5Y cells respectively, and then (1) culture fluid of equal volume was added for 18 h so as to observe there effects on the survival of the SH-SY5Y cells, or (2) 100 micromol/L 6-OHDA was added to observe the effects of intervention on PKC on the survival of the SH-SY5Y cells by using MTT assay. Cell lysis solution with phosphatase inhibitor was used to lyse the culture cells to extract plasma protein. Western blotting was used to detect the expression of phosphorylated PKCdelta. RESULTS: MTT assay showed that all different concentrations (50 - 400 micromol/L) of 6-OHDA significantly and dose-dependently caused cell death with an EC50 of 92 micromol/L. Pretreatment with rottlerin and Bis alone did not influence the survival of the cells significantly,. However, the survival rate of the cells pretreated by Gö6976 alone was 92.3% +/- 3.2% that of the control group (P < 0.01), and the survival rate of the cells pretreated by PMA was 49.5% +/- 1.0% that of the control group (P < 0.01) Pretreatment of rottlerin decreased the death rate of the cells treated with 6-OHDA to 30.4% +/- 1.6% and conferred significant protection against 6-OHDA neurotoxicity by 57% +/- 6% compared to that of the cells treated by 6-OHDA alone (P < 0.01). However, Bis and Gö6976 did not affect the 6-OHDA-induced cell damage. Pretreatment of PMA increased the death rate of the cells treated with 6-OHDA to 67.1% +/- 2.2% and significantly aggravated 6-OHDA-induced cell toxicity by 66% +/- 9% (P < 0.01). Western blotting showed that 6-OHDA administration increased the expression of phosphorylated PKCdelta, pretreatment with Rottlerin inhibited such increase, PMA promoted such increase, and Bis and Gö6976 did not influence such increase. CONCLUSION: Inhibition of PKCdelta phosphorylation with rottlerin ameliorates the neurotoxicity evoked by 6-OHDA, and activation of PKCdelta phosphorylation by PMA aggravates neurotoxicity, which implicating that this kinase participates in the 6-OHDA-induced neurotoxicity and Parkinsonian neurodegeneration.