Objective The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells. Methods The primary NP cells were harvested from male SpragueDawley rats (8-10 weeks old); the hypoxia inducible factor 1α (HIF-1α), HIF-1β, matrix metalloproteinase 2 (MMP-2), andcollagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-β-galactosidase (SA-β-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells prol iferation. Results Immunocytochemical staining showed that NP cells expressed HIF-1α, HIF-1β, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-β-gal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P lt; 0.001). And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (Plt; 0.001). The flow cytometry showed that the G1 phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P lt; 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P lt; 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA. Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.
ObjectiveTo investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.MethodsThe skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.ResultsAfter identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.ConclusionInhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.
ObjectiveTo observe and investigate the effect of HIF-2α in the process of neovascularization of proliferative diabetic retinopathy (PDR).MethodsRetrospective clinical study. From July 2014 to July 2015, 60 eyes of 57 PDR patients diagnosed in Ophthalmology Department of Affiliated Hospital of Qingdao University were included in the study. Twenty-eight eyes of 27 patients received intravitreal injections of 0.5 mg ranibizumab (0.05 ml) at 2-7 days before surgery (ranibizumab group) and other 32 eyes of 30 patients did not (group without ranibizumab). Eighteen eyes of 18 patients with epiretinal membranes were included as controls. Pathological specimens of PDR fibrovascular membrane and premacular membrane were obtained during vitrectomy. The immunohistochemical staining and real-time PCR (RT-PCR) were used to detecting the expression of HIF-2α, Dll4 and VEGF. Kruskal-wallis test was used to compare the expression differences of correlation factors between groups. Spearman correlation analysis was used to analyze the relationship between the two variables.ResultsThe immunohistochemical staining revealed that there were positive expression of HIF-2α, Dll4 and VEGF in all PDR membranes, regardless of the injection of the ranibizumab. The levels of HIF-2α, Dll4 and VEGF protein in the group without ranibizumab were higher than those of the ranibizumab group (t=4.36, 6.01, 4.82; P=0.000, 0.008, 0.016). RT-PCR showed that the differences of the mRNA expression of HIF-2α, Dll4 and VEGF were all statistically significant among the PDR patients and controls (H=18.81,19.60, 20.50; P=0.000, 0.000, 0.000). The expression of HIF-2α, Dll4 and VEGF in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR patients,the expression of HIF-2α, Dll4 and VEGF of the group without ranibizumab was higher than that of the ranibizumab group. The spearman correlation analysis showed that the expression of mRNA between HIF-2α and Dll4, HIF-2α and VEGF were both significantly correlated (r=0.95, 0.87; P＜0.05).ConclusionsThe expression of HIF-2α in the PDR membranes was higher than that of the controls. It is positively correlated with the expression of the DLL4 and VEGF.
Objective To observe the influence of the expression of CD18 on the neutrophile and the leukocyte adhesion to retinal vascular endothelium by hypoxia-inducible factor-1 alpha (HIF-1alpha;) in early diabetic retinopathy rats. Methods Male Sprague-Dawley rats received intraperitoneal injection of streptozotocin to induce diabetes model. 18 diabetic rats were divided into 3 groups randomly after 2 months of diabetes induction, including diabetic group (group B), HIF-1alpha; anti-sense oligonucleotides (ASODN) injection group (group C) and HIF-1alpha; sense oligonucleotides (SODN) injection group (group D), the age and weigh matched health rats were chosen as control group (group A), with 6 rats in each group. Then group A and B rats received 5% glucose solution caudalis veins injection, group C and group D rats received HIF-1alpha; ASODN and HIF-1alpha; SODN caudalis veins injection, respectively(025 mg/kg).The level of CD18 on the neutrophil isolated from the peripheral blood was measured by flow cytometry. Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Results The percentage of CD18 positive neutrophil cell was（44.93plusmn;3.60）% in group B，（18.66plusmn;1.52）% in group A，（31.66plusmn;4.72）% in group C，（51.00plusmn;5.66）% in group D. Compared with each other groups，the differences are statistically significant （F=42.46, Plt;0.001）. The number of positive staining cells of retinal leukocyte was （46.16plusmn;10.68）in group A，（133.83plusmn;20.43）in group B,（99.83plusmn;9.28）in group C，（121.33plusmn;10.23） in group C. Compared group B with group C，the number of positive staining cells raised about 2.89 times；compared group B with group C and D，the differences are statistically significant （P=0.12，95% confidence interval -3.69~28.69）. Conclusions In vivo, HIF-1alpha; can decreased the expression of CD18 on neutrophils from diabetic ratsprime; peripheral blood and the collection of retinal leukostasis in the diabetic animals. HIF-1alpha; may serve as a therapeutic target for the treatment and/or prevention of early diabetic retinopathy. （Chin J Ocul Fundus Dis，2008，24：268-271）
Objective To investigate the effect of small interfering RNA(siRNA) targeting hypoxia inducible factor1alpha; (HIF1alpha;) and vascular endothelial growth factor (VEGF) on expression of VEGF in human vascular endothelial cells. Methods HIF-1alpha; siRNA recombinant plasmid was constructed. Human vascular ndothelial cells were cultured in vitro and divided into normoxia group (20% O2) and hypoxia group (1% O2). Hypoxia group was then divided into control group, vector group, HIF-1alpha; group (HIF-1alpha; siRNA), VEGF group ( VEGF165 siRNA) and cotransfection group (HIF-1alpha; siRNA+VEGF165 siRNA). LipofectamineTM 2000 (LF2000) mediated vector plasmid was transfected to cells in each group except the control group. The expression of HIF-1alpha; siRNA and VEGF165 siRNA recombinant plasmid were identified by reverse transcriptasepolymerase chain reaction (RT-PCR). The expression of VEGF mRNA and protein were detected by RTPCR and immunocytochemical method. Results The expression of HIF-1alpha; siRNA and VEGF165 si RNA recombinant plasmid were detected 24 hours after transfected. The expression of VEGF mRNA and protein was faint in the normoxia group, but increased obviously in hypoxia group. The expression of VEGF mRNA and protein in the HIF1alpha;, VEGF and cotransfection groups were lower than which in the control group. Cotransfection group showed the highest inhibitory effect. Conclusion HIF-1alpha; and VEGF165 siRNA can effectively inhibit the expression of VEGF in human vascular endothelial cells.
Objective To investigate the effects of matrine on cell proliferation and expression of connective tissue growth factor( CTGF) and hypoxia inducible factor-1α( HIF-1α) of human lung fibroblast ( WRC-5) in normoxia ( 21% O2, 74% N2 , 5% CO2 ) and hypoxia ( 1% O2, 94% N2 , 5% CO2 )conditions. Methods MRC-5 cells were cultured and divided into differrent groups interfered with different dose of Matrine ( final concentration of 0 ～3. 2 mmol / L) in normoxia or hypoxia for 24 h. Cells were dividedinto 8 groups according to culture conditions, ie. normoxiagroup( N0 group) , normoxia + matrine 0. 2 mmol / L group( N0. 2 group) , normoxia + matrine 0. 4 mmol / L group( N0. 4 group) , normoxia + matrine 0. 8 mmol / L group( N0. 8 group) , hypoxia group( H0 group) , hypoxia + matrine 0. 2 mmol /L group( H0. 2 group) , hypoxia +matrine 0. 4 mmol /L group( H0. 4 group) , and hypoxia + matrine 0. 8 mmol / L group( H0. 8 group) . The MTT assay was used to measure the cell proliferation activity. Western-blot assay was used to examine the expression of CTGF and HIF-1α. Results Hypoxia promoted the cell proliferation in all groups( P lt;0. 05) .Matrine inhibited the proliferation of WRC-5 cells in a concentration-dependent manner in hypoxia or normoxia conditions( P lt;0. 05) . The expression of CTGF andHIF-1αwas lower in normoxia and higher in hypoxia( P lt;0. 01) . Matrine inhibited the expression of CTGF and HIF-1αin a concentration-dependent manner in hypoxiaand normoxia( P lt;0. 05) . Conclusion Matrine can inhibit the cell proliferation and the expression of CTGF and HIF-1αof WRC-5 cells in normoxia and hypoxia in a concentration-dependent manner.
Autophagy is a lysosome dependent, conservative material degradation process, which exists in all eukaryotic cells and plays import roles in many pathophysiology process. Erectile dysfunction (ED) is a common male disease with multiple etiology. In recent years, more and more evidences have demonstrated that autophagy has a close relation to ED, therefore, we combine previous study to classify ED by hypoxia, aging, diabetes and other causes, and review the advances of autophagy in ED.
Objective To study the mechanisms and treatment of ischemia /reperfusion injury, expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were measured, the effect on suppression of ICAM-1 and VCAM-1 by the pyrrolidine dithiocarbamate (PDTC) were investigated. Methods Endothelial cells were divided into 3 groups, hypoxia group: endothelial cells were exposed in hypoxia condition, then returned to reoxygenation condition; the PDTC group: PDTC was added to the endothelial cells in the culture media before exposing to hypoxia condition; control group: endothelial cells underwent treatment. Confocal microscopy was used to detect expression of ICAM-1 and VCAM-1. Results ICAM-1 and VCAM-1 expression were low in endothelial cells of control group, and increased in hypoxia group . ICAM-1 and VCAM-1 expression of endothelial cells in PDTC group werelower than those in hypoxia group , but higher than those in control group. Conclusions It seems that hypoxia/ reoxygenation can activate the endothelial cells and increase the expression of cell adhesion molecules. PDTC can decrease the expression of ICAM-1 and VCAM-1. PDTC may prove benificial in the treatment of ischemia /reperfusion injury.
ObjectiveTo investigate the anti-apoptotic effect of ginsenoside Rg1 in neonatal rats with hypoxia ischemia brain damage (HIBD), and to explore the possible signaling pathway involved in anti-apoptosis. MethodsForty-eight 10-day-old Sprague Dawley (SD) rats (weighing 17-21 g, male or female) were randomly allocated into 4 groups (12 rats in each group): sham-operation group (sham group), HIBD group (HI group), HIBD+ginsenoside Rg1 group (HI+Rg1 group), and HIBD+ginsenoside Rg1+U0126 group (HI+Rg1+U0126 group). SD rats in HI group, HI+Rg1 group, and HI+Rg1+U0126 group underwent ligation of the right common carotid artery (CCA) and hypoxic ventilation (8%O2+92%N2) for 2.5 hours to prepare the HIBD model, and rats in sham group underwent only separation of the right CCA. SD rats in HI+Rg1+U0126 group received intraventricular injection of 5 μL phosphate buffer saline (PBS) containing U0126 (25 μg/kg) at 1 hour before HIBD, and rats in the other three groups received intraventricular injection of PBS at the same time. The rats in HI+Rg1 group and HI+Rg1+U0126 group received intraperitoneal injection of 0.1 mL normal saline (NS) containing Rg1 (40 mg/kg) at immediate after HIBD, while rats in HI group and sham group received intraperitoneal injection of 0.1 mL NS at immediate after HIBD. At 4 and 24 hours after HIBD, the right hemisphere and hippocampus were collected to detect the protein expression and distribution of extracellular signal-related protein kinase 1/2 (Erk1/2), phospho-Erk1/2 (p-Erk1/2), hypoxia inducible factor 1α (HIF-1α), and cleaved Caspase-3 (CC3) by Western blot and immunohistochemistry staining. TUNEL staining was used to evaluate neural apoptosis in situ. ResultsWestern blot results showed that there were expressions of Erk1/2, p-ERK1/2, HIF-1α, and CC3 proteins at 4 and 24 hours after HIBD in each group. The expressions of HIF-1α and CC3 protein at 4 and 24 hours, and expression of p-Erk1/2 protein at 4 hours were significantly increased in HI group when compared with sham group (P < 0.05). When compared with HI group, the expressions of p-Erk1/2 and HIF-1α protein in HI+Rg1 group were significantly increased (P < 0.05), while the expression of CC3 protein was significantly decreased at 4 and 24 hours (P < 0.05). When compared with HI+Rg1 group, the expressions of p-Erk1/2 and HIF-1α proteins in HI+Rg1+U0126 group were significantly decreased (P < 0.05), while expression of CC3 protein was significantly increased at 4 and 24 hours (P < 0.05). There was no significant difference in Erk1/2 protein expression between groups at different time points (P > 0.05). Immunohistochemistry staining displayed that HIF-1α and CC3 proteins mainly distributed in the nucleus and cytoplasma, while Erk1/2 and p-Erk1/2 proteins mainly distributed in the cytoplasma. The expression levels of protein by immunohistochemistry results were similar to the results by Western blot. TUNEL staining showed that the apoptotic neurons were characterized by yellow or brown particle in the nucleus. The apoptotic index (AI) of neurons at 4 and 24 hours was significantly increased in HI group when compared with sham group (P < 0.05), and the AI of neurons was significantly decreased in HI+Rg1 group when compared with HI group and HI+Rg1+U0126 group at 24 hours (P < 0.05). ConclusionRg1 could enhance HIBD induced HIF-1α expression and inhibit activation of Caspase-3 by Erk1/2 signaling pathway, and play an anti-apoptotic role in neonatal rats with HIBD.
Objective To investigate the preventive effect of simvastatin，a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor，on hypoxic pulmonary hypertension and the relation between it and the angiotensin Ⅱ receptor-1（AT1R） expression in pulmonary arteriole.Methods Thirty male Sprague-Drawley rats were randomly allocated into three groups：a control group，a hypoxic group and a simvastatin preventive group.The animal model of hypoxic pulmonary hypertension was established by exposing the rats to normobaric hypoxic condition（8 h×6 d×3 w），and the preventive group were treated with simvastatin 10 mg/kg before hypoxic processing while the control and hypoxic groups were treated with sodium chloride.The mean pulmonary pressure（mPAP），serum cholesterol concentration，right ventricular hypertrophy index [RV/（LV+S）]，percentage of the wall thickness in the external diameter（WT%），percentage of the wall area in the total vascular area（WA%），and the AT1R expression in pulmonary arterioles were measured.Results When compared with the hypoxic group，in the preventive group，the mPAP and RV/（LV+S）obviously reduced [（22.6±3.86）mm Hg vs （29.3±2.27）mm Hg，（25.13±0.75）% vs （33.18±1.58）%，Plt;0.01 respectively]，the indices of wall thickness of rat pulmonary arteriole and area also decreased significantly [WT%：（15.98±1.96）% vs （25.14±1.85）%；WA%：（54.60±3.94）% vs 74.77±4.52）%；Plt;0.01 respectively]，and the positive degree of AT1R still lessened noticeably（1.23±0.09 vs 1.57±0.13，Plt;0.01）.All of the indices above in the hypoxic group increased markedly compared with the control group（Plt;0.01 respectively）.However，the differences of serum cholesterol among three groups were not significant（Pgt;0.05）.Conclusions Simvastatin can suppress the expression of AT1R in pulmonary vessel and prevent hypoxic pulmonary hypertension.