BACKGROUND:The dynamic changes of hypoxia inducible factor-1 alpha (HIF-α) and inducible nitric oxide synthase (iNOS) genes in the pulmonary artery wall during the process of hypoxic pulmonary hypertension (HPH) development need to be investigated.OBJECTIVE: This study was to observe the gene expressions of HIF-α and iNOS in the pulmonary artery wall at the different hypoxic time points, and to investigate their effects in HPH development.DESIGN: Controlled observation animal experiment.SETTING: Department of Respiration, Second Hospital Affiliated to Nanhua University.MATERIALS: Forty healthy male Wistar rats of clean grade, aged 6-8 weeks, with body mass of (220±10) g were involved in this study. They were randomized into control group (n =8) and hypoxia group (n =32). Four time points, i.e.3, 7, 14, and 21 days after hypoxia were set for the animals in the hypoxia group, 8 rats at each time point.METHODS: This study was carried out in the Institute of Oncology, Nanhua University between August 2004 and December 2005. Rats in the hypoixa group were treated according to the method reported by Li et al. Hypoxia treatment was omitted for the rats in the control group. At each time point, the rats were anesthetized, and then a micro-catheter was inserted into the right jugular vein and connected to a multichannel physiologic recorder, which was used for detecting the mean pulmonary arterial pressure (mPAP). The heart of each euthanized rat was taken out, and its right ventricle (RV), and left ventricle and septum (LV+S) were weighted. Right ventrical hypertrophy index (RVHI) reflected right ventricle hypertrophy degree. Right upper lung tissue of rat was harvested for haematoxylin & eosin (HE) staining and elastic fiber staining. Pathological image analysis software was used to determine pulmonary arterial wall area/total vascular area, lumina area/total vascular area, smooth muscle cell density in the media of pulmonary arteriole, and media thickness of pulmonary arteriole, which were used as remodeling indexes of pulmonary arteriole. HIF-1α and iNOS in the pulmonary arteriole were performed in situ hybridization and immunohistochemical detection. The mean absorbance of pulmonary arteriole wall was used as the relative content of mRNA expression and protein level.MAIN OUTCOME MEASURES: ①Changes in mPAP, RV hypertrophy degree and remodeling indexes of pulmonary arteriole of rats. ② Expressions of HIF-α and iNOS as well as their correlations with mPAP and remodeling indexes of pulmonary arteriole.RESULTS: All the 40 Wistar rats were involved in the final analysis. ①At hypoxia 7 days, mPAP was significantly higher than that of control group (P ＜ 0.05). mPAP reached to the high level at hypoxia 14 days, and then maintained at this level. ② At hypoxia 14 days, RVHI was higher than that of control group (P ＜ 0.05). ③ At hypoxia 7 days, pulmonary arteriole wall was thickened, and lumina became narrowed. There were significant differences in pulmonary arterial wall area/total vascular area, lumina area/total vascular area between hypoxia group and control group (P ＜ 0.05). At hypoxia 14 days, smooth muscle cell density in the intima-media of pulmonary arteriole, and intima-media thickness of pulmonary arteriole were significantly increased (P ＜ 0.05). At hypoxia 21 days, lumina was further narrowed, and obvious smooth muscle hyperplasy was found. HIF-1α and iNOS mRNA expression presented weak-positive in the control group; The relative content of HIF-1α mRNA did not significantly alter at hypoxia 3 and 7 days, but was obviously increased at hypoxia 14 days ,and after this, it maintained at high level. iNOS mRNA was markedly higher than that in the control group at hypoxia 3 days, reached its peak at hypoxia 7 days, was close to the level in the control group at hypoxia 14 days, and enhanced again at hypoxia 21 days, but it was still lower than that at hypoxia 3 days. ⑤HIF-α was mainly found in the intima and media, while iNOS was found in the whole layer of vessels. iNOS was weakly expressed in the intima and media of pulmonary vessels in the control group. No obvious difference in iNOS existed at hypoxia 3 days as compared with control group. iNOS was obviously expressed in media and intima at hypoxia 7 days. Vascular media was thickened at hypoxia 14 days, and the expression of iNOS was enhanced. iNOS was not found in the vascular adventitia in the control group, but was found in the vascular adventitia in the hypoxia group. ⑥mPAP was positively correlated with pulmonary vascular remodeling (r =0.976, P ＜ 0.01 ), and HIF-1α mRNA was positively correlated with iNOS protein(r =0.927, P ＜ 0.05).CONCLUSION: Both HIF-1α and iNOS exert effects in the process of HPH development of rats, and HIF-1α and iNOS gene expression may be mutually regulated.

Objective To examine the effect of simvastatin treatment on Parkinson's disease rats induced by lipopolysaccharide (LPS) and its mechanism.Methods The LPS-PD model was established by injection of LPS (5 mg/mL) into the right substantia nigra compacta (SNC),and rats were randomly divided into control group,LPS-model group and simvastatin treatment group with 15 rats in each group.Rats in the simvastatin treatment group was intraperitoneally administered simvastatin (5 mg/kg) before,and daily for 14 days after surgery,while the control group and LPS-model group received same volume normal saline and LPS respectively.Ionized calcium binding adaptor molecule 1 (Iba-1)-positive cells and the expression of tyrosine hydroxylase (TH),tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the SNC were detected by immunohistochemistry,Western blotting and enzyme-linked immunosorbent assay,respectively.The effect of simvastatin in the PD model was also examined in behavioral tests.Results The LPS-model group exhibited typical animal PD behaviors.Compared with the control group,the LPS-model group exhibited a decreased number of DA neurons,and comparison of the intact side to reduce 81.13％ (P＜0.01) in the SNC,as well as increases in the Iba-1-positive cell number,iNOS,IL-1β and TNF-α expression (P＜0.05).These effects were inhibited by simvastatin treatment (P＜0.05).Conclusion Simvastatin mediates a protective effect on dopaminergic neurons in the SNC in the LPS-PD model,possibly by inhibiting glial cells (astrocytes and microglia) activation,and playing an anti-inflammatory role,thus improving substantia nigra function.

Objective To observe the protective effect of growth differentiation factor 11(GDF11) on myocardial injury and the changes of myocardial apoptosis in type 2 diabetic C57BL/6J mice.Methods Sixty male C57BL/6J mice weighing 20-25 g were randomly divided into three groups: control group (control), type 2 diabetes mellitus group (DM) and GDF11 intervention group (DM + GDF11).To establish mouse model of type 2 diabetes, the mice were fed with high fat and high sugar diet for 4 weeks, and i.p.injected consecutively three times of streptozotocin (STZ) in a dose of 60 mg/kg.After the continuous high-fat and high-sugar diet for 4 weeks, the cardiac function was detected by small animal ultrasound, TUNEL staining was used to detect the apoptosis in myocardium, and the expressions of cleaved-caspase-3, Bcl-2, Bax were measured.Results Diabetic injury significantly reduced the left ventricular ejection fraction and left ventricular short axis shortening rate, and increased myocardial apoptosis.Recombinant GDF11 protein significantly improved cardiac function and reduced myocardial apoptosis.Conclusions Exogenous GDF11 can significantly reduce myocardial apoptosis and improve heart function after diabetic injury.

Objective:To investigate the neuroprotective effects of simvastatin on lipopolysaccharide (LPS)-induced rat model ofParkinson's disease(PD) and the mechanisms involved.Methods:Hemiparkinsonian rat models were induced by stereotaxieal injection ofLPS in the right substantia nigra compacta.After2 weeks of simvastatin treatment, rotational behavior test was performed after the intraperitoneal injection of apomorphine.Expression of tyroxine hydroxylase (TH) and glial fibrillary acidic protein were analyzed through immunohistochemical staining of substantia nigra and striatum, and the level ofTNF-α was evaluated using enzyme-linked immunosorbent assay.Results:Comparing with untreated group, behavioral symptoms of the rats were significantly less in the rats that received simvastatin treatment.TheTH positive cell count in substantia nigra and striatum were significantly increased(P<0.05) andTNF-α expression was significantly decreased(P<0.05) in simvastatin group compared to untreated group.Conclusions:Simvastatin could effectively inhibit the activation of astrocytes, reduceTNF-α expression, and exert anti-inflammatory effects, and thus protect the dopaminergic neurons in substantia nigra and striatum of the rat model ofPD.

Objective To explore the effects of 14-3-3 γ gene on dopaminergic neurons of PD rat model.Methods 6 hydroxydopamine (6-OHDA) was injected into the corpus striatum of 60 SD rats to establish PD model.A week later,Ad/14-3-3 γ was injected into the corpus striatum of 16 rats,while PBS and Ad-LacZ were injected into the corpus striatum of 16 and 28 rats,respectively,as control.X-gal dyeing was utilized to examine the LacZ reporter gene expression in the corpus striatum at 3 day,2 week and 6 week.Real-time PCR was utilized to test the expression level of 14-3-3 γmRNA at 2 weeks after Ad/14-3-3 γ injection.Immunohistochemistry technique was used to detect TH positive neurons and fibers in the corpus striatum and substantial nigra.Western blotting was performed to check the expression of 14-3-3 γ in the corpus striatum at 2 weeks and caspase-3 at 6 veeks after Ad/14-3-3 γ injection.High performance liquid chromatography (HPLC) method was used to examine the contents of DA and DOPAC in the corpus striatum.The rats underwent rotational ethological examination at 1,2 and 6 weeks after the second injection.Results The expression of β-gal,which showed the successful LacZ gene transfection,was found in the corpus striatum of LacZ groups.The 14-3-3 γ mRNA and protein expression in the corpus striatum were significantly higher in the Ad/14-3-3 γ group than in the other two groups.The TH-positive cell ratio of substania nigra to contralateral area in the lesion side was 0.44±0.17 and the optical density (OD) of TH-positive fibers was 0.62±0.14 in the Ad/14-3-3 γ group,both higher than those in PBS group (0.16±0.13 and 0.36±0.15) and LacZ group (0.15±0.09 and 0.30±0.11) (all P＜0.01).The contents of DA and DOPAC in the lesion sides of corpus striatum were increased in the Ad/14-3-3 γ group [(248± 116)ng/g and (752±177) ng/g] than in PBS group [(106±35) ng/g and (724±159) ng/g] and LacZ group [(136±49) ng/g and (765±163) ng/g] (P＜0.01).The DA and DOPAC ratios of the lesion side of corpus striatum to the contralateral side were 0.37±0.14 and 0.38±0.17 in the Ad/14-3-3 γgroup,higher than those in PBS group (0.15±0.08 and 0.13±0.10,respectively) and LacZ group (0.19±0.11 and 0.16±0.09 (all P＜0.01).The expression level of caspase-3 was decreased in Ad/14-3-3 γ group than in PBS and LacZ groups at 6 weeks after the second injection.The turns/min induced by apomorphine in Ad/14-3-3 γ group were 9.4 ± 2.5 at 1 week after the Ad/14-3-3 γinjection,and reduced to 4.6±2.2 at 6 weeks later.While in PBS and LacZ group,the turns were 14.5±4.9 and 13.8±3.5 at 1 week after the second injection,6 weeks later rised to 18.7±5.2 and 20.6± 6.7 respectively,significantly higher than those in Ad/14-3-3 γ group (all P＜0.01).Conclusions 14-3-3γ gene transfer has a protective effect on the dopaminergic neurons and it may be a promising candidate gene for curing PD.

Objective The study was designed to observe influence of simvastatin on lung tissue angiogenesis and the gene expression of vascular endothelial growth factor(VEGF) and platelet factor 4(PF4) of rats with bleomycin (BLM)-induced pulmonary fibrosis. Methods Ninety-six healthy male SD rats were divided into four groups by random number table, including normal control group (A), bleomycin group (B), prednisone acetate treatment group (C) and simvastatin treatment group (D). Lung tissue of rats in each group was detected as specimens. HYP was detected by digestion method. Angiogenesis, VEGF and PF4 protein expression were determined by immunohistochemical method (SP). Expression of VEGF and PF4 mRNA were respectively detected by RT-PCR assay. Results (1)HYP content of group C, D was lower than the group B, which was statistical significance (P <0.01). (2)MVD and the expression of VEGF in group B, C and D was higher than that in group A. PF4 expression of group B, C and D were lower than that of group A (P < 0.01). MVD and the expression of VEGF of group D were lower than those of group B, the expression of PF4 of group D was higher than that in group B (P < 0.05). Conclusion Mechanism of simvastatin on pulmonary fibrosis may be related to regulate the expression of VEGF and PF4 in lung tissue, inhibit pathological angiogenesis.

Objective : To investigate the effects and mechanism of small ubiquitin-like modifier ( SUMO) specific proteinase-1 (SENP-1) on chronic intermittent hypoxia ( CIH) induced myocardial injury in rats． Methods : 32 male SD rats were randomly divided into : control group，CIH group，negative control adeno-associated virus interven- tion group (AAV-shNC) and SENP-1 shRNA adeno-associated virus intervention group (AAV-shSENP-1) ，with 8 rats in each group．After 6 weeks of CIH induction，echocardiography was performed．The levels of cardiac troponin I (cTNI) ，creatine kinase MB isoenzyme ( CKMB) ，myoglobin (Mb) ，lactate dehydrogenase (LDH) in serum and malondialdehyde (MDA) ，uperoxide dismutases ( SOD) ，glutathione ( GSH) ，interleukin( IL) -1 β , IL-6 and tumor necrosis factor-α(TNF-α) in myocardial tissue were detected by ELISA．The pathological changes of myocardial tis- sue was observed by HE staining．The reactive oxygen species ( ROS) level in myocardial tissue was detected by DCFH-DA fluorescence probe labeling．The small ubiquitin-like modifier (SUMO) level of hypoxia inducible factor- 1 α (HIF-1 α) protein in myocardial tissue was detected by kit．The mRNA and protein levels of SENP-1 and HIF- 1 α in myocardial tissue were detected by qRT-PCR and Western blot. Results : Compared with the control group， the pathological damage of myocardial tissue in CIH group was serious，the levels of left ventricular end diastolic diameter (LVEDD) ，left ventricular end systolic dimension (LVESD) and serum cTNI，CKMB，Mb and LDH signif- icantly increased (P＜0. 05) ，and the levels of ROS，MDA，IL-1 β , IL-6，TNF-α and the mRNA and protein levels of SENP-1 and HIF-1α in myocardial tissue also significantly increased (P ＜0. 05 ) ，while the levels of LVEF， LVFS，serum GSH and SOD significantly decreased (P ＜0. 05) ，and the SUMOylates level of HIF-1α protein in myocardial tissue also significantly decreased (P ＜0. 05 ) ．Compared with CIH group，AAV-shSENP-1 group had less myocardial pathological damage，the levels of LVEDD，LVESD and serum cTNI，CKMB，Mb and LDH signifi- cantly decreased (P＜0. 05) ，and the levels of ROS，MDA，IL-1 β, IL-6，TNF-α and the mRNA and protein levels of SENP-1 and HIF-1α in myocardial tissue also significantly decreased (P＜0. 05) ，the levels of LVEF，LVFS，serum GSH and SOD significantly increased (P＜0. 05) ，and the SUMOylates level of HIF-1α protein in myocardial tissue also significantly decreased (P＜0. 05) ． Conclusion Inhibition of SENP-1 expression can alleviate CIH induced myocarditis and oxidative stress in rats，improve myocardial injury and cardiac dysfunction，and its mechanism may be related to the improvement of HIF-1α SUMOylates level，thus inhibiting HIF-1α expression.