Exogenous spermine inhibits the proliferation of human pulmonary artery smooth muscle cells caused by chemically-induced hypoxia via the suppression of the ERK1/2- and PI3K/AKT-associated pathways.

Pulmonary vascular remodeling is a significant pathological feature of hypoxia-induced pulmonary hypertension (HPH), while pulmonary artery smooth muscle cell (PASMC) proliferation plays a leading role in pulmonary vascular remodeling. Spermine (Sp), a polyamine, plays a critical role in periodic cell proliferation and apoptosis. The present study was conducted to observe the association between hypoxia-induced PASMC proliferation and polyamine metabolism, and to explore the effects of exogenous Sp on PASMC poliferation and the related mechanisms. In the present study, PASMCs were cultured with cobalt chloride (CoCl2) to establish a hypoxia model, and Sp at various final concentrations (0.1, 1, 10 and 100 µM) was added to the medium of PASMCs 40 min prior to the induction of hypoxia. Cell proliferation was measured by 3-(4,5-dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay, cell counting kit-8 assay and 5-bromo­2'­deoxyuridine (BrdU) incorporation assay. Cell cycle progression was determined by flow cytometry, and the protein expression levels of spermidine/spermine N1-acetyltransferase (SSAT; the key enzyme in the terminal degradation of polyamine), ornithine decarboxylase (ODC; the key enzyme of polyamine biosynthesis), cyclin D1 and p27 were measured by western blot analysis. The results revealed that the proliferation of the PASMCs cultured with CoCl2 at 50 µM for 24 h markedly increased. The expression of ODC was decreased and the expression of SSAT was increased in the cells under hypoxic conditions. Exogenous Sp at concentrations of 1 and 10 µM significantly inhibited hypoxia-induced PASMC proliferation, leading to cell cycle arrest at the G1/G0 phase. In addition, Sp decreased cyclin D1 expression, increased p27 expression, and suppressed the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT); however, the above-metioned parameters were not markedly affected by Sp at concentrations of 0.1 or 100 µM. These results suggest that hypoxia disrupts polyamine metabolism, and Sp at concentrations of 1 and 10 µM inhibits the increase in human PASMC proliferation caused by chemically-induced hypoxia via the suppression of the ERK1/2- and PI3K/AKT-associated pathways. This study thus offer new insight into the prevention and treatment of HPH.

[Decreased expression of calcium-sensing receptor involved in the progression of diabetic cardiomyopathy].

OBJECTIVE: To observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats. METHODS: Thirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues. RESULTS: Compared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure. CONCLUSION: These results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.

Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7-21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert's staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

[The protective effect of DR2 activation on hypoxia/reperfusion injury in the neonatal rat cardiomyocytes and related mechanism].

OBJECTIVE: To observe the effect of dopamine receptor (DR2) activation on hypoxia/reperfusion injury (HRI) in the neonatal rat cardiomyocytes, and to explore its mechanism. METHODS: The hypoxia/reperfusion (H/R) injury model was established in primarily cultured neonatal rat cardiomyocytes, and randomly assigned: control, H/R, bromocriptine (Bro) and haloperidol (Hal) groups. The cell apoptosis was detected using inverted microscope, transmission electron microscope and flow cytometry (FCM). The lactate dehydrogenase(LDH) and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell medium were analyzed. The expression of mRNA and protein of caspase-3, caspase-8, caspase-9, Fas, Fas-L, Cyt C and Bcl-2 were detected by RT-PCR and Western blot, respectively. RESULTS: Compared with the control group, apoptosis rate, LDH activity, MDA content and the expression of pro-apoptotic factors and anti-apoptotic factors were increased, but SOD activity was decreased in H/R group. Compared with the H/R group, all index above-mentioned were down-regulated or reversed in Bro-group, and had no obvious differences in Hal-group. CONCLUSION: The neonatal rat cardiomyocytes injury and apoptosis caused by hypoxia/reperfusion can be inhibited with DR2 activation, which mechanism is related to scavenging oxygen radical.

Expression of HMGB1 and RAGE in rat and human brains after traumatic brain injury.

BACKGROUND: Increasing evidence suggests that an inflammatory reaction contributes to the secondary brain injury that plays a critical role in the clinical outcome of patients with traumatic brain injury (TBI). Recently, high-mobility group box 1 (HMGB1) has been identified as a key cytokine in the inflammatory reaction and may represent a new target for the treatment of TBI. However, the expression of HMGB1 during this injury process has not yet been studied. METHODS: In this study, the levels of both HMGB1 and receptor for advanced glycation end products (RAGE) in the rat brain were analyzed by Western blot at different time points after TBI. Immunohistochemistry was also performed to examine the expression pattern of HMGB1 and RAGE in both the rat and the human brain after TBI. RESULTS: In the rat brain, HMGB1 levels significantly declined below the basal level at 6 hours after TBI and then gradually returned to the basal level 2 days later. RAGE expression increased 6 hours after TBI and reached its peak after 1 day; this level then slowly decreased but remained higher than the sham-injury group until 6 days after TBI. In both rat and human brains, HMGB1 either disappeared or was translocated from the nucleus to the cytoplasm at early stages after TBI and then was localized to the cytoplasm of phagocytic microglia at later stages. RAGE expression increased in the region surrounding the contused area after TBI in both rat and human brains. At later stages, RAGE was mainly expressed in microglia. CONCLUSION: HMGB1 is involved in both early and later stages after TBI. Targeting HMGB1 signaling may be a promising therapeutic approach for the treatment of TBI.

Small interfering RNA against the 2C genomic region of coxsackievirus B3 exerts potential antiviral effects in permissive HeLa cells.

Coxsackievirus B3 (CVB3) is the most important causal agent of viral heart muscle disease, but no specific antiviral drug is currently available. Small interfering RNA (siRNA) has been used as an antiviral therapeutic strategy via posttranscriptional gene silencing. In this study, eleven siRNAs were designed to target seven distinct regions of the CVB3 genome including VP1, VP2, VP3, 2A, 2C, 3C, and 3D. All of the siRNAs were individually transfected into HeLa cells, which were subsequently infected with CVB3. The impacts of RNA interference (RNAi) on viral replication were evaluated using five measures: cytopathic effect (CPE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 50% tissue culture infectious dose (TCID(50)), real-time RT-PCR, and Western blot. Five of the eleven siRNAs were highly efficient at inhibiting viral replication. This was especially true for siRNA-5, which targeted the ATPase 2C. However, antiviral activity varied significantly among siRNA-9, -10, and -11 even though that they all targeted the 3D region. Our results revealed several effective targets for CVB3 silencing, and provided evidence that sequences except CRE within the 2C region may also be potential targets for CVB3-specific siRNAs design. These data supported a potential role of RNA interference in future antiviral intervention therapies.

[Immunohistochemical expression of HO-1 in traumatic human brain tissue].

OBJECTIVE: To investigate the expression of heme oxygenase-1 (HO-1) at different intervals and to provide evidence for estimation on injury intervals after brain contusion in human. METHODS: Twenty-four patients died of serious brain injury were assigned as injury group and 4 patients died of non-brain injury were served as control group. HO-1 expression was analyzed in brain tissue at different time intervals (3 h, 6-9 h, 12-24 h, 36 h-3d, 5-8d, 17-20d) by immunohistochemistry and auto-image analysis system. RESULTS: The level of HO-1 expression started to increase in 3 h after brain contusion compared to the control group (P < 0.05). The level of HO-1 expression highest level in 12-24 h group, and maintained high level in 36 h-3 d, then decreased gradually. CONCLUSION: The expression of HO-1 might be a strong evidence for human brain contusion time estimation.