Downregulation of Nogo-B ameliorates cerebral ischemia/reperfusion injury in mice through regulating microglia polarization via TLR4/NF-kappaB pathway.

Many studies have shown a close association between Nogo-B and inflammation-related diseases. However, uncertainty does exist, regarding Nogo-B function in the pathological progression of cerebral ischemia/reperfusion (I/R) injury. Middle cerebral artery occlusion/reperfusion (MCAO/R) model was utilized in C57BL/6L mice to mimic ischemic stroke in vivo. Using oxygen-glucose deprivation and reoxygenation (ODG/R) model in microglia cells (BV-2) to establish cerebral I/R injury in vitro. Various methods, including Nogo-B siRNA transfection, mNSS and the rotarod test, TTC, HE and Nissl staining, immunofluorescence staining, immunohistochemistry, Western blot, ELISA, TUNEL and qRT-PCR were employed to probe into the effect of Nogo-B downregulation on cerebral I/R injury and the potential mechanisms. A small amount of Nogo-B expression (protein and mRNA) was observed in cortex and hippocampus before ischemia, then Nogo-B expression increased significantly on day 1, reaching the maximum on day 3, remaining stable on day 14 after I/R, and decreasing gradually after 21 days, but it still rose significantly compared with that observed preischemia. Nogo-B down-regulation could markedly reduce the neurological score and infarct volume, improve the histopathological changes and neuronal apoptosis, lower the number of CD86+/Iba1+ cells and the levels of IL-1ß, IL-6, and TNF-α, and raise the density of NeuN fluorescence, the number of CD206+/Iba1+ cells, and the level of IL-4, IL-10 and TGF-ß in brain of MCAO/R mice. Treatment with Nogo-B siRNA or TAK-242 in BV-2 cells could obviously decrease the CD86 fluorescence density and the mRNA expression of IL-1ß, IL-6 and TNF-α, increase CD206 fluorescence density and the mRNA expression of IL-10 after OGD/R injury. In addition, the expression of TLR4, p-IκBα and p-p65 proteins significantly increased in the brain after MCAO/R and BV-2 cells exposed to OGD/R. Treatment with Nogo-B siRNA or TAK-242 prominently reduced the expression of TLR4, p-IκBα and p-p65. Our findings suggest that the down-regulation of Nogo-B exerts protective effect on cerebral I/R injury by modulating the microglia polarization through inhibiting TLR4/NF-κB signaling pathway. Nogo-B may be a potential therapeutic target for ischemic stroke.

Targeted pharmacokinetics and bioinformatics screening strategy reveals JAK2 as the main target for Xin-Ji-Er-Kang in treatment of MIR injury.

BACKGROUND AND PURPOSE: Xin-Ji-Er-Kang (XJEK) is traditional Chinese formula presented excellent protective effects on several heart diseases, but the potential components and targets are still unclear. The aim of this study is to elucidate the effective components of XJEK and reveal its potential mechanism of cardioprotective effect in myocardial ischemia-reperfusion (MIR) injury. EXPERIMENTAL APPROACH: Firstly, the key compounds in XJEK, plasma and heart tissue were analyzed by high resolution mass spectrometry. Bioinformatics studies were also involved to disclose the potential targets and the binding sites for the key compounds. Secondly, to study the protective effect of XJEK on MIR injury and related mechanism, mice subjected to MIR surgery and gavage administered with XJEK for 6 weeks. Cardiac function parameters and apoptosis level of cardiac tissue were assessed. The potential mechanism was further verified by knock down of target protein in vitro. RESULTS: Pharmacokinetics studies showed that Sophora flavescens alkaloids, primarily composed with matrine, are the key component of XJEK. And, through bioinformatic analysis, we speculated JAK2 could be the potential target for XJEK, and could form stable hydrogen bonds with matrine. Administration of XJEK and matrine significantly improved heart function and reduced apoptosis of cardiomyocytes by increasing the phosphorylation of JAK2 and STAT3. The anti-apoptosis effect of XJEK and matrine was also observed on AC16 cells, and could be reversed by co-treatment with JAK2 inhibitor AG490 or knock-down of JAK2. CONCLUSION: XJEK exerts cardioprotective effect on MIR injury, which may be associated with the activation of JAK2/STAT3 signaling pathway.

Xin-Ji-Er-Kang Alleviates Isoproterenol-Induced Myocardial Hypertrophy in Mice through the Nrf2/HO-1 Signaling Pathway.

Xin-Ji-Er-Kang (XJEK) inhibited cardiovascular remodeling in hypertensive mice in our previous studies. We hypothesized that XJEK may prevent isoproterenol (ISO)-induced myocardial hypertrophy (MH) in mice by ameliorating oxidative stress (OS) through a mechanism that may be related to the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1(HO-1) pathways. Forty SPF male Kunming mice were randomized into 5 groups (n = 8 mice per group): control group, MH group, MH + different doses of XJEK (7.5 g/kg/day and 10 g/kg/day), and MH + metoprolol (60 mg/kg/day). On the eighth day after drug treatment, electrocardiogram (ECG) and echocardiography were performed, the mice were sacrificed, and blood and heart tissues were collected for further analysis. XJEK administration markedly ameliorated cardiovascular remodeling (CR), as manifested by a decreased HW/BW ratio and CSA and less collagen deposition after MH. XJEK administration also improved MH, as evidenced by decreased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and ß-myosin heavy chain (ß-MHC) levels. XJEK also suppressed the decreased superoxide dismutase (SOD) and catalase (CAT) activities and increased malondialdehyde (MDA) levels in serum of mice with MH. XJEK-induced oxidative stress may be related to potentiating Nrf2 nuclear translocation and HO-1 expression compared with the MH groups. XJEK ameliorates MH by activating the Nrf2/HO-1 signaling pathway, suggesting that XJEK is a potential treatment for MH.

Long-term exposure to copper induces mitochondria-mediated apoptosis in mouse hearts.

Copper is a trace element necessary for the normal functioning of organisms, but excessive copper contents may be toxic to the heart. The goal of this study was to investigate the role of excessive copper accumulation in mitochondrial damage and cell apoptosis inhibition. In vivo, the heart copper concentration and cardiac troponin I (c-TnI) and N-terminal forebrain natriuretic peptide (NT-pro-BNP) levels increased in the copper-laden model group compared to those of the control group. Histopathological and ultrastructural observations revealed that the myocardial collagen volume fraction (CVF), perivascular collagen area (PVCA) and cardiomyocyte cross-sectional area (CSA) were markedly elevated in the copper-laden model group compared with the control group. Furthermore, transmission electron microscopy (TEM) showed that the mitochondrial double-layer membrane was incomplete in the copper-laden model groups. Furthermore, cytochrome C (Cyt-C) expression was downregulated in mitochondria but upregulated in the cytoplasm in response to copper accumulation. In addition, Bcl-2 expression decreased, while Bax and cleaved caspase-3 levels increased. These results indicate that copper accumulation in cardiomyocyte mitochondria induces mitochondrial injury, and Cyt-C exposure and induces apoptosis, further resulting in heart damage.

Xin-Ji-Er-Kang protects myocardial and renal injury in hypertensive heart failure in mice.

BACKGROUND: Xin-Ji-Er-Kang (XJEK) as a herbal formula of traditional Chinese medicine (TCM) has shown the protective effects on myocardial function as well as renal function in mouse models of myocardial infarction. HYPOTHESIS/PURPOSE: We investigated the effects of XJEK on cardiovascular- and renal-function in a heart failure mouse model induced by high salt (HS) and the associated mechanisms. STUDY DESIGN: For the purpose of assessing the effects of XJEK on a hypertensive heart failure model, mice were fed with 8% high salt diet. XJEK was administered by oral gavage for 8 weeks. Cardiovascular function parameters, renal function associated biomarkers and XJEK's impact on renin-angiotensin-aldosterone system (RAAS) activation were assessed. To determine the underlying mechanism, the calpain1/junctophilin-2 (JP2)/sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) pathway was further studied in AC16 cells after angiotensin II-challenge or after calpastatin small interfering RNA (siRNA) transfection. RESULTS: Mice on HS-diet exhibited hypertensive heart failure along with progressive kidney injury. Similar to fosinopril, XJEK ameliorated hypertension, cardiovascular-and renal- dysfunction in mice of HS-diet group. XJEK inhibited HS-induced activation of RAAS and reversed the abnormal expression pattern of calpain1and JP2 protein in heart tissues. XJEK significantly improved cell viability of angiotensin II-challenged AC16 cells. Moreover, XJEK's impact on calpain1/JP2 pathway was partly diminished in AC16 cells transfected with calpastatin siRNA. CONCLUSION: XJEK was found to exert cardiovascular- and renal protection in HS-diet induced heart failure mouse model. XJEK inhibited HS-diet induced RAAS activation by inhibiting the activity and expression of calpain1 and protected the junctional membrane complex (JMC) in cardiomyocytes.

Effects of oligomeric grape seed proanthocyanidins on L-NAME-induced hypertension in pregnant mice: Role of oxidative stress and endothelial dysfunction.

The aim of this study was to investigate the effects of Grape Seed Proanthocyanidins (GSP) on Nω-Nitro-L-arginine methyl ester-induced hypertension in pregnant mice. Fifty Kunming mice were randomized into control, control + GSP, model, and model + GSP. Three weeks later, the artery systolic blood pressure was examined and the related pathological changes were detected. Aorta relaxation function was assessed by aorta ring apparatus. Blood urea nitrogen and serum creatinine were measured by an automatic biochemistry analyzer. Colorimetric analysis, enzyme-linked immunosorbent assay, immunofluorescence, and western blot were applied to detect related indicator in serum, cardiac, and kidney tissues. The results showed that GSP treatment for 3 weeks could improve cardiovascular and kidney remodeling indexes and decrease blood urea nitrogen and serum creatinine content in serum, as well as could ameliorate oxidative stress status and endothelial dysfunction. Therefore, it is for the first time found that GSP exerts protective effect against Nω-Nitro-L-arginine methyl ester-induced hypertension in pregnant mice, which provided a theoretical basis for potential application in the clinic.

[ROG,GATA3 and T-bet mRNA levels in peripheral blood mononuclear cells in patients with chronic hepatitis B].

OBJECTIVE: To investigate the role of ROG, GATA3 and T-bet in the progression of chronic hepatitis B (CHB). METHODS: The mRNA levels of ROG, GATA3 and T-bet in peripheral blood mononuclear cells (PBMCs) from 135 patients with CHB (including 45 mild cases, 42 moderate cases, and 48 severe cases) and 15 healthy control subjects were detected by real-time quantitative PCR. RESULTS: The levels of T-bet mRNA in the PBMCs were significantly higher in CHB patients than in the healthy controls (P<0.05), and also differed significantly between the 3 groups of CHB patients (P<0.05). ROG mRNA levels were significantly higher in severe cases of CHB than in the healthy controls and mild and moderate CHB cases (P<0.05), but were similar among the latter 3 groups (P>0.05). The mRNA level of GATA3 in the PBMCs were significantly higher in moderate and severe CHB cases than in the healthy controls and mild CHB cases (P<0.05). The T-bet/GATA3 ratio was significantly greater in the 3 CHB groups than in the control group (P<0.05) but comparable between the 3 CHB groups (P>0.05). ROG levels were not correlated with GATA3 levels or T-bet/GATA3 ratio in the CHB cases. CONCLUSIONS: The mRNA levels of ROG, GATA3 and T-bet in the PBMCs are obviously up-regulated in CHB patients and these 3 genes may participate in the progression of CHB. ROG plays an important role in correcting and maintaining the new balance of Th1/Th2.

Influence of chronic HBV infection on superimposed acute hepatitis E.

AIM: To investigate the influence of chronic hepatitis B virus (HBV) infection [based on the status of hepatitis B e antigen (HBeAg), HBV DNA, and cirrhosis] on superimposed acute hepatitis E. METHODS: A total of 294 patients were recruited from the Department of Infectious Diseases of the Third Affiliated Hospital, Sun Yat-sen University, from January 2003 to January 2012. The patients were classified into two groups: an HBV + hepatitis E virus (HEV) group (a group with chronic HBV infection that was superinfected with acute hepatitis E, n = 118) and an HEV group (a group with acute hepatitis E, n = 176). We retrospectively analyzed and compared the clinical features of the two groups. Statistical analyses were performed using the χ(2) test or Fisher's exact test for categorical variables and the Student's t test for continuous variables. A P value < 0.05 was considered statistically significant. RESULTS: The peak values of prothrombin time, serum total bilirubin, and Model for End-Stage Liver Disease scores were significantly higher in the HBV + HEV group. More patients in the HBV + HEV group had complications (39.8% vs 16.5%, P = 0.000) and developed liver failure (35.6% vs 8.5%, P = 0.000). Additionally, the mortality of the HBV + HEV group was significantly higher (20.3% vs 7.4%, P = 0.002). Further analysis of the HBV + HEV group showed that there were no significant differences in complication occurrence, liver failure incidence, or mortality between patients with different HBeAg and HBV DNA statuses. However, in patients with underlying cirrhosis, complication occurrence and liver failure incidence significantly increased. In total, 12.7% of the patients in the HBV + HEV group received anti-HBV treatment, but this therapy failed to reduce mortality in patients who developed liver failure. CONCLUSION: The presence of underlying cirrhosis in chronic HBV infection results in more severe clinical outcomes with superimposed acute hepatitis E. Anti-HBV treatment cannot improve the prognosis of liver failure caused by HBV-HEV superinfection.

[Construction and expression of non-structural protein gene 3-4b of HCV 1b based on the adenoassociated virus vector].

OBJECTIVE: To clone 1b type of HCV NS3-4b Gene and express in HEK 293 cells, lay the foundation for further study of the HCV NS3-4b recombinant adeno-associated virus vaccine and its dendritic cell vaccine. METHODS: HCV 1b patients' serum was collected, and full length NS3-4b segment was amplified by RT-PCR and cloned into adeno-associated virus' expression vector pAAV. CMV. EGFP in order to express in HEK 293 cells. At last, it was validated whether express or not by Western Blot. RESULTS: The 1b type gene NS3-4b were amplified and consistent to the expected size (2838 bp), the recombinant plasmid has been confirmed its successful restructured by double enzyme and sequencing, at last, Western Blot map can see objective protein expression after it transfect HEK 293 cells. CONCLUSION: The adeno-associsted virus recombination HCV NS3-4b plasmid have successfully constructed and it can express in eukaryotic cells.