Plasmacytoid Dendritic Cells Mediate Myocardial Ischemia/Reperfusion Injury by Secreting Type I Interferons.

Background We previously demonstrated that ischemically injured cardiomyocytes release cell-free DNA and HMGB1 (high mobility group box 1 protein) into circulation during reperfusion, activating proinflammatory responses and ultimately exacerbating reperfusion injury. We hypothesize that cell-free DNA and HMGB1 mediate myocardial ischemia-reperfusion injury by stimulating plasmacytoid dendritic cells (pDCs) to secrete type I interferon (IFN-I). Methods and Results C57BL/6 and interferon alpha receptor-1 knockout mice underwent 40 minutes of left coronary artery occlusion followed by 60 minutes of reperfusion (40'/60' IR) before infarct size was evaluated by 2,3,5-Triphenyltetrazolium chloride-Blue staining. Cardiac perfusate was acquired in ischemic hearts without reperfusion by antegrade perfusion of the isolated heart. Flow cytometry in pDC-depleted mice treated with multiple doses of plasmacytoid dendritic cell antigen-1 antibody via intraperitoneal injection demonstrated plasmacytoid dendritic cell antigen-1 antibody treatment had no effect on conventional splenic dendritic cells but significantly reduced splenic pDCs by 60%. pDC-depleted mice had significantly smaller infarct size and decreased plasma interferon-α and interferon-ß compared with control. Blockade of the type I interferon signaling pathway with cyclic GMP-AMP synthase inhibitor, stimulator of interferon genes antibody, or interferon regulatory factor 3 antibody upon reperfusion similarly significantly attenuated infarct size by 45%. Plasma levels of interferon-α and interferon-ß were significantly reduced in cyclic GMP-AMP synthase inhibitor-treated mice. Infarct size was significantly reduced by >30% in type I interferon receptor monoclonal antibody-treated mice and interferon alpha receptor-1 knockout mice. In splenocyte culture, 40'/0' cardiac perfusate treatment stimulated interferon-α and interferon-ß production; however, this effect disappeared in the presence of cyclic GMP-AMP synthase inhibitor. Conclusions Type I interferon production is stimulated following myocardial ischemia by cardiogenic cell-free DNA/HMGB1 in a pDC-dependent manner, and subsequently activates type I interferon receptors to exacerbate reperfusion injury. These results identify new potential therapeutic targets to attenuate myocardial ischemia-reperfusion injury.

Role of endoplasmic reticulum oxidase 1α in H9C2 cardiomyocytes following hypoxia/reoxygenation injury.

Endoplasmic reticulum (ER) oxidase 1α (ERO1α) is a glycosylated flavoenzyme that is located on the luminal side of the ER membrane, which serves an important role in catalyzing the formation of protein disulfide bonds and ER redox homeostasis. However, the role of ERO1α in myocardial hypoxia/reoxygenation (H/R) injury remains largely unknown. In the present study, ERO1α expression levels in H9C2 cardiomyocytes increased following H/R, reaching their highest levels following 3 h of hypoxia and 6 h of reoxygenation. In addition, H/R induced apoptosis, and significantly increased expression levels of ER stress (ERS) markers 78 kDa glucose­regulated protein and C/EBP homologous protein. Moreover, the genetic knockdown of ERO1α using short hairpin RNA suppressed cell apoptosis, caspase­3 activity, expression levels of cleaved caspase­12 and cytochrome c in the cytoplasm. Overall, this suggested that ERO1α knockdown may protect against H/R injury. The ERS activator tunicamycin (TM) was used to counteract the ERO1α­induced reduction in ERS; however, the percentage of apoptotic cells and the level of mitochondrial damage did not change. In conclusion, the results from the present study suggested that ERO1α knockdown may protect H9C2 cardiomyocytes from H/R injury through inhibiting intracellular ROS production and increasing intracellular levels of Ca2+, suggesting that ERO1α may serve an important role in H/R.

Lazaroid U83836E protects the heart against ischemia reperfusion injury via inhibition of oxidative stress and activation of PKC.

Oxidative stress has been demonstrated to be important during myocardial ischemia/reperfusion injury (MIRI). The lazaroid U83836E, which combines the amino functionalities of the 21­aminosteroids with the antioxidant ring portion of vitamin E, is a reactive oxygen species scavenger. The aim of the current study was to investigate the effect of U83836E on MIRI and its mechanisms of action. Rat hearts were subjected to 30 min ligation of the left anterior descending coronary artery, followed by 2 h reperfusion. The results demonstrated that at 5 mg/kg, U83836E markedly protected cardiac function in ischemia/reperfusion rat models, decreased the malondialdehyde content and creatinine kinase activity, while increasing superoxide dismutase and glutathione peroxidase activity. Additionally, U83836E significantly decreased the histological damage to the myocardium, reduced the area of myocardial infarction in the left ventricle and modified the mitochondrial dysfunction. Furthermore, U83836E enhanced the translocation of protein kinase Cε (PKCε) from the cytoplasm to the membrane. However, the cardioprotective effects of U83836E were reduced in the presence of the PKC inhibitor, chelerythrine (1 mg/kg). Therefore, the results of the present study suggest that U83836E has a potent protective effect against MIRI in rat models through the direct anti­oxidative stress mechanisms and the activation of PKC signaling.

Effect of artesunate supplementation on bacterial translocation and dysbiosis of gut microbiota in rats with liver cirrhosis.

AIM: To evaluate the effect of artesunate (AS) supplementation on bacterial translocation (BT) and gut microbiota in a rat model of liver cirrhosis. METHODS: Fifty-four male Sprague-Dawley rats were randomly divided into a normal control group (N), a liver cirrhosis group (M) and a liver cirrhosis group intervened with AS (MA). Each group was sampled at 4, 6 and 8 wk. Liver cirrhosis was induced by injection of carbon tetrachloride (CCl4), intragastric administration of 10% ethanol, and feeding a high fat diet. Rats in the MA group were intragastrically administered with AS (25 mg/kg body weight, once daily). Injuries of the liver and intestinal mucosa were assessed by hematoxylin-eosin or Masson's trichrome staining. Liver index was calculated as a ratio of the organ weight (g) to body weight (g). The gut microbiota was examined by automated ribosomal intergenic-spacer analysis of fecal DNA. BT was assessed by standard microbiological techniques in the blood, mesenteric lymph nodes (MLNs), liver, spleen, and kidney. RESULTS: Compared to group N, the body weight was reduced significantly in groups M and MA due to the development of liver cirrhosis over the period of 8 wk. The body weight was higher in group MA than in group M. The liver indices were significantly elevated at 4, 6 and 8 wk in groups M and MA compared to group N. AS supplementation partially decreased the liver indices in group MA. Marked histopathologic changes in the liver and small intestinal mucosa in group M were observed, which were alleviated in group MA. Levels of pro-inflammatory interleukin-6 and tumor necrosis factor-α were significantly elevated at 8 wk in ileal homogenates in group M compared to group N, which were decreased after AS supplementation in group MA. The dysbiosis of gut microbiota indicated by the mean diversity (Shannon index) and mean similarity (Sorenson index) was severe as the liver cirrhosis developed, and AS supplementation had an apparent intervention effect on the dysbiosis of gut microbiota at 4 wk. The occurrence of BT was increased in the liver of group M compared to that of group N. AS supplementation reduced BT in group MA at 8 wk. BT also occurred in the MLNs, spleen, and kidney, which was reduced by AS supplementation. BT was not detected in the blood in any group. CONCLUSION: Dysbiosis of gut microbiota, injury of intestinal mucosal barrier and BT occurred as liver cirrhosis progressed, which might enhance inflammation and aggravate liver injury. AS may have other non-antimalarial effects that modulate gut microbiota, inhibit BT and alleviate inflammation, resulting in a reduction in CCl4, alcohol and high fat-caused damages to the liver and intestine.

Artesunate alleviates hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats.

The current study was performed in order to explore the effect of artesunate (Art) on experimental hepatic fibrosis and the potential mechanism involved. Art, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb Artemisia Annua, is a safe and effective antimalarial drug. Hepatic fibrosis was induced in SD rats by multiple pathogenic factors. Rats were treated concurrently with Art (28.8 mg/kg) given daily by oral gavage for 6 or 8 weeks to evaluate its protective effects. Our data demonstrated that Art treatment obviously attenuated hepatic fibrosis, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM). Art remarkably decreased endotoxin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels as well. Art significantly downregulated protein and mRNA expression of α-smooth muscle actin (α-SMA), toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88) and transforming growth factor beta 1 (TGF-ß1). Art also significantly inhibited the nuclear transcription factor kappa B p65 (NF-κB p65) translocation into the nucleus. In addition, there were no remarkable differences between the N group and the NA group. In conclusion, we found that Art could alleviate hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats, suggesting that Art may be a potential candidate for the therapy of hepatic fibrosis.

Study on the interaction between ligustrazine and 12-tungstophosphoric acid using resonance Rayleigh scattering and resonance nonlinear scattering spectra, and its analytical applications.

In an HCl medium (pH 1.5), ligustrazine (2,3,5,6-tetramethylpyrazine, TMP) reacted with 12-tungstophosphoric acid (TP) to form a 3 : 1 ion-association complex. As a result, the intensities of resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS) were greatly enhanced and new scattering spectra appeared. The maximum RRS, SOS and FDS wavelengths of the ion-association complexes were located at 379, 738 and 395 nm, respectively. The scattering intensity increments (ΔIRRS , ΔISOS and ΔIFDS ) were directly proportional to the concentration of ligustrazine within certain ranges. The detection limits (3σ) of RRS, SOS and FDS were 1.6, 3.2 and 2.8 ng/mL. Optimal conditions for the RRS method and factors influencing the method were discussed, and the structure of the ion-association complex and the reaction mechanism were investigated. Transmission electron microscopy (TEM) was used to characterize the structures of the ion-association complex. Based on the ion-association reaction and its spectral response, a rapid, simple and sensitive RRS method for the determination of TMP was developed. It was applied to the determination of TMP in tablet and urine samples with satisfactory results.

Electrochemical sensors using gold submicron particles modified electrodes based on calcium complexes formed with alizarin red S for determination of Ca(2+) in isolated rat heart mitochondria.

A simple glassy carbon electrode (GCE) modified with gold submicron particles (AuSPs), characterized by a mean diameter of about0.15-0.20µm has been developed. Herein, the complexation reaction of Ca(2+) with alizarin red S (ARS), in 0.1M KOH, has been followed by electrochemical methods using the modified electrode which is able to catalyze the electro-reduction of ARS. When the stoichiometry ratio of Ca(2+) and ARS is 1:2, a new reduction peak at a higher negative potential of -0.975V appeared, and the peak of ARS at -0.815V disappeared. The peak current of ARS in alkaline solution is proportional to the concentration of Ca(2+) in the range 6.0×10(-7)-1.2×10(-4)M with a limit of detection (LOD) of 5.1×10(-7)M. Furthermore, the complex site of Ca(2+) with ARS was analysized by the experimental UV-vis and infrared spectrums and those calculated electronic and vibrational spectroscopies with density functional theory (DFT). The good accordance between theoretical and experimental data confirms that chelation of calcium ion preferentially occurs at the deprotonated catechol site. Then, we implemented an electrochemical assay for the investigation of Ca(2+) in preparations of isolated rat heart mitochondria, which demonstrates the submicron particles modified electrode is a simple and rapid sensor for determining the Ca(2+) in the biological samples.

[Protective effect of tanshinol on the hepatopulmonary syndrome in rat].

OBJECTIVE: To explore the mechanism of tanshinol on alleviate the inflammatory injury of lung tissue in rat hepatopulmonary syndrome (HPS). METHODS: SD rats were randomly divided into normal control group (n = 8), hepatopulmonary syndrome (HPS) group (n = 11) and tanshinol intervention group (n = 9). HE staining was used to observe the histopathology changes of pulmonary and hepatic tissues, and to count the number of macrophages in lung tissues. The activity of alanine transferase (ALT) and concentrations of endotoxin, tumor necrosis factor-a (TNF-alpha) and homocystein (Hcy) in plasma were detected. The concentrations of TNF-alpha, nitric oxide (NO) and malondialdehyde (MDA) and the activity of inducible nitric oxide synthase (iNOS) in the lung tissues were measured, respectively. RESULTS: Thickened alveolar septum and increased macrophages were observed in lungs in HPS rat. After administered with tanshinol, the pulmonary pathological changes were alleviated and the number of macrophages in lung tissue was decreased compared with HPS group. The activity of ALT and the concentrations of endotoxin, TNF-alpha and Hcy in plasma ,and TNF-alpha, iNOS, NO and MDA in lung tissue in HPS group were higher than those of normal control group; meanwhile, those tanshinol group were less those that of HPS group. CONCLUSION: Tanshinol may play an important role in delaying the development of HPS through protecting liver or directly antagonizing the effect of intestinal endotoxemia so as to alleviate the inflammatory reaction in lung tissue.

[A method for primary culture of pulmonary microvascular endothelial cells].

OBJECTIVE: To explore a simple and practical method of primarily culturing rat pulmonary microvascular endothelial cells (PMECs) in vitro, and observe the cell growth status and identify the PMECs. METHODS: Wistar rats (n=40, aged 4-5 weeks) were sacrificed to take the lung tissue. After removal of pleura, the peripheral lung tissues were cut into pieces (1 mm(3)) in aseptic condition. The endothelial cells were cultured in the DMEM medium containing heparin sodium and in the RPMI1640 medium supplemented with special additives or not, respectively. Cell growth and morphology was observed under an inverted microscope. The expression of CD31 in cells was detected by immunofluorescence staining. RESULTS: After incubation for 24 hours, PMECs in the medium containing special additives were the most in number and purity compared with the other two culture systems. At 24 hours, endothelial cells migrated from the lung tissue, and at 14 days, the cells aggregated and grew obviously, exhibiting a polygon shape, being tightly arranged and paving the base of Petri dish. After sub-culturing, the cells spread much more and most cells became spindle shaped, which showed a tendency of endothelial cell angiogenesis in vitro. CD31 was positive in immunofluorescence staining. CONCLUSION: The adherent culture method of tissue explants in the medium added by the special additives was proved to a good method to obtain a high-purity rat PMECs in vitro.

Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome.

OBJECTIVE: This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development. METHODS: The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB. RESULTS: The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlation between these proteins and GRP78. CONCLUSIONS: Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications.

Expression of the 78 kD glucose-regulated protein is induced by endoplasmic reticulum stress in the development of hepatopulmonary syndrome.

OBJECTIVE: This study is to explore the role of 78 kD glucose-regulated protein (GRP78) in the development of hepatopulmonary syndrome (HPS) in rats. METHODS: The rat model of liver cirrhosis and HPS were induced with multiple pathogenic factors. Hematoxylin and eosin (H & E) staining was performed to detect the pathological changes of the lung and liver tissues. The levels of alanine transferase (ALT), endotoxin, and tumor necrosis factor-α (TNF-α) in plasma and TNF-α and malondialdehyde (MDA) in lung tissues were detected. RT-PCR and Western blotting were conducted to detect the mRNA and protein expression levels of GRP78 in lungs. RESULTS: The plasma endotoxin level was gradually increased as HPS developed, and the mRNA and protein expression levels of GRP78 in lungs were also increased as the disease progressed. The levels of ALT and TNF-α in plasma and the contents of TNF-α and MDA in lung tissues were gradually increased along with the disease progression, with a strong positive correlation. Compared with controls, the plasma TNF-α level and the mRNA and protein expression levels of GRP78 in lung tissues were significantly higher in rats with HPS. The levels of endotoxin and ALT in plasma and the level of MDA in lungs were significantly higher in rats with HPS than controls. CONCLUSIONS: The increased GRP78 expression is indicative of endoplasmic reticulum stress response during HPS, which may play an important role in the disease pathogenesis.

Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris.

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.