Chronic high­salt intake induces cardiomyocyte autophagic vacuolization during left ventricular maladaptive remodeling in spontaneously hypertensive rats.

The role of autophagy in high-salt (HS) intake associated hypertensive left ventricular (LV) remodeling remains unclear. The present study investigated the LV autophagic change and its association with the hypertensive LV remodeling induced by chronic HS intake in spontaneously hypertensive rats (SHR). Wistar Kyoto (WKY) rats and SHR were fed low-salt (LS; 0.5% NaCl) and HS (8.0% NaCl) diets and were subjected to invasive LV hemodynamic analysis after 8, 12 and 16 weeks of dietary intervention. Reverse transcription-quantitative PCR and western blot analysis were performed to investigate the expression of autophagy-associated key components. The LV morphologic staining was performed at the end of the study. The rat H9c2 ventricular myoblast cell-associated experiments were performed to explore the mechanism of HS induced autophagic change. A global autophagy-associated key component, as well as increased cardiomyocyte autophagic vacuolization, was observed after 12 weeks of HS intake. During this period, the heart from HS-diet-fed SHR exhibited a transition from compensated LV hypertrophy to decompensation, as shown by progressive impairment of LV function and interstitial fibrosis. Myocardial extracellular [Na+] and the expression of tonicity-responsive enhancer binding protein (TonEBP) was significantly increased in HS-fed rats, indicating myocardial interstitial hypertonicity by chronic HS intake. The global autophagic change and overt deterioration of LV function were not observed in LS-fed SHR and HS-fed WKY rats. The study of rat H9c2 cardiomyocytes demonstrated a cytosolic [Na+] elevation-mediated, reactive oxygen species-dependent the autophagic change occurred when exposed to an increased extracellular [Na+]. The present findings demonstrated that a myocardial autophagic change participates in the maladaptive LV remodeling induced by chronic HS intake in SHR, which provides a possible target for future intervention studies on HS-induced hypertensive LV remodeling.

[Research progress on pesticide residues of Panacis Quinquefolii Radix].

Panacis Quinquefolii Radix is the dry root of Panax quinquefolium, which is a perennial plant of Araliaceae. The plant has a long growth cycle and serious growth barrier problem, which leads to the use of pesticides. As a result, the pesticide residues in Panacis Quinquefolii Radix are arousing great concern. This paper reviews the research findings on the investigation, detection methods, content analysis and risk assessment of pesticide residues in Panacis Quinquefolii Radix since 1993, and compares the pesticide residue limit standards of different countries and regions. The pesticide residues in Panacis Quinquefolii Radix have been changing from organochlorines with high toxicity to triazines and triazoles with low toxicity. The pesticide residues are generally low, while the pollution of pentachloronitrobenzene and other pesticides still exist. The detection method has evolved from chromatography to chromatography-mass spectrometry. There are no reports of health risks caused by pesticide residues of Panacis Quinquefolii Radix. Pesticide residue is a major factor restricting the sound development of Panacis Quinquefolii Radix industry in China. Therefore, we suggest to improve the registration of pesticides applied to the plant, popularize mature ecological planting mode and supporting technology, and strengthen the research on the risk assessment and limit standard of pesticide residue in Panacis Quinquefolii Radix.

[Research progress on pesticide residues of Lonicera Japonica Flos].

Lonicera Japonica Flos is the dried bud or nascent flower of Lonicera japonica(Caprifoliaceae). The plant suffers from various diseases and pests in the growth period and thus pesticides are often used. As a result, the resultant pesticide residues in Lonicera Japonica Flos have aroused great concern. This review summarized the investigation, detection methods, content analysis, and risk assessment of pesticide residues in Lonicera Japonica Flos since 1996, and compared the maximum residue limits among different countries and regions. The results showed that the pesticide residues were detected in Lonicera Japonica Flos from different production areas, and only some exceeded the limits. The residual pesticides have changed from organochlorines to new types such as tebuconazole and nitenpyram. The detection method has upgraded from chromatography to chromatography-mass spectrometry. Most pesticide residues will not cause health risks, except carbofuran. Pesticide residues limit the development of Lonicera Japonica Flos industry in China. In practice, we should improve the drug registration of Lonicera Japonica Flos, promote ecological prevention and control technology, and formulate and promote pesticide residue limit standard of Lonicera Japonica Flos.

[Effect of iron death inhibitor on hypoxia/reoxygenation injury of cardiomyocytes and its mechanism].

OBJECTIVE: To investigate the effects of ferrostatin-1 (Fer-1) on cardiomyocyte hypoxia/reoxygenation injury and its mechanisms. METHODS: The original generation of myocardial cells were extracted from 1～3 d newborn SD rats, which were randomly divided into normal control group (control), hypoxia reoxygenation (H/R) group and hypoxia reoxygenation + iron death inhibitors group (H/R + Fer-1). After 52 h of culture, cells in H/R group were added with 4 mmol/L Na2S2O4 solution. After 1 h of hypoxia, cells were reoxygenated with DMEM medium containing 10% calf serum for 3 h.The H/R+ Fer-1 group was pretreated with Fer-1 (2 µmol/L) for 24 h and then subjected to hypoxia and reoxygenation. The release rate of lactate dehydrogenase (LDH) was measured by UV spectrophotometry, the cell survival rate was measured by CCK-8 method, SOD was measured by xanthine oxidase method, MDA was measured by chemical coloration, and the changes of mitochondrial membrane potential and reactive oxygen species (ROS) were observed by immunofluorescence. Western blot was used to detect the expressions of ACSL4 and GPX4. RESULTS: Compared with the control group, the cell activity, SOD release and MMP level were decreased (P＜0.05), the levels of LDH, MDA and ROS were increased (P＜0.05), the protein expression of ACSL4 was increased (P＜0.05), and the protein expression of GPX4 was decreased (P＜0.05) in H/R group. Compared with the H/R group, the cell activity, SOD release and MMP level were increased (P＜0.05), the level of LDH, MDA and ROS were decreased (P＜0.05), the protein expression of ACSL4 was decreased (P＜0.05), and the protein expression of GPX4 was increased (P＜0.05) in H/R+Fer-1 group. CONCLUSION: Fer-1 can inhibit the production of intracellular reactive oxygen species by regulating ACSL4 and GPX4, thereby alleviating the hypoxia and reoxygenation injury of primary cardiomyocytes caused by iron death.

Pseudolaric Acid B Attenuates High Salt Intake-Induced Hypertensive Left Ventricular Remodeling by Modulating Monocyte/Macrophage Phenotypes.

BACKGROUND Studies in ApoE knockout mice have shown that pseudolaric acid B (PB) can act as an immunomodulatory drug and attenuate atherosclerosis progression by modulating monocyte/macrophage phenotypes. Our previous study demonstrated that high salt intake could shift the phenotype of monocytes/macrophages to an inflammatory phenotype, and that this shift was related to hypertension and hypertensive left ventricular (LV) remodeling. However, no comprehensive assessment of the effects of PB on hypertensive LV remodeling has been conducted. MATERIAL AND METHODS In this study, RAW264.7 macrophages cultured with different concentrations of NaCl were used to investigate the modulating effects of PB on macrophage phenotype. Furthermore, N-nitro-L-arginine methyl ester hypertensive mice were used to investigate the modulating effects of PB on monocyte phenotype. LV remodeling was investigated by echocardiography. LV morphologic staining (for cardiomyocyte hypertrophy and collagen deposition) was performed at the time of sacrifice. RESULTS The results showed that PB significantly improved the viability of RAW264.7 cells, suppressed their phagocytic and migration abilities, and inhibited their phenotypic shift to M1 macrophages. In addition, the blood pressure of PB-treated mice was significantly decreased relative to that of control mice. Furthermore, after PB treatment, the percentage of Ly6Chi monocytes was significantly decreased while that of Ly6Clo monocytes was apparently increased. Moreover, PB preserved LV function and alleviated myocardial fibrosis and cardiomyocyte hypertrophy as measured at the end of the experimental period. The transfer of monocytes from PB-treated mice to hypertensive mice achieved the same effects. CONCLUSIONS Together, these findings indicate that PB exerts its protective effects on hypertensive LV remodeling by modulating monocyte/macrophage phenotypes and warrants further investigation.

[The expression of calcium-sensing receptor in rats with acute myocardial infarction and its effect on cells apoptosis].

OBJECTIVE: To investigate the change of calcium sensing receptor (CaSR) expression at different time in rat tissue with acute myocardial infarction (AMI) and its effect on cardiomyocyte apoptosis. METHODS: The healthy Wistar rats were randomly divided into Sham and AMI groups, the rat myocardial infarction model was established by ligating left anterior descending coronary artery. The changes of cardiac morphology and hemodynamics were detected at 1, 2 and 4 weeks,respectively. The expressions of CaSR mRNA and protein in myocardial tissue were detected by RT-PCR and Western blot, respectively. The expressions of Bax, Bcl-2, caspase-3 and caspase-9 proteins were detected by Western blot. The serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) activity and cardiac troponin (cTnT) were determined. The apoptosis of cardiomyocytes were tested by TUNEL staining. RESULTS: Compared with the sham group, the expressions of CaSR mRNA and protein, the apoptosis index were increased significantly with the development of AMI (P＜0.05). The ultrastructural damage of cardiomyocytes was serious; the levels of LVSP, +dp/dtmax and -dp/dtmax were decreased,while the levels of LVEDP was increased (P＜0.05); In AMI group, the cTnT level, CK and LDH activities were all increased (P＜0.05). With the development of myocardial infarction, the cTnT level and CK activity were gradually decreased, while the activity of LDH was not significantly changed. The expressions of promote apoptosis-related Bax, caspase-3 and caspase-9 were significantly increased, and the expression of inhibited apoptosis-related protein(factor)Bcl-2 was significantly decreased (P＜0.05). CONCLUSION: With the development of myocardial infarction,the expressions of CaSR mRNA and protein,the apoptosis index in rat myocardial tissue were increased with time prolongation after AMI. The increased expression of CaSR is involved in rat myocardial infarction, which is related with apoptosis.

The Dynamics of Circulating Monocyte Subsets and Intra-Plaque Proliferating Macrophages during the Development of Atherosclerosis in ApoE-/- Mice.

To detect the development of monocytes and proliferative macrophages in atherosclerosis of ApoE-/- mice, we randomly assigned 84 ApoE-/- mice fed western diet or chow diet. On weeks 2, 4, 6, 8, 10, and 12 after fed high-fat diet or normal chow diet, animals were euthanized (n = 7 for each group at each time point). Flow cytometry methods were used to analyze the proportions of circulation monocyte subsets. The macrophage and proliferative macrophage accumulation within atherosclerotic plaques was estimated by confocal florescence microscopy. Plasma levels of total cholesterol and triglyceride were measured by ELISA kit. The plaques of aortic sinus were stained with Oil Red O. The percent of Ly6Chi circulation monocyte, the density of proliferation macrophage, the total plasma cholesterol and triglyceride levels, the lesion area of ApoE-/- mice were consistently elevated in chow diet throughout the trial. The total plasma cholesterol and triglyceride levels, the lesion area were elevated in western diet group with age, and they were always higher than the chow diet group. The Ly6Chi monocytes and proliferative macrophages reached a plateau at 8 weeks and 6 weeks; despite continued high-triglyceride high-cholesterol diet the percent did not significantly change. Interestingly, the density of macrophage did not change significantly over age in western and chow diet groups. Our results provide a dynamic view of Ly6Chi monocyte subset, the density of macrophage and proliferation macrophage change during the development and progression of atherosclerosis, which is relevant for designing new treatment strategies targeting mononuclear phagocytes in this model.

Effects of allisartan isoproxil on blood pressure and target organ injury in patients with mild to moderate essential hypertension.

Evidence has shown that angiotensin II type 1 receptor antagonists have lower blood pressure and have target organ protective effects, but this is not the case for the drug allisartan isoproxil. The aim of this study was to evaluate the effects of allisartan isoproxil on blood pressure and target organ injury in patients with mild to moderate essential hypertension.In total, 80 essential hypertensive participants were randomly divided into an allisartan group and a nifedipine group (n = 40 per group), and their blood pressure was measured once per month for 6 months. A 2-dimensional echocardiogram was performed at baseline and at the end of the study. The serum levels of renal injury indexes, endothelial function markers, inflammatory factors, blood biochemical assays and urinary measurements were determined at baseline and at 6 months.At the end of the study, both systolic and diastolic blood pressure were significantly decreased in the allisartan group compared with baseline and showed the same antihypertensive effect as the nifedipine group. Meanwhile, the left ventricular remodeling, 24-hours levels of urinary microalbumin, endothelial dysfunction, and arterial stiffness were all significantly improved compared with that of the baseline and the nifedipine group (all P < .05).The present study showed that allisartan isoproxil had favorable blood pressure lowering and heart, renal, and endothelial protective effects in patients with mild to moderate essential hypertension.

Effects of a low salt diet on isolated systolic hypertension: A community-based population study.

Evidence has shown that long-term sodium reduction can not only reduce blood pressure, but also provide cardiovascular benefits. To date, there is little evidence related to the effects of salt reduction on isolated systolic hypertension (ISH).A total of 126 hypertensive patients were divided into an ISH group (n = 51) and a non-ISH (NISH) group (n = 75). The members of each group were then randomly assigned to low sodium salt (LSSalt) or normal salt (NSalt) diets for 6 months. Their blood pressure was measured every 2 months. Serum plasma renin-angiotensin activity, blood biochemical assays and urinary measurements were determined at the baseline and at the end of the 6 months.At the end of the study, the mean systolic blood pressure (SBP) of the ISH LSSalt group had significantly decreased by 10.18 mm Hg (95% confidence interval (CI): 3.13 to 17.2, P = .006) compared with that of the ISH NSalt group, while the mean SBP only decreased by 5.10 mm Hg (95% CI: -2.02 to 12.2, P = .158) in the NISH LSSalt group compared with that of the NISH NSalt group. The mean diastolic blood pressure (DBP) had no significant differences in the ISH and NISH groups. No obvious renin angiotensin system activation was found after LSSalt intervention. Regarding the urinary excretion of electrolytes and blood biochemical assays, the LSSalt treatment had the same effects on the ISH group as on the NISH group.The present study showed that the SBP of ISH patients was significantly decreased with the LSSalt intervention, while neither the SBP of the NISH patients nor the DBP of either group were similarly decreased, which indicated that ISH patients were more sensitive to salt restriction.

Discordance Between VASP Phosphorylation and Platelet Aggregation in Defining High On-Clopidogrel Platelet Reactivity After ST-Segment Elevation Myocardial Infarction.

To investigate potential clinical characteristics associated with discordance between platelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) flow cytometry (FCM) assay and light transmission aggregometry (LTA) in defining high on-clopidogrel platelet reactivity (HPR) after ST-segment elevation myocardial infarction (STEMI). In this study, platelet responsiveness was measured by the above 2 methods simultaneously on day 1 and on day 6 of STEMI onset in 90 consecutive patients who underwent primary percutaneous coronary intervention. The FCM-derived platelet reactivity index and LTA-derived platelet aggregation rate were both significantly reduced after dual antiplatelet therapy on day 6. Multiple variable-adjusted logistic regression analysis revealed that smoking (odds ratio [OR]: 4.507, 95% confidence interval [CI]: 1.123-18.09, P = .034) and onset-to-admission time (per 1 hour increase, OR: 1.196, 95% CI: 1.023-1.398, P = .025) both were independent predictors for the discordance between the 2 methods. Additionally, improved correlation and concordance was observed in nonsmokers compared with smokers. Our data show that smoking and prolonged onset-to-admission time are associated with discordance between platelet VASP-P and LTA in defining HPR after STEMI, which should be considered when planning personalized antiplatelet therapy.

[Analyze moisture transformation and transport rules during processing of Paeoniae Radix Alba by using low-field NMR].

Moisture status and content during the processing of Paeoniae Radix Alba were studied by nuclear magnetic resonance (NMR) and nuclear magnetic resonance imaging (MRI) to investigate the changes of transverse relaxation time (T 2) and MRI images during boiling and drying processes of Paeoniae Radix Alba. The results showed that water in Paeoniae Radix Alba fresh products was major of free water, and in the boiling process, the content of free water increased whereas the content of bound water declined. At the end of boiling, content of free water reached over 90%. During the drying process, T 2 moved to the left, and moisture mobility was reduced. The MRI image directly showed that moisture transfer was outside-in process for both increase and decrease. At the end of drying, remaining moisture was mainly present in inner layer of Paeoniae Radix Alba. Quality and appearance were affected by the change of moisture during processing process of medicinal herbs. NMR and MRI could provide direct reference evidence for its moisture changes, and the results of this study could provide direct references and technical support for optimization of processing process of root medicinal materials and evaluation of Chinese herbal pieces.

[A new benzamide derivative from rice fermentation of Streptomyces sp. CPCC 202950].

Eight compounds were isolated from the rice fermentation of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography over silica, Sephadex LH-20, flash C18, and reversed-phase HPLC. Their structures were identified as 3-[(3'-amino-3'-oxoprop-1'-en-2'-yl)oxy]benzamide (1), m-hydroxybenzamide (2), leptosphaepin (3), 5-methyluracil (4), feruloylamide (5), p-hydroxyphenylacetoamide (6), vanillamide (7), cyclo (L-val-L-ala) (8). Among them, 1 was a new benzamide analogue, and 2 was a new natural product. In the preliminary assays, none of the compounds 1-8 exhibited obvious inhibition of HIV-1 protease activity, and toxic with the Hela, HepG2, and U2OS cells. (IC50 > 10 µmol•L⁻¹).

VEGF-C-mediated cardiac lymphangiogenesis in high salt intake accelerated progression of left ventricular remodeling in spontaneously hypertensive rats.

High salt (HS) diet can accelerate the progress of hypertensive left ventricular (LV) remodeling. But the detailed mechanism remains poorly understood. We hypothesized HS intake could impact cardiac lymphangiogenesis through tonicity-responsive enhancer binding protein (TonEBP)/vascular endothelial growth factor-C (VEGF-C) signaling pathway which might play an important role in HS intake accelerated LV remodeling. Eight-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were randomized to 0.5% NaCl (Low salt, LS) and 8% NaCl (high salt, HS) diets for 12 weeks. LV remodeling was determined by echocardiography. LV invasive hemodynamic analysis and morphologic staining (cardiomyocyte hypertrophy, collagen deposition, TonEBP expression, macrophage infiltration and lymphatic density) were performed at the time of sacrifice. The blood pressure of SHR-HS group was significantly increased compared to SHR-LS and WKY groups. Meanwhile, The LV chamber size was markedly enlargement, LV function apparently compromised accompanied with a severe macrophage infiltration, and fibrosis in the perivascular and interstitium of LV compared with SHR-LS group. Furthermore, the expression levels of VEGF-C, TonEBP, and lymphatic markers in SHR-HS group were significantly increased parallel with apparent lymphangiogenesis compared with SHR-LS group. Our work indicates that TonEBP/VEGF-C signaling pathway was up-regulated in HS intake accelerated hypertensive LV remodeling process that may be valuable for further investigation.

Overexpression of VEGF-C attenuates chronic high salt intake-induced left ventricular maladaptive remodeling in spontaneously hypertensive rats.

Recent studies have shown that the tonicity-responsive enhancer binding protein (TonEBP)/vascular endothelial growth factor-C (VEGF-C) signaling pathway-induced lymphangiogenesis provides a buffering mechanism for high salt (HS) intake-induced elevation of blood pressure (BP). Moreover, blocking of TonEBP/VEGF-C signaling by mononuclear phagocyte depletion can induce salt-sensitive hypertension in rats. We hypothesized that HS intake could have an impact on cardiac lymphangiogenesis, and regulation of VEGF-C bioactivity, which is largely through the main receptor for VEGFR-3, may modulate HS intake-induced left ventricular remodeling. We demonstrated upregulation of TonEBP, increased macrophage infiltration, and enhanced lymphangiogenesis in the left ventricles of spontaneously hypertensive rats (SHR) that were fed a HS diet (8.0% NaCl). Then, retrovirus vectors capable of overexpression (ΔNΔC/VEGF-C/Cys152Ser, used for overexpressing VEGF-C) and blocking (VEGFR-3-Rg, used for trapping of bioactive VEGF-C) of VEGF-C and control vector (pLPCX) were intravenously administered to SHR from week 9 of a 12-wk HS loading period. At the end of the HS challenge, overexpression of VEGF-C led to enhanced cardiac lymphangiogenesis, decreased myocardial fibrosis, and macrophage infiltration, preserved left ventricular functions, as well as decreased blood pressure level compared with the HS group and the control vector-treated HS group. In contrast, systemic blocking of VEGF-C was associated with elevation of blood pressure level and an exacerbation of hypertensive left ventricular remodeling, as indicated by increased fibrosis and macrophage infiltration, and diminished lymphangiogenesis. Hence, our findings highlight that VEGF-C/VEGFR-3 is a promising therapeutic target to attenuate hypertensive left ventricular remodeling induced by HS intake, presumably via blood pressure-dependent and -independent mechanisms.

Decreased circulating microRNA-223 level predicts high on-treatment platelet reactivity in patients with troponin-negative non-ST elevation acute coronary syndrome.

To investigate the relationship between circulating microRNA 223 (miR-223) levels and clopidogrel responsiveness in patients with coronary heart disease. A total of 62 consecutive patients with troponin-negative non-ST elevation acute coronary syndrome (NSTE-ACS) scheduled for elective percutaneous coronary intervention were enrolled. The plasma circulating miR-223 levels were quantified by real-time PCR, and platelet reactivity was determined by platelet reactivity index (PRI), measured by vasodilator-stimulated phosphoprotein (VASP) phosphorylation flow cytometry after 300 mg (for at least 24 h) or 75 mg clopidogel (for at least 5 days) plus aspirin treatment. All subjects were dichotomized according to PRI median (normal-responders: PRI ≤ 56.3%, n = 31 and low-responders: PRI > 56.3%, n = 31). Compared with normal-responders, circulating miR-223 level was significantly decreased in low-responders (P = 0.007). In addition, miR-223 level was statistically correlated with PRI (Spearman r = -0.379, P = 0.002). Stepwise binary logistic regression analysis revealed that among factors that potentially influence platelet reactivity (CYP2C19*2/*3 loss-of-function genotypes, use of calcium channel blockers/proton-pump inhibitors, age, diabetes and smoking), decreased circulating miR-223 level was the only independent predictor for the presence of PRI-determined lower responders (OR 0.111, 95% CI 0.018-0.692, P = 0.019). Our data suggest that circulating miR-223 may serve as a novel biomarker for assessment of clopidogrel responsiveness in troponin-negative NSTE-ACS patients.

Spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments.

BACKGROUND: Recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. Mineralocorticoid receptor (MR) has been reported as a target to regulate macrophage polarization. The present work was designed to investigate the therapeutic potential of MR antagonism in bleomycin-induced acute lung injury and fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: We first demonstrated the expression of MR in magnetic bead-purified Ly6G-/CD11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (BALF) from C57BL/6 mice. Then, a pharmacological intervention study using spironolactone (20 mg/kg/day by oral gavage) revealed that MR antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor ß1, and interleukin-1ß at mRNA and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by Masson' trichrome staining) in bleomycin treated (2.5 mg/kg, via oropharyngeal instillation) male C57BL/6 mice. Moreover, serial flow cytometry analysis in blood, BALF and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating Ly6C(hi) monocyte expansion, and reduce alternative activation (F4/80+CD11c+CD206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (F4/80+CD11c-) remained unaffected by spironolactone during investigation. CONCLUSIONS/SIGNIFICANCE: The present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of MR-mediated circulating monocyte and alveolar macrophage phenotype switching.

Modulation of mononuclear phagocyte inflammatory response by liposome-encapsulated voltage gated sodium channel inhibitor ameliorates myocardial ischemia/reperfusion injury in rats.

BACKGROUND: Emerging evidence shows that anti-inflammatory strategies targeting inflammatory monocyte subset could reduce excessive inflammation and improve cardiovascular outcomes. Functional expression of voltage-gated sodium channels (VGSCs) have been demonstrated in monocytes and macrophages. We hypothesized that mononuclear phagocyte VGSCs are a target for monocyte/macrophage phenotypic switch, and liposome mediated inhibition of mononuclear phagocyte VGSC may attenuate myocardial ischemia/reperfusion (I/R) injury and improve post-infarction left ventricular remodeling. METHODOLOGY/PRINCIPAL FINDINGS: Thin film dispersion method was used to prepare phenytoin (PHT, a non-selective VGSC inhibitor) entrapped liposomes. Pharmacokinetic study revealed that the distribution and elimination half-life of PHT entrapped liposomes were shorter than those of free PHT, indicating a rapid uptake by mononuclear phagocytes after intravenous injection. In rat peritoneal macrophages, several VGSC α subunits (NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaVX, Scn1b, Scn3b and Scn4b) and ß subunits were expressed at mRNA level, and PHT could suppress lipopolysaccharide induced M1 polarization (decreased TNF-α and CCL5 expression) and facilitate interleukin-4 induced M2 polarization (increased Arg1 and TGF-ß1 expression). In vivo study using rat model of myocardial I/R injury, demonstrated that PHT entrapped liposome could partially suppress I/R injury induced CD43+ inflammatory monocyte expansion, along with decreased infarct size and left ventricular fibrosis. Transthoracic echocardiography and invasive hemodynamic analysis revealed that PHT entrapped liposome treatment could attenuate left ventricular structural and functional remodeling, as shown by increased ejection fraction, reduced end-systolic and end-diastolic volume, as well as an amelioration of left ventricular systolic (+dP/dt max) and diastolic (-dP/dt min) functions. CONCLUSIONS/SIGNIFICANCE: Our work for the first time demonstrates the therapeutic potential of VGSC antagonism via liposome mediated monocyte/macrophage targeting in acute phase after myocardial I/R injury. These results suggest that VGSCs in mononuclear phagocyte system might be a novel target for immunomodulation and treatment of myocardial I/R injury.

Antiangiogenic triterpenes isolated from Chinese herbal medicine Actinidia chinensis Planch.

Actinidia chinensis Planch. is a famous Chinese herbal medicine to treat many diseases such as cancers. Triterpenes, polyphenols and anthraquinones are normally considered as the main constituents for its effects. In this study, eleven known triterpenes were isolated from the root of Actinidia chinensis., and were examined for its antiangiogenic activities. Their structures were elucidated by comprehensive spectroscopic methods, including IR, UV, HR-ESI-MS, and 1D and 2D NMR techniques. The eleven compounds are following: 2α,3α,19-trihydroxyurs-12-en-28-oic acid (1), 2α,3ß-dihydroxyurs-12-en-28-oic acid (2), 2α,3α,23-trihydroxyurs-12-en-28-oic acid (3), asiatic acid (4), ursolic acid (5), 2α,3ß,19,24-tetrahydroxyurs-12-en-28-oic acid (6), 2α,3ß,19-trihydroxyolean-12-en-28-oic acid (7), 2α,3α,24-trihydroxyolean-12-en-28-oic acid (8), oleanolic acid (9), 3ß-O-acetyloleanolic acid (10), 2α,23-dihydroxylmicromeric acid (11). All these compounds were evaluated with respect to their antiangiogenic activities utilizing the assays of human umbilical vein endothelial cells (HUVEC) proliferation and tube formation and Ursolic acid (used as control) and compounds 2, 3, 4, and 8 exhibited significant, dose-dependently, antiangiogenic activity in the tested concentration range. Our findings suggest that antitumor action of Actinidia chinensis Planch. is partly via inhibiting tumor angiogenesis by triterpenes, and compounds 2, 3, 4, and 8 as the novel potential antiangiogenic agents are worthy of further translational research.

[Construction of recombinant rat δNδC/VEGF-C/Cys152Ser retrovirus vector and its expression in RAW 264.7 cells].

AIM: To construct a retroviral vector containing rat δNδC/VEGFand verify its expression in RAW 264.7 cells. METHODS: The ddVEGF-C gene was amplified by polymerase chain reaction (PCR) from pSecTag-ddVEGF-C and cloned into pLPCX vector. After the procedure of PCR, double enzyme digestion analysis and DNA sequencing, the recombinant plasmid was transfected into packaging cells PT67 using Lipofectamine(TM); 2000, and the positive clones were collected by means of puromycin selection and detected for the viral titer. The expression of ddVEGF-C mRNA and protein in the RAW264.7 cells was determined by RT-PCR and Western blotting, respectively. RESULTS: PCR and double enzyme digestion analysis demonstrated that the recombinant pLPCX-ddVEGF-C was successfully constructed by displaying two positive bands of 382 bp and 6 191 bp as expected. Viral titer was 2×10(7); CFU/mL. RT-PCR and Western blotting showed the expression of ddVEGF-C at the mRNA and protein levels in RAW 264.7 cells. CONCLUSION: The recombinant vector pLPCX-ddVEGF-C has been successfully constructed as well as PT67 packaging cells expressing stably ddVEGF-C, which provides a potential tool for further VEGF-C related study.

Antiangiogenic polyketides from Peperomia dindygulensis Miq.

Two new polyketides: 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen- 4-one (2), together with eleven known compounds: 4-hydroxy-2-[(3,4-methylenedioxy- phenyl)tridecanoyl] cyclohexane-1,3-dione (3), oleiferinone (4), 4-hydroxy-2-[(3,4- methylenedioxyphenyl)undecanoyl]cyclohexane-1,3-dione (5), 4-hydroxy-2-[(11-phenyl- undecanoyl)cyclohexane-1,3-dione (6), proctorione C (7), surinone C (8), 5-hydroxy- 7,8,4'-trimethoxyflavone (9), 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10), 5-hydroxy- 7,3',4'-trimethoxyflavone (11), 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) and cepharanone B (13) were isolated from the whole plant of Peperomia dindygulensis Miq. Their structures were elucidated by spectroscopic methods, including 2D-NMR techniques. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cell (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.