Exploring the mechanism of luteolin by regulating microglia polarization based on network pharmacology and in vitro experiments.

Neuroinflammation manifests following injury to the central nervous system (CNS) and M1/M2 polarization of microglia is closely associated with the development of this neuroinflammation. In this study, multiple databases were used to collect targets regarding luteolin and microglia polarization. After obtaining a common target, a protein-protein interaction (PPI) network was created and further analysis was performed to obtain the core network. Molecular docking of the core network with luteolin after gene enrichment analysis. In vitro experiments were used to examine the polarization of microglia and the expression of related target proteins. A total of 77 common targets were obtained, and the core network obtained by further analysis contained 38 proteins. GO and KEGG analyses revealed that luteolin affects microglia polarization in regulation of inflammatory response as well as the interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways. Through in vitro experiments, we confirmed that the use of luteolin reduced the expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, p-NFκBIA (p-IκB-α), p-NFκB p65, and MMP9, while upregulating the expression of Arg-1 and IL-10. This study reveals various potential mechanisms by which luteolin induces M2 polarization in microglia to inhibit the neuroinflammatory response.

Based on network pharmacology and in vitro experiments to prove the effective inhibition of myocardial fibrosis by Buyang Huanwu decoction.

Among cardiovascular diseases, myocardial fibrosis (MF) is a major pathological change underlying heart failure and is associated with a high mortality rate. However, the molecular mechanism underlying MF has remained elusive. Buyang Huanwu decoction (BYHWD), a traditional Chinese medicine (TCM) formula for cardiovascular diseases, exhibits good anti-inflammatory and blood-activating properties. In the present study, we studied the MF inhibitory effect of BYHWD using network pharmacology and experimental validation. We used several databases to collect information on MF and related drugs and finally obtained cross-targets for BYHWD and MF. After that we got protein-protein interaction (PPI) network and performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses to obtain key signaling pathways for further study. After screening, interleukin (IL)-6, IL-1ß, and matrix metallopeptidase 9 (MMP9) were selected for in vitro experiments, which included cell cycle studies, cell migration rate, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and western blotting (WB). Finally, molecular docking was performed to validate the results. We found 299 common targets between BYHWD and MF. In total, 75 core targets of the PPI core network were selected for enrichment analysis, and the IL-17 signaling pathway, which is intricately linked to inflammation, was speculated to be involved. Accordingly, in vitro experiments were performed. Altogether, our findings indicated that BYHWD can affect the function of cardiac fibroblasts and reduce the expression of inflammatory factors in rats. In summary, BYHWD can inhibit MF by reducing the expression of inflammatory factors and affecting the IL-17 signaling pathway.

Enhanced Migration of Bone Marrow-Derived Mesenchymal Stem Cells with Tetramethylpyrazine and Its Synergistic Effect on Angiogenesis and Neurogenesis After Cerebral Ischemia in Rats.

Bone marrow-derived mesenchymal stem cells (BMSCs) hold great promise for treating ischemic stroke owing to their capacity to secrete various trophic factors with potent angiogenic and neurogenic potentials. However, the relatively poor migratory capacity of BMSCs toward infarcted regions limits effective therapies for the treatment of stroke. The combination of BMSCs and pharmacological agent can promote the migration of BMSCs toward infarcted regions and improve the therapeutic effects after stroke. In this study, we aimed to investigate whether BMSCs combined with tetramethylpyrazine (TMP) enhanced BMSC migration into the ischemic brain, which had better therapeutic effect in the treatment of stroke. In a rat stroke model, we found that combination treatment significantly upregulated ischemic brain stromal-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) expressions, and promoted BMSCs homing toward the ischemic regions than BMSC monotherapy. Moreover, BMSCs combined with TMP synergistically increased the expression of vascular endothelial growth factor and brain-derived neurotrophic factor, promoted angiogenesis and neurogenesis, and improved functional outcome after stroke. These results suggest that combination treatment could not only enhance the migration of BMSCs into the ischemic brain but also act in a synergistic way to potentiate endogenous repair processes and functional recovery after ischemic stroke.

Assessment of the association between serum uric acid levels and the incidence of hypertension in nonmetabolic syndrome subjects: A prospective observational study.

The purpose of this study was to examine the association between serum uric acid (sUA) and the incidence of hypertension in nonmetabolic syndrome (non-MetS) subjects.This was a prospective observational study including 23,525 subjects who had been followed up for at least 5 years. A logistic regression model was used to assess independent risk factors associated with hypertension. An area under the receiver operating characteristic curve (auROC) was generated, and a nomogram was developed to assess diagnostic ability of sUA and the sUA-based score.We enrolled 11,642 subjects, and 763 (6.55%) were diagnosed with hypertension at the 5-year follow-up. Subjects were classified into 4 groups based on the sUA quarter. Using Q1 as the reference group, Q2, Q3, and Q4 were found to show a higher risk for the development of hypertension with odds ratio of 1.51 (1.15, 1.98), 1.72 (1.30, 2.27), and 2.27 (1.68, 3.06), respectively (P < .001) after adjusting for other known confounding variables. Interaction analysis showed that there was no significant difference between subgroups stratified on the basis of sex, age, body mass index, fasting plasma glucose, and high-density lipoprotein cholesterol except triglycerides (P = .006). The auROCs for sUA and the sUA-based score were 0.627 (0.607, 0.647) and 0.760 (0.742, 0.777), respectively. A nomogram comprising independent risk factors was developed to predict the 5-year risk of hypertension for each subject.High sUA was significantly associated with the incidence of hypertension in non-MetS subjects adjusting for confounders.

Preconditioning of bone marrow-derived mesenchymal stromal cells by tetramethylpyrazine enhances cell migration and improves functional recovery after focal cerebral ischemia in rats.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) is one of the new therapeutic strategies for treating ischemic stroke. However, the relatively poor migratory capacity of BMSCs toward infarcted regions limited the therapeutic potential of this approach. Pharmacological preconditioning can increase the expression of CXC chemokine receptor 4 (CXCR4) in BMSCs and enhance cell migration toward the injury site. In the present study, we investigated whether tetramethylpyrazine (TMP) preconditioning could enhance BMSCs migration to the ischemic brain and improve functional recovery through upregulating CXCR4 expression. METHODS: BMSCs were identified by flow cytometry analysis. BMSCs migration was evaluated in vitro by transwell migration assay, and CXCR4 expression was measured by quantitative reverse transcription-polymerase chain reaction and western blot analysis. In rats with focal cerebral ischemia, the neurological function was evaluated by the modified neurological severity score, the adhesive removal test and the corner test. The homing BMSCs and angiogenesis were detected by immunofluorescence, and expression of stromal cell-derived factor-1 (SDF-1) and CXCR4 was measured by western blot analysis. RESULTS: Flow cytometry analysis demonstrated that BMSCs expressed CD29 and CD90, but not CD34 and CD45. TMP pretreatment dose-dependently induced BMSCs migration and CXCR4 expression in vitro, which was significantly inhibited by AMD3100, a CXCR4 antagonist. In rat stroke models, we found more TMP-preconditioned BMSCs homing toward the infarcted regions than nonpreconditioned cells, leading to improved neurological performance and enhanced angiogenesis. Moreover, TMP-preconditioned BMSCs significantly upregulated the protein expression of SDF-1 and CXCR4 in the ischemic boundary regions. These beneficial effects of TMP preconditioning were blocked by AMD3100. CONCLUSION: TMP preconditioning enhances the migration and homing ability of BMSCs, increases CXCR4 expression, promotes angiogenesis, and improves neurological performance. Therefore, TMP preconditioning may be an effective strategy to improve the therapeutic potency of BMSCs for ischemic stroke due to enhanced BMSCs migration to ischemic regions.

Leptin protects cardiomyocytes from serum-deprivation-induced apoptosis by increasing anti-oxidant defence.

1. Leptin, an important adipose-derived hormone, can be associated with cardiac pathophysiology; however, the role of leptin in cardiomyocyte apoptosis is poorly understood. The present study examines serum-deprivation-induced apoptosis in primary cultured cardiomyocytes treated with leptin. 2. Cardiomyocytes were subjected to serum deprivation in the presence or absence of leptin (5 or 50 nmol/L) for 48 h. Apoptosis was determined by Hoechst 33258 and Annexin V-FITC/propidium iodide dual staining. Cell viability, malondialdehyde (MDA) content, caspase 3 activation, and the expression and enzyme activity of superoxide dismutase (SOD) were measured. Small interference RNA (siRNA) targeting SOD1 and SOD2 were used to knockdown their expression and measure apoptosis. 3. Serum deprivation caused nearly 30% of apoptosis in cardiomyocytes, and an approximately 60% decrease in cell viability. The mRNA levels and the activated form of caspase 3 were greatly increased. In the presence of leptin, the apoptotic rate was reduced to approximately 15%, cell viability was increased and the activation of caspase 3 was partially inhibited. Additionally, the augmented lipid peroxidation (MDA formation) was abolished, and the impaired activities of SOD1 and SOD2 were restored by leptin. The mRNA expression of SOD2, but not SOD1, was stimulated by leptin. Transfection with siRNA that cause deficiency of either SOD1 or SOD2 attenuated the anti-apoptotic effects of leptin. 4. The results suggest that leptin inhibits serum-deprivation-induced apoptosis in cardiomyocytes by activating SOD. The present study outlines the direct actions of leptin in cardiac disorders that are related to elevated leptin levels.

[Nordihydroguaiaretic acid partially inhibits inflammatory responses after focal cerebral ischemia in rats].

The aim of the present study is to investigate the role of nordihydroguaiaretic acid (NDGA) on inflammatory cells accumulation after focal cerebral ischemia and the underlying mechanism. Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion (MCAO) followed by 72 h of reperfusion. NDGA (5 and 10 mg/kg) was administered intraperitoneally 30 min, 2, 24, 48 h after reperfusion, respectively. The brain injuries were observed by neurological and histological examination. Endogenous IgG exudation, neutrophils and macrophages/microglia accumulation, and intercellular adhesion molecule-1 (ICAM-1) protein expression were determined by immunohistochemistry 72 h after reperfusion. ICAM-1 mRNA was determined by RT-PCR 72 h after reperfusion. The catalysates of 5-lipoxygenase (5-LOX), leukotriene B4 (LTB4) and cysteinyl leukotrienes (CysLTs), were evaluated by ELISA 3 h after reperfusion. The results showed that NDGA ameliorated neurological dysfunction, decreased infarct volume, and inhibited endogenous IgG exudation, neutrophils infiltration, ICAM-1 mRNA and protein expression 72 h after reperfusion. Moreover, NDGA reduced the levels of LTB4 and CysLTs 3 h after reperfusion. However, NDGA did not reduce the accumulation of macrophages/microglia 72 h after reperfusion. These results suggest that NDGA decreases neutrophil infiltration in the subacute phase of focal cerebral ischemia via inhibiting 5-LOX activation.

Minocycline inhibits 5-lipoxygenase expression and accelerates functional recovery in chronic phase of focal cerebral ischemia in rats.

AIMS: We previously reported that minocycline attenuates acute brain injury and inflammation after focal cerebral ischemia, and this is partly mediated by inhibition of 5-lipoxygenase (5-LOX) expression. Here, we determined the protective effect of minocycline on chronic ischemic brain injury and its relation with the inhibition of 5-LOX expression after focal cerebral ischemia. MAIN METHODS: Focal cerebral ischemia was induced by 90 min of middle cerebral artery occlusion followed by reperfusion for 36 days. Minocycline (45 mg/kg) was administered intraperitoneally 2h and 12h after ischemia and then every 12h for 5 days. Sensorimotor function was evaluated 1-28 days after ischemia and cognitive function was determined 30-35 days after ischemia. Thereafter, infarct volume, neuron density, astrogliosis, and 5-LOX expression in the brain were determined. KEY FINDINGS: Minocycline accelerated the recovery of sensorimotor and cognitive functions, attenuated the loss of neuron density, and inhibited astrogliosis in the boundary zone around the ischemic core, but did not affect infarct volume. Minocycline significantly inhibited the increased 5-LOX expression in the proliferated astrocytes in the boundary zone, and in the macrophages/microglia in the ischemic core. SIGNIFICANCE: Minocycline accelerates functional recovery in the chronic phase of focal cerebral ischemia, which may be partly associated with the reduction of 5-LOX expression.

Antagonism of endothelin-1 inhibits hypoxia-induced apoptosis in cardiomyocytes.

Apoptosis is well documented to be a common feature of many pathological processes of the heart. Exogenous endothelin-1 (ET-1) has been shown to be proapoptotic or antiapoptotic, depending on ET-1 concentration, cell type, and the ratio of ETA/ETB receptor subtypes. The role of endogenous ET-1 in cardiomyocyte apoptosis, however, is not clarified. This study observed the effects of the ETA-receptor antagonists BQ610 and BQ123 and the ETB-receptor antagonist BQ788 on hypoxia-induced apoptosis in primary cultured neonatal rat cardiomyocytes. Hypoxic apoptosis was induced by incubating cardiomyocytes in serum-free medium under 3% O2 and 5% CO2 for 24 h and evaluated by TUNEL analysis and flow cytometry. TUNEL analysis showed that the apoptotic cardiomyocytes constituted 24.2% +/- 2.2% of the total cells under hypoxic conditions. Treatment with BQ610 (5 micromol/L) significantly reduced the apoptosis rate to 13.2% +/- 3.7% (data from 4 independent experiments, p < 0.01 vs. hypoxia). Flow cytometry showed that the percentage of apoptotic cells positively stained with annexin V and propidium iodide was 42.76% +/- 4.44% (n = 12) in cultures subjected to hypoxia. BQ123 at 0.04, 0.2, and 1.0 micromol/L dose-dependently reduced the apoptosis rate to 34.00% +/- 10.35% (n = 6, p < 0.05), 30.38% +/- 8.28% (n = 6, p < 0.01), and 22.89% +/- 4.19% (n = 6, p < 0.01), respectively. In contrast, BQ788 did not affect hypoxic apoptosis. These findings suggested that endogenous ET-1 contributed to hypoxia-induced apoptosis in cultured cardiomyocytes, which was mediated by ETA receptors, but not by ETB receptors.