Jianhua Fu

Baicalin attenuates proinflammatory cytokine production in oxygen-glucose deprived challenged rat microglial cells by inhibiting TLR4 signaling pathway.

Baicalin, a flavonoid compound isolated from Scutellariae radix, has been shown to possess a number of pharmacological effects. The aim of the present study was to observe the inhibitory effects of baicalin on the activation of microglial cells induced by oxygen-glucose deprivation (OGD) and the specific mechanisms by which these effects are mediated. Cultured rat primary microglial cells were exposed to baicalin at final concentrations of 10 μg/ml, 20 μg/ml and 40 μg/ml during 4h of OGD. The effects of baicalin on (i) cell viability; (ii) secretion of proinflammatory cytokines; (iii) Tlr4 mRNA expression; (iv) p-c-jun, p-ERK1/2, p-JNK, p-p38, TRAF6 and p-IκB-α levels; and (v) co-localization of TLR4 and MyD88 were evaluated using the Cell Counting Kit-8 (CCK-8), enzyme-linked immunosorbent assays (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), western blot and double-labeled immunofluorescence staining, respectively. OGD increased cell viability and release of TNF-α, IL-1β, IL-6 and IL-8, these effects were suppressed by baicalin. Baicalin also attenuated the OGD-induced increases in Tlr4 mRNA expression. In addition, high dose of baicalin reduced TRAF6 levels remarkably. Furthermore, baicalin also downregulated phosphorylation of IκB-α, c-jun, ERK1/2, JNK, p38 and inhibited the OGD-induced transfer of MyD88 from cytoplasm to membrane in microglial cells. The results show that baicalin can inhibit OGD-induced production of inflammatory factors in microglial cells by attenuating inflammatory factors and regulating the TLR4 signaling pathways.

Baicalin protects rat brain microvascular endothelial cells injured by oxygen-glucose deprivation via anti-inflammation.

Baicalin, which is isolated from Scutellariae Radix, has been evidenced to possess several pharmacological effects. The present study focuses on the in vitro protective effect of baicalin on oxygen-glucose deprivation (OGD) injured brain microvascular endothelial cells (BMECs) via anti-inflammation and mechanisms against BMECs damaged by OGD. Cultured primary rat BMECs were exposed to baicalin at the concentrations of 100μM (high dose) and 10μM (low dose) for 6h after a 2h OGD. The effects of baicalin were evaluated in terms of (i) cell viability; (ii) lactate dehydrogenase (LDH) leakage rate; (iii) levels of TNF-α, IL-1β, IL-6 in culture media; (iv) protein expressions of p-MEK6, p-MEK1/2, p-ERK, p-IκBα, NF-κB p65, p-IKKα, p-IKKβ and p-p38; and (v) nuclear translocation of NF-κB p65 and p-IκBα. The results showed that OGD treatment could reduce cell viability, increase LDH leakage rate, increase the levels of TNF-α, IL-1β and IL-6 in the culture media. These effects were suppressed by baicalin with high or low dose. In addition, baicalin could notably down-regulate the phosphorylation of proteins in MAPK signaling pathway such as p-MRK1/2, p-ERK and p-p38. While low dose of baicalin could significantly suppress the phosphorylation of proteins in NF-кB signaling pathway such as p-IKKα, p-IKKβ and p-IκBα. Furthermore, baicalin at 10μM could remarkably inhibit nuclear transcriptional activity triggered via NF-κB p65 and p-IκBα in BMECs. In conclusion, baicalin displays a protective effect on OGD-injured BMECs in vitro by attenuating inflammatory factors via down-regulated the MAPK and NF-κB signaling pathway.

Hawthorn leaves flavonoids decreases inflammation related to acute myocardial ischemia/reperfusion in anesthetized dogs.

ObjectiveTo investigate the effects and mechanisms of hawthorn leaves flavonoids (HLF) on acute myocardial ischemia/reperfusion in anesthetized dogs.MethodsThe acute ischemia models were prepared by ligating left anterior descending (LAD) artery for 60 min. Qualified 15 male dogs were randomly divided into 3 groups with 5 in each group: blank control (treated with normal saline 3 mL/kg) group, HLF low dosage (5 mg/kg) group and high dosage (10 mg/kg) group, with an once injection through a femoral vein 5 min before reperfusion. Epicardial electrocardiogram was adopted to measure the scope and degree of myocardial ischemia. Simultaneously, neutrophil infiltration in infarct (Inf) and remote site (RS) of myocardial tissue was measured by myeloperoxidase (MPO) activity assay. The serum interleukin-1 (IL-1) and tumor necrosis factorα (TNF-α) content were quantified by radioimmuno-assay. Furthermore, expression of G protein-coupled receptor kinase 2 (GRK2) and nuclear factor κB (NF-κB) in Inf and RS tissue were detected by Western blotting technique.ResultsIschemia and reperfusion increased the MPO activity and IL-1 and TNF-α content. HLF (10 and 5 mg/kg) could significantly decrease the degree and scope of myocardial ischemia; markedly inhibit the increase of MPO activity, and IL-1 and TNF-α content induced by myocardial ischemia/infarction. Furthermore, HLF increased GRK2 expression and inhibited NF-κB expression in Inf tissue.ConclusionHLF could improve the situation of acute myocardial ischemia and inhibit the inflammation in anesthetized dogs, which might be due to its increasing effect on the GRK2 and NF-κB expressions.

Circulating MicroRNA Profiles Differ between Qi-Stagnation and Qi-Deficiency in Coronary Heart Disease Patients with Blood Stasis Syndrome

We compared the circulating microRNA profiles of Qi-stagnation (QSB) and Qi-deficiency (QDB) in coronary heart disease (CHD) patients with blood stasis syndrome. Twenty-nine CHD patients were divided into QSB group and QDB group. The analysis was carried out through comparing their circulating microRNA profiles and the following bioinformatics analysis. The number of differential miRNAs in QDB group was much more than that in QSB group. Functional annotations of the differentially expressed miRNAs target genes in the QSB group and QDB group were, respectively, related to regulation of cellular component organization, regulation of glucose metabolic process, and so forth and protein kinase cascade, phosphate metabolic process, and so forth. KEGG pathway analysis showed that the process Qi-deficiency was associated with phagocytosis including endocytosis and mTOR signaling pathway. Specifically, pathway of cell adhesion molecules played the crucial role in the pathological process of Qi-stagnation, with a unique upregulation except for pathways associated with cancer signal. MicroRNA-gene-net analysis indicated that let-7c, miR-4487, miR-619, miR-8075, miR-6735, and miR-32-5p and miR-17-5p, miR-130a, and miR 320 family had the most important and extensive regulatory function for Qi-stagnation syndromes and Qi-deficiency syndromes, respectively. Differentially expressed miRNAs and concerned pathways suggest different molecular mechanisms that may mediate the pathological process of QSB and QDB syndromes.

Shuangshen Ningxin Capsule, a Traditional Chinese Medicinal Preparation, Alleviates Myocardial Ischemia through Autophagy Regulation.

Shuangshen Ningxin capsule (SSNX), a modern Chinese formula, has been used to treat cardiovascular diseases in Eastern Asia. Our study focuses on the autophagy regulation of SSNX against coronary artery injuries. Myocardial infarction model was established in Chinese miniswines (CMS) by coronary artery balloon injury. SSNX was administered to the CMS for 8 weeks with 4 mg/kg or 16 mg/kg. Myocardial cells were incubated with 20% SSNX medicated serum for 2 hours. Assays were performed to detect the effects of SSNX on (i) coronary artery diameter by angiography, (ii) hemodynamics by noninvasive hemodynamic monitoring system, (iii) plaque burden and plaque volume by intravenous ultrasound (iv) coronary artery histology by H&E staining, (v) autophagosome by transmission electron microscopy, (vi) lactate dehydrogenase (LDH) leakage, and (vii) Beclin-1 and LC3-I/II expressions by Western blot. The results showed that CMS treated with SSNX exhibited the correction for the disturbed cardiac hemodynamics, increase of coronary artery diameter, reduction of high plaque burden and plaque volume, and decrease of LDH. The inhibitory effect of SSNX on CMS autophagy was demonstrated by the reduction of autophagosome and the downregulation of beclin-1 and LC3-I/II. SSNX may protect coronary artery and increase the stability of plaque through the suppression of myocardial cellular autophagy, which suggests the potentially therapeutic effect of SSNX on ischemic cardiovascular disease.

Extract of Paris polyphylla Simth protects cardiomyocytes from anoxia-reoxia injury through inhibition of calcium overload.

ObjectiveTo assess any direct effect of extract of Paris polyphylla Simth (EPPS), a Chinese plant, on a cardiomyocyte subject to ischemia-reperfusion injury and to further elucidate its protective effect against myocardium ischemia on the cellular level.MethodsNeonatal rat cardiomyocytes were isolated and subjected to an anoxia-reoxia injury simulating the ischemia-reperfusion injury in vivo in the presence or absence of EPPS or diltizem, a positive control. The lactate dehydrogenase (LDH) activities in culture supernatants and cell viabilities were analyzed using the enzymatic reaction kinetics monitoring-method and MTT method, respectively. Free intracellular calcium concentrations and activities of Na+-K+ ATPase and Ca2+ ATPase in cells were also measured with laser confocal microscopy and the inorganic phosphorus-transformation method, respectively.ResultsIn cardiomyocytes subject to anoxia-reoxia injury, EPPS at 50–400 mg/L showed a concentration-dependent inhibition on LDH leakage and maintenance of cell viability, and the effect was significant at 275 and 400 mg/L (both P<0.01). In addition, EPPS at 275 and 400 mg/L significantly inhibited the increase in intracellular free calcium (both P<0.01) as well as decreased the activities of Na+-K+ ATPase and Ca2+ ATPase (P<0.01, P<0.05).ConclusionsEPPS prevents anoxia-reoxia injury in neonatal rat cardiomyocytes in vitro by preservation of Na+-K+ ATPase and Ca2+ ATPase activities and inhibition of calcium overload. The direct protective effect on cardiomyocytes may be one of the key mechanisms that underlie the potential therapeutic benefit of EPPS against myocardium ischemia.

Ginsenoside Rg1 protects starving H9c2 cells by dissociation of Bcl-2-Beclin1 complex.

BackgroundAutophagy can result in cellular adaptation, as well as cell survival or cell death. We investigated how ginsenoside Rg1(G-Rg1) regulates the relationship between autophagy and apoptosis induced by continuous starvation.MethodsH9c2 cells under continuous starvation were treated with or without ginsenoside Rg1, and autophagy and apoptosis related proteins were assessed over a continuous time course by Western blot. Dynamic fluorescence intensity of green fluorescent protein (GFP)-LC3 was used to assess autophagosome formation by live cell imaging. Cyan fluorescent protein (CFP) -Beclin1(BECN1) and yellow fluorescent protein (YFP) -Bcl-2 were co-transfected into cells to observe ginsenoside Rg1 regulation of BECN1/Bcl-2 interaction using Fluorescence Resonance Energy Transfer (FRET). Immunoprecipitation was also used to assess BECN1/Bcl-2 interaction over a continuous time course.ResultsIn H9c2 cells, starvation induced both apoptosis and autophagy. Cell apoptosis was significantly attenuated in ginsenoside Rg1-treated conditions, while autophagy was promoted. Ginsenoside Rg1 weakened the interaction between Beclin1 and Bcl-2, inhibiting apoptosis while promoting autophagy. Our results suggest that autophagy is beneficial to starved cardiac cells over a period of time. Furthermore, we describe the effect of ginsenoside Rg1 on the relationship between autophagy and apoptosis during starvation.ConclusionsOur findings provide valuable evidence for employing ginsenoside Rg1 as a specific promoter of autophagy and inhibitor of apoptosis.

Transcriptomic Analysis of Myocardial Ischemia Using the Blood of Rat.

Myocardial ischemia is a pathological state of heart with reduced blood flow to heart and abnormal myocardial energy metabolism. This disease occurs commonly in middle aged and elderly people. Several studies have indicated that the rat was an appropriate animal model used to study myocardial ischemia. In this study, in order to gain insights into the pathogenesis of myocardial ischemia, we sequenced the transcriptomes of three normal rats as control and the same number of myocardial ischemia rats. We sequenced the genomes of 6 rats, including 3 cases (myocardial ischemia) and 3 controls using Illumina HiSeq 2000. Then we calculated the gene expression values and identified differentially expressed genes based on reads per kilobase transcriptome per million (RPKM). Meanwhile we performed a GO enrichment analysis and predicted novel transcripts. In our study, we found that 707 genes were up-regulated and 21 genes were down-regulated in myocardial ischemia rats by at least 2-fold compared with controls. By the distribution of reads and the annotation of reference genes, we found 1,703 and 1,552 novel transcripts in cases and controls, respectively. At the same time, we refined the structure of 9,587 genes in controls and 10,301 in cases. According to the results of GO term and pathway analysis of differentially expressed genes, we found that the immune response, stimulus response, response to stress and some diseases may be associated with myocardial ischemia. Since many diseases, especially immune diseases, are associated with myocardial ischemia, we should pay more attention to the complications which might result from myocardial ischemia.

Synergistic neuroprotective effect of microglial‑conditioned media treated with geniposide and ginsenoside Rg1 on hypoxia injured neurons

The synergistic mechanism underlying the effects of multi‑component combined drug use for complex diseases remains to be fully elucidated. Microglial activation following ischemia can either affect neural survival or cause neuronal injury. The aim of the present study was to determine the synergistic effect of geniposide and ginsenoside Rg1, based on microglial‑neuronal communication. N2a neuronal cells were divided into the following seven groups: Control group; normal cultured microglial cells in conditioned medium (N‑MG‑CM) group; oxygen‑glucose deprivation (OGD) model group; OGD‑injured MG‑CM (I‑MG‑CM) group; geniposide‑treated MG‑CM (G‑MG‑CM) group; ginsenoside Rg1‑treated MG‑CM (R‑MG‑CM) group; and combination‑treated MG‑CM (C‑MG‑CM) group. A series of assays were used to detect the effects of the different MG‑CM on neurons in terms of: (i) cell viability, determined using a Cell Counting Kit‑8; (ii) lactate dehydrogenase (LDH) leakage rate; (iii) expression of NMDAR1 and activated caspase‑3, detected using western blotting; (iv) mitochondrial transmembrane potential, determined by JC‑1; and (v) mitochondrial ultrastructural features, determined using electron microscopy. The experimental results demonstrated that MG‑CM including the integrated use of geniposide and ginsenoside Rg1 significantly protected neuronal cell viability and inhibited LDH leakage, suppressed the expression of N‑methyl‑D‑aspartate receptor subunit 1 and activated caspase‑3, increased the mitochondrial transmembrane potential and improved the mitochondrial ultrastructure. MG‑CM from separately used geniposide or ginsenoside Rg1 demonstrated differential neuroprotection at different levels. These findings revealed that the synergistic drug combination of geniposide and ginsenoside Rg1 in the treatment of stroke is a feasible approach for use.

Metabonomics Study of Heart Homogenates from Myocardial Infarction Rats Using Liquid Chromatography/Time of Flight Mass Spectrometry

In this study, a metabonomics analysis of heart homogenates from myocardial ischemic rats was performed by LC–TOF–MS. Hydrophilic interaction chromatography (HILIC) was used to separate the endogenous metabolites in heart homogenates. Partial least squares to latent structure-discriminant analysis (PLS-DA) was used for data analysis. Good separations were observed between the normal and model groups and 15 potential biomarkers were identified. The major disturbed metabolic pathways were purine metabolism, pyrimidine metabolism, urea cycle, and energy metabolism. The results demonstrated that a metabonomics approach based on HILIC-MS was useful for studying metabolic mechanism on target tissue of the myocardial infarction rat.