Jianxun Liu

EFFECTS OF CROCIN ON REPERFUSION-INDUCED OXIDATIVE/NITRATIVE INJURY TO CEREBRAL MICROVESSELS AFTER GLOBAL CEREBRAL ISCHEMIA

This paper studied the effects of crocin, a pharmacologically active component of Crocus sativus L., on ischemia/reperfusion (I/R) injury in mice cerebral microvessels. Transient global cerebral ischemia (20 min), followed by 24 h of reperfusion, significantly promoted the generation of nitric oxide (NO) and malondialdehyde (MDA) in cortical microvascular homogenates, as well as markedly reduced the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) and promoted the activity of nitric oxide synthase (NOs). Reperfusion for 24 h led to serous edema with substantial microvilli loss, vacuolation, membrane damage and mitochondrial injuries in cortical microvascular endothelial cells (CMEC). Furthermore, enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and decreased expression of matrix metalloproteinase-9 (MMP-9) were detected in cortical microvessels after I (20 min)/R (24 h). Reperfusion for 24 h also induced membrane (functional) G protein-coupled receptor kinase 2 (GRK2) expression, while it reduced cytosol GRK2 expression. Pretreatment with crocin markedly inhibited oxidizing reactions and modulated the ultrastructure of CMEC in mice with 20 min of bilateral common carotid artery occlusion (BCCAO) followed by 24 h of reperfusion in vivo. Furthermore, crocin inhibited GRK2 translocation from the cytosol to the membrane and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels. We propose that crocin protects the brain against excessive oxidative stress and constitutes a potential therapeutic candidate in transient global cerebral ischemia.

RNA interference-mediated downregulation of Beclin1 attenuates cerebral ischemic injury in rats

AbstractAim:To test the role of the Beclin 1-dependent autophagy pathway in brain damage during cerebral ischemia.Methods:Focal cerebral ischemia was established in rats using a middle cerebral artery occlusion (MCAO) model. A lentiviral vector-associated RNA interference (RNAi) system was stereotaxically injected into the ipsilateral lateral ventricle to reduce Beclin1 expression. We measured the ipsilateral infarct volume, autophagosome formation, neurogenesis and apoptosis, all of which could be modulated by Beclin1 RNAi.Results:On the 14th day after MCAO, Beclin1 downregulation by RNAi increased the population of neural progenitor cells (BrdU+-DCX+), newborn immature cells (BrdU+-Tuj-1+) and mature neurons (BrdU+-MAP-2+), and reduced the apoptosis of immature neurons (caspase-3+-DCX+ and caspase-3+-Tuj-1+) surrounding the ischemic core of the ipsilateral hemisphere. Furthermore, RNAi-mediated downregulation of Beclin1 decreased infarct volume and inhibited histological injury and neurological deficits.Conclusion:RNAi-mediated downregulation of Beclin1 improves outcomes after transient MCAO.

Berberine alleviates ox-LDL induced inflammatory factors by up-regulation of autophagy via AMPK/mTOR signaling pathway

BackgroundInflammation induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Recently, roles of autophagy against inflammation in the process of atherosclerosis have drawn increasing attention. Here, we tested the possible molecular mechanisms by which berberine confers an anti-inflammatory effect in macrophages by upregulation of autophagy.MethodsJ774A.1 macrophages were incubated with various doses of ox-LDL for various times. We evaluated the inflammatory factors and autophagy proteins (LC3II/LC3I, and SQSTM1/p62) to ascertain the optimal dose and time. Ox-LDL–induced inflammatory factors and autophagy in J774A.1 cells were tested by the AimPlex multiplex assay, Western blotting, confocal microscopy, and transmission electron microscopy in the presence of berberine or chloroquine (CQ). Adenosine 5’-monophosphate-activated protein kinase (AMPK) inhibitor compound C was used to evaluate the AMPK/mTOR signaling pathway.ResultsBerberine dose- and time-dependently reduced ox-LDL–induced inflammation and increased the ratio of LC3II/LC3I, and SQSTM1/p62 in J774A.1 cells. CQ significantly attenuated the berberine-induced autophagy and anti-inflammation. In addition, berberine increased the ratio of p-AMPK/AMPK and decreased the ratio of p-mTOR/mTOR. AMPK inhibitor compound C abolished berberine-induced autophagy and promoted p-mTOR/mTOR expression in J774A.1 cells.ConclusionBerberine treatment inhibits inflammation in J774A.1 cells by inducing autophagy, which is mediated through activation of the AMPK/mTOR signaling pathway. Importantly, this study provides new insight into berberine’s molecular mechanism and its therapeutic potential in the treatment of atherosclerosis.

Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury

Ischemic heart disease is a leading cause of death in human population and protection of myocardial infarction (MI) associated with ischemia-reperfusion (I/R) remains a challenge. MG53 is an essential component of the cell membrane repair machinery that protects injury to the myocardium. We investigated the therapeutic value of using the recombinant human MG53 (rhMG53) protein for treatment of MI. Using Langendorff perfusion of isolated mouse heart, we found that I/R caused injury to cardiomyocytes and release of endogenous MG53 into the extracellular solution. rhMG53 protein was applied to the perfusion solution concentrated at injury sites on cardiomyocytes to facilitate cardioprotection. With rodent models of I/R-induced MI, we established the in vivo dosing range for rhMG53 in cardioprotection. Using a porcine model of angioplasty-induced MI, the cardioprotective effect of rhMG53 was evaluated. Intravenous administration of rhMG53, either prior to or post-ischemia, reduced infarct size and troponin I release in the porcine model when examined at 24h post-reperfusion. Echocardiogram and histological analyses revealed that the protective effects of rhMG53 observed following acute MI led to long-term improvement in cardiac structure and function in the porcine model when examined at 4weeks post-operation. Our study supports the concept that rhMG53 could have potential therapeutic value for treatment of MI in human patients with ischemic heart diseases.

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.

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway

Aim:To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI).Methods:Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg−1·d−1, ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg−1·d−1, ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting.Results:Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg−1·d−1) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor LY294002. Treatment with Rd (25, 50, and 100 μmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of LY294002.Conclusion:Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.

The Cardioprotective Effects of Citric Acid and L-Malic Acid on Myocardial Ischemia/Reperfusion Injury

Organic acids in Chinese herbs, the long-neglected components, have been reported to possess antioxidant, anti-inflammatory, and antiplatelet aggregation activities; thus they may have potentially protective effect on ischemic heart disease. Therefore, this study aims to investigate the protective effects of two organic acids, that is, citric acid and L-malic acid, which are the main components of Fructus Choerospondiatis, on myocardial ischemia/reperfusion injury and the underlying mechanisms. In in vivo rat model of myocardial ischemia/reperfusion injury, we found that treatments with citric acid and L-malic acid significantly reduced myocardial infarct size, serum levels of TNF-α, and platelet aggregation. In vitro experiments revealed that both citric acid and L-malic acid significantly reduced LDH release, decreased apoptotic rate, downregulated the expression of cleaved caspase-3, and upregulated the expression of phosphorylated Akt in primary neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation injury. These results suggest that both citric acid and L-malic acid have protective effects on myocardial ischemia/reperfusion injury; the underlying mechanism may be related to their anti-inflammatory, antiplatelet aggregation and direct cardiomyocyte protective effects. These results also demonstrate that organic acids, besides flavonoids, may also be the major active ingredient of Fructus Choerospondiatis responsible for its cardioprotective effects and should be attached great importance in the therapy of ischemic heart disease.

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.

MicroRNA-384-5p regulates ischemia-induced cardioprotection by targeting phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (PI3K p110δ)

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. This study identified 16 differentially expressed miRNAs in ischemic myocardium of rats using TaqMan Low Density Array. In addition, bioinformatics analyses, such as Gene ontology and Pathway assays, were applied to determine the apoptosis pathway, only regulated by miR-384-5p, and all the associated target genes (PIK3CD, PPP3CA, PPP3CB, PPP3R1, CASP3 and IL1A). These target genes, besides PIK3CB, were shown to be significantly up-regulated by qRT-PCR assay, which further suggested that PIK3CD, PPP3CA, PPP3R1, CASP3, IL1A could be regulated by miR-384-5p. MTT, Western blot, qRT-PCR and luciferase assays were used to investigate the role of miR-384-5p in myocardial ischemia. We found that cleaved caspase3 expression was up-regulated by miR-384-5p and down-regulated by miR-384-5p inhibitor suggesting that apoptosis pathway was regulated by miR-384-5p. We also found that miR-384-5p suppressed cell viability while miR-384-5p inhibitor improved it, confirming H9c2 cell survival was affected by miR-384-5p. In addition, the PIK3CD protein level in H9c2 cells was up-regulated by miR-384-5p inhibitor. We found that miR-384-5p expression level decreased and PIK3CD protein level increased in both ischemic myocardium of rats and hypoxic H9c2 cells, and that miR-384-5p suppress PIK3CD expression through a miR-384-5p binding site within the 3′ untranslational region of PIK3CD. These results show that miR-384-5p, an important protecting factor, plays a significant role in cardioprotection by regulating PIK3CD in myocardial ischemia.

Pharmacochemistry and integrated pharmacokinetics of six alkaloids after oral administration of Huang-Lian-Jie-Du-Tang decoction

Pharmacochemistry and integrated pharmacokinetics of six alkaloids (groenlandicine, berberine, palmatine, epiberberine, jatrorrhizine, and columbamine) after oral administration of Huang-Lian-Jie-Du-Tang (HLJDT) decoction were investigated in this paper. The method of plasma pharmacochemistry was applied to predict the potential bioactive components in HLJDT decoction. Based on the accurate molecular weight, 10 components including 2 flavonoids (baicalin and wogonoside), 1 iridoid glycoside (geniposide), and 7 alkaloids (above-mentioned 6 alkaloids and coptisine) were structurally identified. Then, integrated pharmacokinetics of the alkaloids in Sprague–Dawley rats after oral administration of HLJDT decoction was investigated by HPLC method. The results showed that the pharmacokinetic behaviors of the alkaloids were different although their chemical structures were similar. This study developed a method to predict the potential bioactive components in HLJDT decoction and research the pharmacokinetic behaviors of the potential bioactive components.