Dunaliella salina Alga Protects against Myocardial Ischemia/Reperfusion Injury by Attenuating TLR4 Signaling.

Myocardial ischemia/reperfusion (I/R) injury is marked by rapid increase in inflammation and not only results in myocardial apoptosis but also compromises the myocardial function. Dunaliella salina (D. salina), a halophilic unicellular microalga, has been used as a provitamin A carotenoid supplement and color additive. Several studies have reported that D. salina extract could attenuate lipopolysaccharides-induced inflammatory effects and regulate the virus-induced inflammatory response in macrophages. However, the effects of D. salina on myocardial I/R injury remain unknown. Therefore, we aimed to investigate the cardioprotection of D. salina extract in rats subjected to myocardial I/R injury that was induced by occlusion of the left anterior descending coronary artery for 1 h followed by 3 h of reperfusion. Compared with the vehicle group, the myocardial infarct size significantly decreased in rats that were pre-treated with D. salina. D. salina significantly attenuated the expressions of TLR4, COX-2 and the activity of STAT1, JAK2, IκB, NF-κB. Furthermore, D. salina significantly inhibited the activation of caspase-3 and the levels of Beclin-1, p62, LC3-I/II. This study is the first to report that the cardioprotective effects of D. salina may mediate anti-inflammatory and anti-apoptotic activities and decrease autophagy through the TLR4-mediated signaling pathway to antagonize myocardial I/R injury.

Lumbrokinase regulates endoplasmic reticulum stress to improve neurological deficits in ischemic stroke.

Ischemic stroke is characterized by the loss of cerebral blood flow, which frequently leads to neurological deficits. Tissue plasminogen activator is the only therapeutic agent approved to treat ischemic stroke but increases the risk of intracranial hemorrhage and mortality. The fibrinogen-depleting agent lumbrokinase has been used to improve myocardial perfusion in symptomatic stable angina and to prevent secondary ischemic stroke. Lumbrokinase is highly fibrin-specific and only active in the presence of fibrin. Therefore, lumbrokinase has a low risk of hemorrhage due to excessive fibrinolysis. In this study, we aimed to clarify the neuroprotection of lumbrokinase in mice subjected to permanent middle cerebral artery occlusion. Lumbrokinase significantly attenuated infarct volume and improved neurological dysfunction. Lumbrokinase dramatically decreased the expressions of the endoplasmic reticulum (ER) transmembrane receptor protein inositol-requiring enzyme-1 (IRE1) and its downstream transcription factor, XBP-1, caspase-12, and NF-κB activity, thereby significantly inhibiting apoptosis and autophagy and decreasing the NLRP3 inflammasome. Our evidence indicates that post-stroke treatment with lumbrokinase protects against ischemic stroke, thereby regulating ER stress through the collective inhibitory effect of the IRE1 signaling pathways to decrease apoptosis, autophagy, and inflammatory responses. We suggest that lumbrokinase is potential as an adjuvant treatment for ischemic stroke.

Activation of peripheral TRPM8 mitigates ischemic stroke by topically applied menthol.

BACKGROUND: No reports exist as to neuroprotective effects associated with topical activation of transient receptor potential melastatin 8 (TRPM8), a noted cold receptor. In the present study, we identified whether activating peripheral TRPM8 can be an adjuvant therapy for ischemic stroke. METHODS: Menthol, an agonist of TRPM8, was applied orally or topically to all paws or back of the mouse after middle cerebral artery occlusion (MCAO). We used Trpm8 gene knockout (Trpm8-/-) mice or TRPM8 antagonist and lidocaine to validate the roles of TRPM8 and peripheral nerve conduction in menthol against ischemic stroke. RESULTS: Application of menthol 16% to paw derma attenuated infarct volumes and ameliorated sensorimotor deficits in stroke mice induced by MCAO. The benefits of topically applied menthol were associated with reductions in oxidative stress, neuroinflammation and infiltration of monocytes and macrophages in ischemic brains. Antagonizing TRPM8 or Trpm8 knockout dulls the neuroprotective effects of topically application of menthol against MCAO. Immunohistochemistry analyses revealed significantly higher TRPM8 expression in skin tissue samples obtained from the paws compared with skin from the backs, which was reflected by significantly smaller infarct lesion volumes and better sensorimotor function in mice treated with menthol on the paws compared with the back. Blocking conduction of peripheral nerve in the four paws reversed the neuroprotective effects of topical menthol administrated to paws. On the other hand, oral menthol dosing did not assist with recovery from MCAO in our study. CONCLUSION: Our results suggested that activation of peripheral TRPM8 expressed in the derma tissue of limbs with sufficient concentration of menthol is beneficial to stroke recovery. Topical application of menthol on hands and feet could be a novel and simple-to-use therapeutic strategy for stroke patients.

In situ slow-release recombinant growth differentiation factor 11 exhibits therapeutic efficacy in ischemic stroke.

Systemic growth differentiation factor 11 (GDF11) treatment improves the vasculature in the hippocampus and cortex in mice in recent studies. However, systemic application of recombinant GDF11 (rGDF11) cannot cross the brain blood barrier (BBB). Thus, large doses and long-term administration are required, while systemically applied high-dose rGDF11 is associated with deleterious effects, such as severe cachexia. This study tested whether in situ low dosage rGDF11 (1 µg/kg) protects the brain against ischemic stroke and it investigated the underlying mechanisms. Fibrin glue mixed with rGDF11 was applied to the surgical cortex for the slow release of rGDF11 in mice after permanent middle cerebral artery occlusion (MCAO). In situ rGDF11 improved cerebral infarction and sensorimotor function by upregulating Smad2/3 and downregulating FOXO3 expression. In situ rGDF11 was associated with reductions in protein and lipid oxidation, Wnt5a, iNOS and COX2 expression, at 24 h after injury. In situ rGDF11 protected hippocampal neurons and subventricular neural progenitor cells against MCAO injury, and increased newborn neurogenesis in the peri-infarct cortex. Systematic profiling and qPCR analysis revealed that Pax5, Sox3, Th, and Cdk5rap2, genes associated with neurogenesis, were increased by in situ rGDF11 treatment. In addition, greater numbers of newborn neurons in the peri-infarct cortex were observed with in situ rGDF11 than with systemic application. Our evidence indicates that in situ rGDF11 effectively decreases the extent of damage after ischemic stroke via antioxidative, anti-inflammatory and proneurogenic activities. We suggest that in situ slow-release rGDF11 with fibrin glue is a potential therapeutic approach against ischemic stroke.

Paeonol Protects Against Myocardial Ischemia/Reperfusion-Induced Injury by Mediating Apoptosis and Autophagy Crosstalk.

Many studies have shown that crosstalk exists between apoptosis and autophagy, despite differences in mechanisms between these processes. Paeonol, a major phenolic compound isolated from Moutan Cortex Radicis, the root bark of Paeonia × suffruticosa Andrews (Paeoniaceae), is widely used in traditional Chinese medicine as an antipyretic, analgesic and anti-inflammatory agent. In this study, we investigated the detailed molecular mechanisms of the crosstalk between apoptosis and autophagy underlying the cardioprotective effects of paeonol in rats subjected to myocardial ischemia/reperfusion (I/R) injury. Myocardial I/R injury was induced by occlusion of the left anterior descending coronary artery (LAD) for 1 h followed by 3 h of reperfusion. Paeonol was intravenously administered 15 min before LAD ligation. We found that paeonol significantly improved cardiac function after myocardial I/R injury and significantly decreased myocardial I/R-induced arrhythmia and mortality. Paeonol also significantly decreased myocardial infarction and plasma LDH activity and Troponin-I levels in carotid blood after I/R. Compared with vehicle treatment, paeonol significantly upregulated Bcl-2 protein expression and significantly downregulated the cleaved forms of caspase-8, caspase-9, caspase-3 and PARP protein expression in the I/R injured myocardium. Myocardial I/R-induced autophagy, including the increase of Beclin-1, p62, LC3-I, and LC3-II protein expression in the myocardium was significantly reversed by paeonol treatment. Paeonol also significantly increased the Bcl-2/Bax and Bcl-2/Beclin-1 ratios in the myocardium after I/R injury. The cardioprotective role of paeonol during I/R injury may be due to its mediation of crosstalk between apoptotic and autophagic signaling pathways, which inhibits apoptosis and autophagic cell death.

Exogenous GDF11 attenuates non-canonical TGF-ß signaling to protect the heart from acute myocardial ischemia-reperfusion injury.

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor beta 1 (TGF-ß1) superfamily that reverses age-related cardiac hypertrophy, improves muscle regeneration and angiogenesis, and maintains progenitor cells in injured tissue. Recently, targeted myocardial delivery of the GDF11 gene in aged mice was found to reduce heart failure and enhance the proliferation of cardiac progenitor cells after myocardial ischemia-reperfusion (I-R). No investigations have as yet explored the cardioprotective effect of exogenous recombinant GDF11 in acute I-R injury, despite the convenience of its clinical application. We sought to determine whether exogenous recombinant GDF11 protects against acute myocardial I-R injury and investigate the underlying mechanism in Sprague-Dawley rats. We found that GDF11 reduced arrhythmia severity and successfully attenuated myocardial infarction; GDF11 also increased cardiac function after I-R, enhanced HO-1 expression and decreased oxidative damage. GDF11 activated the canonical TGF-ß signaling pathway and inactivated the non-canonical pathways, ERK and JNK signaling pathways. Moreover, administration of GDF11 prior to reperfusion protected the heart from reperfusion damage. Notably, pretreatment with the activin-binding protein, follistatin (FST), inhibited the cardioprotective effects of GDF11 by blocking its activation of Smad2/3 signaling and its inactivation of detrimental TGF-ß signaling. Our data suggest that exogenous GDF11 has cardioprotective effects and may have morphologic and functional recovery in the early stage of myocardial I-R injury. GDF11 may be an innovative therapeutic approach for reducing myocardial I-R injury.

Sirt1 Activation by Post-ischemic Treatment With Lumbrokinase Protects Against Myocardial Ischemia-Reperfusion Injury.

Lumbrokinase is used as an oral supplement to support and maintain healthy cardiovascular function, and to treat cardiovascular diseases in clinical for more than 10 years. Up until now, the mechanism of the cardioprotective effects of post-ischemic treatment with lumbrokinase has remained unclear. We therefore investigated the signaling pathways involved in the amelioration of myocardial ischemia-reperfusion (I-R) injury in rats treated with lumbrokinase 20 min after myocardial ischemia. Compared to vehicle-treated rats, post-ischemic treatment with lumbrokinase was associated with significant reductions in myocardial I-R-induced arrhythmias and myocardial damage, and an improvement in cardiac function. Moreover, lumbrokinase significantly upregulated levels of silent information regulator 1 (Sirt1). In addition, lumbrokinase significantly increased manganese-dependent superoxide dismutase expression, decreased Cleaved-Caspase-3 expression, and induced deacetylation of FoxO1. On the other hand, lumbrokinase also significantly downregulated levels of succinate dehydrogenase, cytochrome c oxidase, nuclear factor kappa B (NF-κB) and elevated levels of microtubule-associated protein light chain 3. Notably, the cardioprotective effects of lumbrokinase were abolished by administration of the specific Sirt1 inhibitor EX527. These findings demonstrate that post-ischemic treatment with lumbrokinase attenuates myocardial I-R injury through the activation of Sirt1 signaling, and thus enhances autophagic flux and reduces I-R-induced oxidative damage, inflammation and apoptosis.

Phellinus linteus Mycelium Alleviates Myocardial Ischemia-Reperfusion Injury through Autophagic Regulation.

The incidence of myocardial ischemia-reperfusion (IR) injury is rapidly increasing around the world and this disease is a major contributor to global morbidity and mortality. It is known that regulation of programmed cell death including apoptosis and autophagy reduces the impact of myocardial IR injury. In this study, the cardioprotective effects and underlying mechanisms of Phellinus linteus (Berk. and Curt.) Teng, Hymenochaetaceae (PL), a type of medicinal mushroom, were examined in rats subjected to myocardial IR injury. The left main coronary artery of rats was ligated for 1 h and reperfused for 3 h. The arrhythmia levels were monitored during the entire process and the infarct size was evaluated after myocardial IR injury. Furthermore, the expression levels of proteins in apoptotic and autophagic pathways were observed. Pretreatment with PL mycelium (PLM) significantly reduced ventricular arrhythmia and mortality due to myocardial IR injury. PLM also significantly decreased myocardial infarct size and plasma lactate dehydrogenase level after myocardial IR injury. Moreover, PLM administration resulted in decreased caspase 3 and caspase 9 activation and increased Bcl-2/Bax ratio. Phosphorylation level of AMPK was elevated while mTOR level was reduced. Becline-1 and p62 levels decreased. These findings suggest that PLM is effective in protecting the myocardium against IR injury. The mechanism involves mediation through suppressed pro-apoptotic signaling and regulation of autophagic signaling, including stimulation of AMPK-dependent pathway and inhibition of beclin-1-dependent pathway, resulting in enhancement of protective autophagy and inhibition of excessive autophagy.

Lumbrokinase attenuates myocardial ischemia-reperfusion injury by inhibiting TLR4 signaling.

Lumbrokinase, a novel antithrombotic agent, purified from the earthworm Lumbricus rubellus, has been clinically used to treat stroke and cardiovascular diseases. However, inflammatory responses associated with the cardioprotective effect of lumbrokinase remain unknown. In this study, the signaling pathways involved in lumbrokinase-inhibited expressions of inflammation mediators were investigated in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left main coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals were treated with/without lumbrokinase and the severities of I-R-induced arrhythmias and infarction were compared. Lumbrokinase inhibited I-R-induced arrhythmias and reduced mortality, as well as decreased the lactate dehydrogenase levels in carotid blood. Lumbrokinase also inhibited the enhancement of I-R induced expressions of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and matrix metalloproteinase (MMP)-9 through toll-like receptor 4 (TLR4) signaling pathway. Moreover, our results demonstrated that stimulation with lumbrokinase decreases the phosphorylation of JNK, IκB, and NF-κB. These findings suggested that lumbrokinase is a potent cardioprotective drug in rats with I-R injury. The cardioprotective effects of lumbrokinase may be correlated with its inhibitory effect on the I-R-induced expressions of COX-2, iNOS and MMP-9, mediated by TLR4 signaling through JNK and NF-κB pathways.

Suppressing cardiac vagal modulation and changing sleep patterns in rats after chronic ischemic stroke injury.

Chronic autonomic function and sleep architecture changes in patients post-stroke are not well understood. Using wireless polysomnographic recordings, this study aimed to investigate the long-term effects on sleep patterns and autonomic function in free moving rats after middle cerebral artery occlusion (MCAO). The sleep pattern and heart rate variability (HRV) of Wistar-Kyoto rats (WKY) were analyzed. After 7-10days, the rats were divided into two groups: an MCAO group (n=8) and a sham surgery group (n=8). Compared with shams, MCAO rats showed decreased accumulated quiet sleep (QS) time over 24h during the 3rd week. The time percentage, duration and delta power of QS were also significantly decreased in the MCAO group during the dark period. Compared with baseline, there were significant increases in the parasympathetic-associated HRV measures in the sham group, including the total power (TP), high frequency power (HF) and lower frequency power (LF), throughout the post-operative weeks (primarily the 2nd and 3rd weeks), reflecting a developmental increase of parasympathetic modulation; the normalized LF and the LF-HF ratio were unaffected. In great contrast, however, most of the HRV measures in the MCAO group were not significantly changed. Therefore, this study showed that the long-term effects of ischemic stroke injury involve retardation of the establishment of parasympathetic enhancement and disturbance of the normal sleep-wake cycle.

Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine.

AIMS: The aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC). MAIN METHODS: Nerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. KEY FINDINGS: Amongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose-response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. SIGNIFICANCE: These data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.