Implication of IGF1R signaling in the protective effect of Astragaloside IV on ischemia and reperfusion-induced cardiac microvascular endothelial hyperpermeability.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) causes damage to coronary capillary endothelial barrier and microvascular leakage (MVL), aggravating tissue injury and heart dysfunction. However, the effective strategy for protecting endothelium barrier of cardiac vasculature remains limited. PURPOSE: This study aimed to explore the effect of Astragaloside IV (ASIV) on coronary MVL after cardiac I/R and the underlying mechanism. STUDY DESIGN: Sprague-Dawley (SD) rats were used for assessment of the efficacy of Astragaloside IV in protection of myocardial I/R injury, while human cardiac microvascular endothelial cells were applied to gain more insight into the underlying mechanism. METHODS: Sprague-Dawley rats with or without pretreatment by ASIV at 10 mg/kg were subjected to occlusion of left coronary anterior descending artery followed by reperfusion. Endothelial cells were exposed to hypoxia and re-oxygenation (H/R). The distribution of junction proteins was detected by immunofluorescence staining and confocal microscope, the content of junction proteins was detected by Western blot, the level of adenosine triphosphate (ATP) was detected by ELISA, and the signal pathway related to permeability was detected by siRNA infection. The fluorescence intensity of FITC-albumin and FITC-Dextran was measured to evaluate the permeability of endothelial cells. RESULTS: ASIV exhibited protective effects on capillary damage, myocardium edema, albumin leakage, leucocyte infiltration, and the downregulated expression of endothelial junction proteins after I/R. Moreover, ASIV displayed ability to protect ATP from depletion after I/R or H/R, and the effect of ASIV on regulating vascular permeability and junction proteins was abolished once ATP synthase was inhibited. Notably, ASIV activated the insulin-like growth factor 1 receptor (IGF1R) and downstream signaling after reoxygenation. Knocking IGF1R down abolished the effect of ASIV on restoration of ATP, junction proteins and endothelial barrier after H/R. CONCLUSION: ASIV was potential to prevent MVL after I/R in heart. Moreover, the study for the first time demonstrated that the beneficial role of ASIV depended on promoting production of ATP through activating IGF1R signaling pathway. This result provided novel insight for better understanding the mechanism underlying the potential of ASIV to cope with cardiac I/R injury.

Quantitative Proteomics Reveal That Metabolic Improvement Contributes to the Cardioprotective Effect of T89 on Isoproterenol-Induced Cardiac Injury.

BACKGROUND: T89, a traditional Chinese medicine, has passed phase II, and is undergoing phase III clinical trials for treatment of ischemic cardiovascular disease by the US FDA. However, the role of T89 on isoproterenol (ISO)-induced cardiac injury is unknown. The present study aimed to explore the effect and underlying mechanism of T89 on ISO-induced cardiac injury. METHODS: Male Sprague-Dawley rats received subcutaneous injection of ISO saline solution at 24 h intervals for the first 3 days and then at 48 h intervals for the next 12 days. T89 at dose of 111.6 and 167.4 mg/kg was administrated by gavage for 15 consecutive days. Rat survival rate, cardiac function evaluation, morphological observation, quantitative proteomics, and Western blotting analysis were performed. RESULTS: T89 obviously improved ISO-induced low survival rate, attenuated ISO-evoked cardiac injury, as evidenced by myocardial blood flow, heart function, and morphology. Quantitative proteomics revealed that the cardioprotective effect of T89 relied on the regulation of metabolic pathways, including glycolipid metabolism and energy metabolism. T89 inhibited the enhancement of glycolysis, promoted fatty acid oxidation, and restored mitochondrial oxidative phosphorylation by regulating Eno1, Mcee, Bdh1, Ces1c, Apoc2, Decr1, Acaa2, Cbr4, ND2, Cox 6a, Cox17, ATP5g, and ATP5j, thus alleviated oxidative stress and energy metabolism disorder and ameliorated cardiac injury after ISO. The present study also verified that T89 significantly restrained ISO-induced increase of HSP70/HSP40 and suppressed the phosphorylation of ERK, further restored the expression of CX43, confirming the protective role of T89 in cardiac hypertrophy. Proteomics data are available via ProteomeXchange with identifier PXD024641. CONCLUSION: T89 reduced mortality and improves outcome in the model of ISO-induced cardiac injury and the cardioprotective role of T89 is correlated with the regulation of glycolipid metabolism, recovery of mitochondrial function, and improvement of myocardial energy.

The Composite of 3, 4-Dihydroxyl-Phenyl Lactic Acid and Notoginsenoside R1 Attenuates Myocardial Ischemia and Reperfusion Injury Through Regulating Mitochondrial Respiratory Chain.

AIM: 3,4-Dihydroxyl-phenyl lactic acid (DLA) and notoginsenoside R1 (R1) are known to protect ischemia and reperfusion (I/R) injury by targeting Sirtuin1/NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10/the Mitochondrial Complex I (Sirt-1/NDUFA10/Complex I) and Rho-associated kinase/adenosine triphosphate (ROCK/ATP) ATP synthase δ subunit (ATP 5D), respectively. We hypothesized that a composite of the two may exhibit a more potent effect on I/R injury. The study was designed to test this hypothesis. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent left anterior descending artery occlusion and reperfusion, with or without DLA, R1, or a combination of 3,4-dihydroxyl-phenyl lactic acid and notoginsenoside R1 (DR) pretreatment. Heart function, myocardial morphology, myocardial infarct, myocardial blood flow (MBF), apoptosis, vascular diameter, and red blood cell (RBC) velocity in venules were evaluated. Myeloperoxidase (MPO), malondialdehyde (MDA), and 8-oxo-deoxyguanosine (8-OHdG) were assessed. The content of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP), the activity of mitochondrial respiratory chain Complex I and its subunit NDUFA10, the Mitochondrial Complex V (Complex V) and its subunit ATP 5D, Sirt-1, Ras homolog gene family, member A (RhoA), ROCK-1, and phosphorylated myosin light chain (P-MLC) were evaluated. R1 binding to Sirt-1 was determined by surface plasmon resonance. RESULTS: DLA inhibited the expression of Sirt-1, the reduction in Complex I activity and its subunit NDUFA10 expression, the increase in MPO, MDA, and 8-OhdG, and apoptosis. R1 inhibited the increase in the expression of RhoA/ROCK-1/P-MLC, the reduction of Complex V activity and its subunit ATP 5D expression, alleviated F-actin, and myocardial fiber rupture. Both DLA and R1 reduced the myocardial infarction size, increased the velocities of RBC in venules, and improved MBF and heart function impaired by I/R. DR exhibited effects similar to what was exerted, respectively, by DLA and R1 in terms of respiratory chain complexes and related signaling and outcomes, and an even more potent effect on myocardial infarct size, RBC velocity, heart function, and MBF than DLA and R1 alone. CONCLUSION: A combination of 3,4-dihydroxyl-phenyl lactic acid and notoginsenoside R1 revealed a more potent effect on I/R injury via the additive effect of DLA and R1, which inhibited not only apoptosis caused by low expression of Sirt-1/NDUFA10/Complex I but also myocardial fiber fracture caused by RhoA/ROCK-1 activation and decreased expression of ATP/ATP 5D/Complex V.

QiShenYiQi Pills Attenuates Ischemia/Reperfusion-Induced Cardiac Microvascular Hyperpermeability Implicating Src/Caveolin-1 and RhoA/ROCK/MLC Signaling.

Aims: Coronary microvascular hyperpermeability is an important contributor to ischemia or reperfusion (I/R) injury. However, the effective strategy for this insult remains limited. This study aimed to explore the protective effect of the compound Chinese medicine QiShenYiQi Pills (QSYQ) against coronary microvascular hyperpermeability after cardiac I/R with focusing on the underlying mechanism. Methods and Results: Male Sprague-Dawley rats under anesthesia were subjected to occlusion of left coronary anterior descending artery followed by reperfusion. QSYQ was administrated 90 min before ischemia initiation. Human cardiac microvascular endothelial cells (HCMECs) underwent hypoxia or reoxygenation (H/R) challenge with QSYQ administrated 1 h prior to hypoxia. QSYQ exhibited effects on attenuating microvascular damage and albumin leakage after I/R injury, showing a role in maintaining endothelial junctions, caveolae, and collagen in basement membrane (BM) of microvessels. Study using HCMECs disclosed that QSYQ protected endothelial barrier from impairment by H/R, attenuating the decline of respiratory chain complex I and ATP synthase, activation of Src/caveolin-1 and increase of RhoA/ROCK/p-MLC, MMP-9, and CTSS. PP2, a Src inhibitor, partially imitated the effect of QSYQ. Conclusions: The QSYQ was able to prevent I/R-induced cardiac microvascular hyperpermeability via a mechanism involving Src/caveolin-1 and RhoA/ROCK/MLC signaling.

Post-treatment With Qing-Ying-Tang, a Compound Chinese Medicine Relives Lipopolysaccharide-Induced Cerebral Microcirculation Disturbance in Mice.

Objective: Lipopolysaccharide (LPS) causes microvascular dysfunction, which is a key episode in the pathogenesis of endotoxemia. This work aimed to investigate the effect of Qing-Ying-Tang (QYT), a compound Chinese medicine in cerebral microcirculation disturbance and brain damage induced by LPS. Methods: Male C57/BL6 mice were continuously transfused with LPS (7.5 mg/kg/h) through the left femoral vein for 2 h. QYT (14.3 g/kg) was given orally 2 h after LPS administration. The dynamics of cerebral microcirculation were evaluated by intravital microscopy. Brain tissue edema was assessed by brain water content and Evans Blue leakage. Cytokines in plasma and brain were evaluated by flow cytometry. Confocal microscopy and Western blot were applied to detect the expression of junction and adhesion proteins, and signaling proteins concerned in mouse brain tissue. Results: Post-treatment with QYT significantly ameliorated LPS-induced leukocyte adhesion to microvascular wall and albumin leakage from cerebral venules and brain tissue edema, attenuated the increase of MCP-1, MIP-1α, IL-1α, IL-6, and VCAM-1 in brain tissue and the activation of NF-κB and expression of MMP-9 in brain. QYT ameliorated the downregulation of claudin-5, occludin, JAM-1, ZO-1, collagen IV as well as the expression and phosphorylation of VE-cadherin in mouse brain. Conclusions: This study demonstrated that QYT protected cerebral microvascular barrier from disruption after LPS by acting on the transcellular pathway mediated by caveolae and paracellular pathway mediated by junction proteins. This result suggests QYT as a potential strategy to deal with endotoxemia.

Cardiotonic Pills Plus Recombinant Human Prourokinase Ameliorates Atherosclerotic Lesions in LDLR-/- Mice.

AIM: This study was to explore the protective effects of cardiotonic pills (CP) or/and recombinant human prourokinase (proUK)on the atherosclerosis and the potential underlying mechanism. METHODS AND RESULTS: Atherosclerosis was induced in LDLR-/- mice by high fat diet contained 20% lard and 0.5% cholesterol. Daily oral administration of CP (130 mg/kg) or/and intravenous injection of proUK (2.5 mg/kg, twice a week) began at 8 weeks after feeding with high fat diet and continued for 4 weeks. CP alone treatment markedly decreased plasma triglyceride, but did not ameliorate atherosclerosis plaque. No effect was observed for proUK alone on any endpoints tested. CP plus proUK induced a significantly reduction in the atherosclerotic lesions, along with decreased levels of total cholesterol, triglyceride in the plasma. CP plus proUK inhibited the elevated hepatic total cholesterol and triglyceride in high fat diet-fed LDLR-/- mice, up-regulating the expressions of ATP-binding cassette gene 5 and 8, and adipose triglyceride lipase. In the aorta, CP plus proUK inhibited the expression of scavenger receptor A and CD36 in LDLR-/- mice. In addition, we observed that systemic inflammation was inhibited, manifested downregulation of plasma macrophage inflammatory protein-1α and intercellular cell adhesion molecule-1. CONCLUSION: CP plus proUK effectively attenuated atherosclerosis plaque in LDLR-/- mice, which is associated with normalizing the lipid metabolism in the liver and aorta, reducing phagocytosis of receptor-mediated modified-LDL uptake and inhibiting systemic inflammation.

Total Salvianolic Acid Injection Prevents Ischemia/Reperfusion-Induced Myocardial Injury Via Antioxidant Mechanism Involving Mitochondrial Respiratory Chain Through the Upregulation of Sirtuin1 and Sirtuin3.

Sirtuin1 (Sirt1) and Sirtuin3 (Sirt3) are known to participate in regulating mitochondrial function. However, whether Total Salvianolic Acid Injection (TSI) protects against myocardial ischemia/reperfusion (I/R) injury through regulating Sirt1, Sirt3, and mitochondrial respiratory chain complexes is unclear. The aim of this study was to explore the effects of TSI on I/R-induced myocardial injury and the underlying mechanism. Male Sprague-Dawley rats were subjected to 30 min occlusion of the left anterior descending coronary artery followed by 90 min reperfusion with or without TSI treatment (8 mg/kg/h). The results demonstrated that TSI attenuated I/R-induced myocardial injury by the reduced infarct size, recovery of myocardial blood flow, and decreased cardiac apoptosis. Moreover, TSI protected heart from oxidative insults, such as elevation of myeloperoxidase, malondialdehyde, hydrogen peroxide, ROS, as well as attenuated I/R-elicited downregulation of Sirt1, Sirt3, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 10 (NDUFA10), succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), and restoring mitochondrial respiratory chain complexes activity. The in vitro study in H9c2 cells using siRNA transfection further confirmed the critical role of Sirt1 and Sirt3 in the effect of TSI on the expression of NDUFA10 and SDHA. These results demonstrated that TSI attenuated I/R-induced myocardial injury via inhibition of oxidative stress, which was related to the activation of NDUFA10 and SDHA through the upregulation of Sirt1 and Sirt3.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Caffeic acid attenuates rat liver injury after transplantation involving PDIA3-dependent regulation of NADPH oxidase.

The transplanted liver inevitably suffers from ischemia reperfusion (I/R) injury, which represents a key issue in clinical transplantation determining early outcome and long-term graft survival. A solution is needed to deal with this insult. This study was undertaken to explore the effect of Caffeic acid (CA), a naturally occurring antioxidant, on I/R injury of grafted liver and the mechanisms involved. Male Sprague-Dawley rats underwent orthotopic liver transplantation (LT) in the absence or presence of CA administration. In vitro, HL7702 cells were subjected to hypoxia/reoxygenation. LT led to apparent hepatic I/R injury, manifested by deteriorated liver function, microcirculatory disturbance and increased apoptosis, along with increased PDIA3 expression and nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase activity, and membrane translocation of NADPH oxidase subunits. Treatment with CA attenuated the above alterations. siRNA/shRNA-mediated knockdown of PDIA3 in HL7702 cells and rats played the same role as CA not only in inhibiting ROS production and NADPH oxidase activity, but also in alleviating hepatocytes injury. CA protects transplanted livers from injury, which is likely attributed to its protection of oxidative damage by interfering in PDIA3-dependent activation of NADPH oxidase.

Bushen Huoxue Attenuates Diabetes-Induced Cognitive Impairment by Improvement of Cerebral Microcirculation: Involvement of RhoA/ROCK/moesin and Src Signaling Pathways.

Type 2 Diabetes mellitus (T2DM) is closely correlated with cognitive impairment and neurodegenerative disease. Bushen Huoxue (BSHX) is a compound Chinese medicine used clinically to treat diabetes-induced cognitive impairment. However, its underlying mechanisms remain unclear. In the present study, KKAy mice, a genetic model of type 2 diabetes with obesity and insulin resistant hyperglycemia, received a daily administration of BSHX for 12 weeks. Blood glucose was measured every 4 weeks. After 12 weeks, BSHX treatment significantly ameliorated the T2DM related insults, including the increased blood glucose, the impaired spatial memory, decreased cerebral blood flow (CBF), occurrence of albumin leakage, leukocyte adhesion and opening capillary rarefaction. Meanwhile, the downregulation of the tight junction proteins (TJ) claudin-5, occludin, zonula occluden-1 (ZO-1) and JAM-1 between endothelial cells, amyloid-ß (Aß) accumulation in hippocampus, increased AGEs and RAGE, and expression of RhoA/ROCK/moesin signaling pathway and phosphorylation of Src kinase in KKAy mice were significantly protected by BSHX treatment. These results indicate that the protective effect of BSHX on T2DM-induced cognitive impairment involves regulation of RhoA/ROCK1/moesin signaling pathway and phosphorylation of Src kinase.

YangXue QingNao Wan and Silibinin Capsules, the Two Chinese Medicines, Attenuate Cognitive Impairment in Aged LDLR (+/-) Golden Syrian Hamsters Involving Protection of Blood Brain Barrier.

The purpose of the study was to explore the effect and the underlying mechanism of YangXue QingNao Wan (YXQNW) and Silibinin Capsules (SC), the two Chinese medicines, on cognitive impairment in older people with familial hyperlipidaemia. Fourteen month-old female LDLR (+/-) golden Syrian hamsters were used with their wild type as control. YXQNW (0.5 g/kg/day), SC (0.1 g/kg/day), or YXQNW (0.5 g/kg/day) + SC (0.1 g/kg/day) were administrated orally for 30 days. To assess the effects of the two drugs on plasma lipid content and cognitive ability, plasma TC, TG, LDL-C, and HDL-C were measured, and Y maze task was carried out both before and after administration. After administering of the drugs for 30 days, to evaluate the effect of the two drugs on disturbed blood flow caused by hyperlipidemia, the cerebral blood flow (CBF) was measured. To assess blood-brain barrier integrity, albumin leakage in middle cerebral artery (MCA) area was determined. To evaluate the effect of the drugs on impaired microvessels, the number and morphology of microvessels were assessed in hippocampus area. To further evaluate the ultrastructure of microvessels in hippocampus, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were carried out. To assess the profiles of claudin-5 and occludin in hippocampus, we performed immunofluorescence. Finally, to assess the expression of claudin-5, JAM-1, occludin and ZO-1 in hippocampus, western blot was carried out. The results showed that YXQNW, SC, and YXQNW + SC improved cognitive impairment of aged LDLR (+/-) golden Syrian hamsters without lowering plasma TC and LDL-C. YXQNW, SC, and YXQNW + SC attenuated albumin leakage in MCA area and neuronal damage in hippocampus, concomitant with an increase in CBF, a decrease of perivascular edema and an up-regulated expression of claudin-5, occludin and ZO-1. In conclusion, YXQNW, SC, and YXQNW + SC are able to improve cognitive ability in aged LDLR (+/-) golden Syrian hamsters via mechanisms involving maintaining blood-brain barrier integrity. These findings provide evidence suggesting YXQNW or SC as a potential regime to counteract the cognitive impairment caused by familial hypercholesterolemia.

The Contribution of Different Components in QiShenYiQi Pills® to Its Potential to Modulate Energy Metabolism in Protection of Ischemic Myocardial Injury.

Ischemic heart diseases remain a challenge for clinicians. QiShenYiQi pills® (QSYQ) has been reported to be curative during coronary heart diseases with modulation of energy metabolism as one of the underlying mechanisms. In this study, we detected the effect of QSYQ and its components on rat myocardial structure, mitochondrial respiratory chain complexes activity and energy metabolism, and heart function after 30 min of cardiac ischemia, with focusing on the contribution of each component to its potential to regulate energy metabolism. Results showed that treatment with QSYQ and all its five components protected myocardial structure from damage by ischemia. QSYQ also attenuated release of myocardial cTnI, and restored the production of ATP after cardiac ischemia. AS-IV and Rb1, but not Rg1, R1, and DLA, had similar effect as QSYQ in regulation of energy metabolism. These results indicate that QSYQ may prevent ischemia-induced cardiac injury via regulation of energy metabolism, to which each of its components contributes differently.

Ginsenoside Rg1 Ameliorates Rat Myocardial Ischemia-Reperfusion Injury by Modulating Energy Metabolism Pathways.

As a major ingredient of Radix ginseng, ginsenoside Rg1 (Rg1) has been increasingly recognized to benefit the heart condition, however, the rationale behind the role is not fully understood. In vitro study in H9c2 cardiomyocytes has shown the potential of Rg1 to increase ATP content in the cells. We thus speculated that the protective effect of Rg1 on heart ischemia and reperfusion (I/R) injury implicates energy metabolism regulation. The present study was designed to verify this speculation. Male Sprague-Dawley rats were subjected to 30 min of occlusion of left coronary anterior descending artery followed by reperfusion for 90 min. Rg1 (5 mg/kg/h) was continuously administrated intravenously 30 min before occlusion until the end of reperfusion. Myocradial blood flow and heart function were monitored over the period of I/R. Myocardial infarct size, structure and apoptosis, energy metabolism, and change in RhoA signaling pathway were evaluated 90 min after reperfusion. Binding of Rg1 to RhoA was assessed using Surface Plasmon Resonance (SPR). Rg1 prevented I/R-elicited insults in myocardium, including myocardial infarction and apoptosis, decreased myocardial blood flow (MBF) and heart function, and alteration in myocardium structure. Rg1 restored the production of ATP in myocardium after I/R. Rg1 was able to bind to RhoA and down-regulate the activity of RhoA signaling pathway. These results indicated that Rg1 had protective potential against I/R-induced myocardial injury, which may be related to inhibiting myocardial apoptosis and modulating energy metabolism through binding to RhoA.

Ginsenoside Rb1 protects against ischemia/reperfusion-induced myocardial injury via energy metabolism regulation mediated by RhoA signaling pathway.

Cardiac ischemia and reperfusion (I/R) injury remains a challenge for clinicians. Ginsenoside Rb1 (Rb1) has been reported to have the ability to attenuate I/R injury, but its effect on energy metabolism during cardiac I/R and the underlying mechanism remain unknown. In this study, we detected the effect of Rb1 on rat myocardial blood flow, myocardial infarct size, cardiac function, velocity of venule red blood cell, myocardial structure and apoptosis, energy metabolism and change in RhoA signaling pathway during cardiac I/R injury. In addition, the binding affinity of RhoA to Rb1 was detected using surface plasmon resonance (SPR). Results showed that Rb1 treatment at 5 mg/kg/h protected all the cardiac injuries induced by I/R, including damaged myocardial structure, decrease in myocardial blood flow, impaired heart function and microcirculation, cardiomyocyte apoptosis, myocardial infarction and release of myocardial cTnI. Rb1 also inhibited the activation of RhoA signaling pathway and restored the production of ATP during cardiac I/R. Moreover, SPR assay showed that Rb1 was able to bind to RhoA in a dose-dependent manner. These results indicate that Rb1 may prevent I/R-induced cardiac injury by regulation of RhoA signaling pathway, and may serve as a potential regime to improve percutaneous coronary intervention outcome.

Catalpol restores LPS-elicited rat microcirculation disorder by regulation of a network of signaling involving inhibition of TLR-4 and SRC.

LPS-induced microvascular hyperpermeability and hemorrhage play a key role in the development of sepsis, the attenuation of which might be an important strategy to prevent sepsis. However, the current clinical therapies have proven to be inefficient in improving the prognosis for patients with sepsis. Catalpol, an iridoid glycoside extracted from the roots of Rehmannia, has been reported to protect against LPS-induced acute lung injury through a Toll-like receptor-4 (TLR-4)-mediated NF-κB signaling pathway. However, it is still unknown whether catalpol can be an effective treatment to ameliorate the LPS-induced microvascular disorder. The present study aimed to investigate the impact of catalpol on LPS-induced mesenteric microvascular disorder and its underlying mechanism. Male Wistar rats were challenged by infusion of LPS (10 mg·kg-1·h-1) through the left femoral vein for 120 min. Post-treatment with catalpol (10 mg/kg) alleviated the LPS-induced microvascular hyperpermeability and hemorrhage; reduced mortality; ameliorated the alteration in the distribution of claudin-5 and the junctional adhesion molecule-1, as well as the degradation of collagen IV and laminin; and attenuated the increase of TLR-4 level, phosphorylations of Src tyrosine kinase, phosphatidyl inositol 3-kinase, focal adhesion kinase, and cathepsin B activation. In vitro study in human umbilical vein endothelial cells verified these results and further revealed that inhibition of TLR-4 and Src each simulated some, but not all, of the effects that catalpol exerted. Besides, surface plasmon resonance showed that catalpol could directly bind to TLR-4 and Src. These results demonstrated that catalpol was able to ameliorate the LPS-induced microvascular barrier damage and hemorrhage by targeting both TLR-4 and Src, thus attenuating the phosphorylation of Src kinase, phosphatidyl inositol 3-kinase, and focal adhesion kinase, as well as cathepsin B activation.

3, 4-dihydroxyl-phenyl lactic acid restores NADH dehydrogenase 1 α subunit 10 to ameliorate cardiac reperfusion injury.

The present study aimed to detect the role of 3, 4-dihydroxyl-phenyl lactic acid (DLA) during ischemia/reperfusion (I/R) induced myocardial injury with emphasis on the underlying mechanism of DLA antioxidant. Male Spragu-Dawley (SD) rats were subjected to left descending artery occlusion followed by reperfusion. Treatment with DLA ameliorated myocardial structure and function disorder, blunted the impairment of Complex I activity and mitochondrial function after I/R. The results of 2-D fluorescence difference gel electrophoresis revealed that DLA prevented the decrease in NDUFA10 expression, one of the subunits of Complex I. To find the target of DLA, the binding affinity of Sirtuin 1 (SIRT1) to DLA and DLA derivatives with replaced two phenolic hydroxyls was detected using surface plasmon resonance and bilayer interferometry. The results showed that DLA could activate SIRT1 after I/R probably by binding to this protein, depending on phenolic hydroxyl. Moreover, the importance of SIRT1 to DLA effectiveness was confirmed through siRNA transfection in vitro. These results demonstrated that DLA was able to prevent I/R induced decrease in NDUFA10 expression, improve Complex I activity and mitochondrial function, eventually attenuate cardiac structure and function injury after I/R, which was possibly related to its ability of binding to and activating SIRT1.

Levo-tetrahydropalmatine attenuates mouse blood-brain barrier injury induced by focal cerebral ischemia and reperfusion: Involvement of Src kinase.

The restoration of blood flow following thrombolytic therapy causes ischemia and reperfusion (I/R) injury leading to blood-brain barrier (BBB) disruption and subsequent brain edema in patients of ischemic stroke. Levo-tetrahydropalmatine (l-THP) occurs in Corydalis genus and some other plants. However, whether l-THP exerts protective role on BBB disruption following cerebral I/R remains unclear. Male C57BL/6N mice (23 to 28 g) were subjected to 90 min middle cerebral artery occlusion, followed by reperfusion for 24 h. l-THP (10, 20, 40 mg/kg) was administrated by gavage 60 min before ischemia. We found I/R evoked Evans blue extravasation, albumin leakage, brain water content increase, cerebral blood flow decrease, cerebral infarction and neurological deficits, all of which were attenuated by l-THP treatment. Meanwhile, l-THP inhibited tight junction (TJ) proteins down-expression, Src kinase phosphorylation, matrix metalloproteinases-2/9 (MMP-2/9) and caveolin-1 activation. In addition, surface plasmon resonance revealed binding of l-THP to Src kinase with high affinity. Then we found Src kinase inhibitor PP2 could attenuate Evans blue dye extravasation and inhibit the caveolin-1, MMP-9 activation, occludin down-expression after I/R, respectively. In conclusion, l-THP attenuated BBB injury and brain edema, which were correlated with inhibiting the Src kinase phosphorylation.

Salvianolic Acid B Ameliorates Lipopolysaccharide-Induced Albumin Leakage from Rat Mesenteric Venules through Src-Regulated Transcelluar Pathway and Paracellular Pathway.

Lipopolysaccharide (LPS) causes microvascular barrier disruption, leading to albumin leakage from microvessels resulting in a range of disastrous sequels. Salvianolic acid B (SalB) is a major water-soluble component derived from Salvia miltiorrhiza. Previous studies showed its potential to attenuate microvascular barrier dysfunction, but the underlying mechanism is not fully understood. The present study was intended to investigate the impact of SalB on endothelial cell barrier in vivo in rat mesenteric venules as well as in vitro in human umbilical vein endothelial cells (HUVECs), aiming at disclosing the mechanism thereof, particularly the role of Src in its action. Male Wistar rats were challenged by infusion of LPS (2 mg/kg/h) through left femoral vein for 90 min. SalB (5 mg/kg/h) was administrated either simultaneously with LPS or 30 min after LPS infusion through the left jugular vein. Vesicles in venular walls were observed by electron microscopy. HUVECs were incubated with LPS with or without SalB. The expression of Zonula occluden-1 (ZO-1), VE-cadherin, caveolin-1 and Src in HUVECs was assessed by Western blot and confocal microscopy, binding of SalB to Src was measured using Surface Plasmon Resonance and BioLayer Interferometry. Treatment with SalB inhibited albumin leakage from rat mesenteric venules and inhibited the increase of vesicle number in venular endothelial cells induced by LPS. In addition, SalB inhibited the degradation of ZO-1, the phosphorylation and redistribution of VE-cadherin, the expression and phosphorylation of caveolin-1, and phosphoirylation of Src in HUVECs exposed to LPS. Furthermore, SalB was found able to bind to Src. This study demonstrates that protection of SalB against microvascular barrier disruption is a process involving both para- and trans-endothelial cell pathway, and highly suggests Src as the key enzyme for SalB to work.

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain.

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain.

ROCK-dependent ATP5D modulation contributes to the protection of notoginsenoside NR1 against ischemia-reperfusion-induced myocardial injury.

Cardiac ischemia-reperfusion (I/R) injury remains a challenge for clinicians, which initiates with energy metabolism disorder. The present study was designed to investigate the protective effect of notoginsenoside R1 (NR1) on I/R-induced cardiac injury and underlying mechanism. Male Sprague-Dawley rats were subjected to 30-min occlusion of the left coronary anterior descending artery followed by reperfusion with or without NR1 pretreatment (5 mg·kg(-1)·h(-1)). In vitro, H9c2 cells were cultured under oxygen and glucose deprivation/reoxygenation conditions after NR1 (0.1 mM), Rho kinase (ROCK) inhibitor Y-27632 (10 µM), or RhoA/ROCK activator U-46619 (10 nM) administration. Myocardial infarct size, myocardial histology, and cardiac function were evaluated. Myofibril and mitochondria morphology were observed by transmission electron microscopy. F-actin and apoptosis were determined by immunofluorescence and TUNEL staining. ATP and AMP content were assessed by ELISA. Phosphorylated-AMP-activated protein kinase, ATP synthase subunits, apoptosis-related molecules, and the level and activity of ROCK were determined by Western blot analysis. We found that NR1 pretreatment ameliorated myocardial infarction, histological injury, and cardiac function induced by I/R. Furthermore, similar to the effect of Y-27632, NR1 improved H9c2 cell viability, maintained actin skeleton and mitochondria morphology, and attenuated apoptosis induced by oxygen and glucose deprivation/reoxygenation. Importantly, NR1 prevented energy abnormity, inhibited the expression and activation of ROCK, and restored the expression of the mitochondrial ATP synthase δ-subunit both in vivo and in vitro, whereas U-46619 suppressed the effect of NR1. These results prove NR1 as an agent able to prevent I/R-induced energy metabolism disorder via inhibiting ROCK and enhancing mitochondrial ATP synthase δ-subunits, which at least partially contributes to its protection against cardiac I/R injury.