Xiaojin Zhang

Involvement of Reductive Stress in the Cardiomyopathy in Transgenic Mice With Cardiac-Specific Overexpression of Heat Shock Protein 27

Oxidative stress plays an important role in cardiac diseases, which has been well demonstrated, whereas the role of reductive stress has been poorly investigated. We and others have shown previously that heat shock protein 27 (Hsp27) plays a role as an antioxidant. To investigate whether overexpression of Hsp27 could lead to reductive stress and result in cardiomyopathy, we generated transgenic mice with different expression levels of Hsp27. We observed that transgenic mice with high levels of Hsp27 developed cardiomyopathy. The myopathic hearts were under reductive stress, which was evidenced by an increased ratio of reduced glutathione/oxidized glutathione and a decreased level of reactive oxygen species. In addition, upregulated glutathione peroxidase 1 and decreased iron content were revealed in the myopathic hearts. More importantly, inhibition of glutathione peroxidase 1 significantly attenuated the development of cardiomyopathy. The data indicate that the Hsp27-induced cardiomyopathy could be attributed to, at least in part, upregulation of glutathione peroxidase 1. Our findings suggest that reductive stress plays an important role in the development of cardiomyopathy and that Hsp27 may serve as a potential target for the treatment of patients with cardiomyopathy.

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

BACKGROUND Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth. METHODS To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth. RESULTS LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth. CONCLUSION Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Cardiac-specific expression of heat shock protein 27 attenuated endotoxin-induced cardiac dysfunction and mortality in mice through a PI3K/Akt-dependent mechanism.

Cardiac dysfunction is a major consequence of septic shock and may be responsible for the high mortality of sepsis. We have reported that transgenic mice with cardiac-specific overexpression of heat shock protein 27 (Hsp27 Tg) exhibited the protection against doxorubicin-induced cardiac dysfunction. We hypothesized that overexpression of Hsp27 will attenuate cardiac dysfunction during endotoxemia. Hsp27 Tg and age-matched wild-type (WT) mice were injected with LPS. Cardiac function was evaluated by echocardiography, survival rate was carefully monitored, and activities of signaling pathways were determined by immunoblot. LPS administration significantly decreased cardiac function in WT mice. In Hsp27 Tg mice, LPS-induced cardiac dysfunction was significantly attenuated as evidenced by increased ejection fraction (27.3%) and fractional shortening (37.1%), respectively, compared with LPS-treated WT mice. Heat shock protein 27 Tg mice were more resistant to LPS-induced mortality than WT. The levels of phospho-Akt and phospho-glycogen synthase kinase 3&bgr; (phospho-GSK-3&bgr;) in the myocardium were significantly increased in Hsp27 Tg mice compared with WT after LPS administration. Nuclear factor &kgr;B-binding activity was significantly decreased in Hsp27 Tg mice compared with WT mice after LPS challenge. Similar results were observed in in vitro studies using Hsp27-transfected rat cardiomyoblasts. Importantly, phosphoinositide 3-kinase inhibition abolished the protective effect of Hsp27 in LPS-induced cardiac dysfunction and mortality of endotoxemia. Our results suggest that Hsp27 plays an important role in attenuation of cardiac dysfunction and mortality in endotoxemia and that the mechanisms of the protection may involve activation of the PI3K/Akt signaling pathway.

Attenuation of cardiac dysfunction by HSPA12B in endotoxin-induced sepsis in mice through a PI3K-dependent mechanism.

AIMS cardiac dysfunction is a critical manifestation of severe sepsis/septic shock and is responsible for high mortality due to sepsis. Recent evidence suggests that angiogenic factors have a protective effect on sepsis-induced organ damage. Heat shock protein A12B (HSPA12B) is a newly discovered gene that is essential for angiogenesis. We hypothesized that overexpression of HSPA12B would induce protection against endotoxin-induced cardiac dysfunction. METHODS AND RESULTS to evaluate this hypothesis, we generated transgenic mice overexpressing the human hspa12b gene (Tg). Wild-type (WT) littermates served as controls. Tg and WT mice were treated with lipopolysaccharide (LPS) and cardiac function was measured after 6 h. LPS treatment caused cardiac dysfunction in WT mice. In contrast, cardiac function was significantly preserved in Tg mice following LPS administration. LPS increased the expression of vascular cell adhesion molecule-1 (VCAM-1)/intercellular adhesion molecule-1 (ICAM-1) and leucocyte infiltration into the myocardium of WT mice. In Tg mice, LPS-increased VCAM-1/ICAM-1 expression and leucocyte infiltration were significantly attenuated. Overexpression of HSPA12B also prevented the decrement in the activation of phosphatidlyinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling in the myocardium. Importantly, PI3K inhibition with Wortmannin abolished the protection of HSPA12B against LPS-induced cardiac dysfunction. CONCLUSION these results suggest that HSPA12B plays an important role in the attenuation of endotoxin-induced cardiac dysfunction and that the mechanisms involve the preserved activation of PI3K/Akt signalling, resulting in attenuation of LPS-increased expression of VCAM-1/ICAM-1 and leucocyte infiltration into the myocardium.

α-Lipoic acid prevents bupivacaine-induced neuron injury in vitro through a PI3K/Akt-dependent mechanism

BACKGROUND Bupivacaine is an amide type local anesthetic which is widely used for epidural anesthesia and nerve blockade in patients. However, local administration of bupivacaine could cause neuron injury showing transient neurologic symptoms. alpha-Lipoic acid (LA) was shown to protect nerve cells from substance-induced injury. We hypothesized that LA administration could attenuate bupivacaine-induced neurotoxicity. METHODS To evaluate our hypothesis, we treated mouse neuroblastoma N2a cells with LA 30 min before the cells were exposed to bupivacaine. We evaluated cellular injury by examination of cell viability, morphology changes, nuclear condensation, and Annexin V staining. We also examined the levels of intracellular reactive oxygen species (ROS) and activation of PI3K/Akt signaling pathway. In a separate experiment, we determined the effect of Akt inhibition on cell viability in the presence of LA and bupivacaine. RESULTS Bupivacaine treatment significantly induced cell injury as evidenced by decreased cell viability, increased nuclear condensation and Annexin V staining. Administration of LA significantly attenuated bupivacaine-induced cell injury. In addition, LA treatment increased the levels of phospho-Akt and phospho-GSK3beta and attenuated bupivacaine decreased the levels of ROS. More significantly, pharmacological inhibition of Akt abolished the LA-induced protection from bupivacaine-caused cell injury. CONCLUSIONS Our findings suggest that pretreatment of neuroblastoma cells with LA protected neural cells from bupivacaine-induced injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.

Overexpression of HSPA12B protects against cerebral ischemia/reperfusion injury via a PI3K/Akt-dependent mechanism

BACKGROUND AND PURPOSE HSPA12B is a newly discovered member of the Hsp70 family proteins. This study investigated the effects of HSPA12B on focal cerebral ischemia/reperfusion (I/R) injury in mice. METHODS Transgenic mice overexpressing human HSPA12B (Tg) and wild-type littermates (WT) were subjected to 60 min of middle cerebral artery occlusion to induce ischemia and followed by reperfusion (I/R). Neurological deficits, infarct volumes and neuronal death were examined at 6 and 24hrs after reperfusion. Blood-brain-barrier (BBB) integrity and activated cellular signaling were examined at 3 hrs after reperfusion. RESULTS After cerebral I/R, Tg mice exhibited improvement in neurological deficits and decrease in infarct volumes, when compared with WT I/R mice. BBB integrity was significantly preserved in Tg mice following cerebral I/R. Tg mice also showed significant decreases in cell injury and apoptosis in the ischemic hemispheres. We observed that overexpression of HSPA12B activated PI3K/Akt signaling and suppressed JNK and p38 activation following cerebral I/R. Importantly, pharmacological inhibition of PI3K/Akt signaling abrogated the protection against cerebral I/R injury in Tg mice. CONCLUSIONS The results demonstrate that HSPA12B protects the brains from focal cerebral I/R injury. The protective effect of HSPA12B is mediated though a PI3K/Akt-dependent mechanism. Our results suggest that HSPA12B may have a therapeutic potential against ischemic stroke.

α-Lipoic acid attenuates LPS-induced cardiac dysfunction through a PI3K/Akt-dependent mechanism.

Myocardial dysfunction is an important manifestation of sepsis/septic shock. Activation of Phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway has been shown to improve cardiac performance during sepsis/septic shock. We have reported previously that α-lipoic acid (LA) activates PI3K/Akt pathway in neuronal cells. It is possible, therefore, that treatment with LA will attenuate cardiac dysfunction during sepsis/septic shock through a PI3K/Akt-dependent mechanism. To test this possibility, we treated mice with LA prior to lipopolysaccharide (LPS) challenge. Cardiac function was analyzed by echocardiography 6h after LPS challenge. LPS significantly suppressed cardiac function as evidenced by decreases in EF% and FS% in mice. However, LA pretreatment significantly attenuated cardiac dysfunction following LPS challenge. LA pretreatment also improved survival in LPS-challenged mice. Furthermore, LA markedly attenuated the LPS-induced inflammatory response in myocardium, as evidenced by decreases in the upregulation of VCAM-1, ICAM-1 and iNOS, as well as myocardial leucocytes infiltration. Moreover, LPS challenge significantly decreased the phosphorylation levels of Akt and Gsk-3β, which was prevented by LA pretreatment. More importantly, inhibition of PI3K/Akt signaling by Wortmannin (WM) completely abrogated the LA-induced protection in cardiac dysfunction following LPS challenge. Collectively, our results demonstrated that LA improved cardiac function during endotoxemia. The mechanism was through, at least in part, preserved activation of the PI3K/Akt signaling.

RNA interference‐mediated silencing of the polo‐like kinase 1 gene enhances chemosensitivity to gemcitabine in pancreatic adenocarcinoma cells

Gemcitabine is the first‐line chemotherapeutic agent for advanced adenocarcinoma of the pancreas; however, chemoresistance to gemcitabine remains a major cause of failure for the clinical treatment of this disease. Polo‐like kinase 1 (Plk‐1) is highly expressed in pancreatic cancer cell lines and pancreatic tumour tissues, and is involved in a wide variety of cell cycle processes. Nevertheless, its biological role and implication for gemcitabine resistance are not clearly defined. In this study, we used RNA‐interference (RNAi)‐mediated depletion of Plk‐1 to determine its potential for sensitizing pancreatic tumour cells to gemcitabine. We showed that the level of Plk‐1 protein was correlated significantly with gemcitabine resistance in human pancreatic adenocarcinoma cells and that overexpression of Plk‐1 reduced sensitivity to gemcitabine in these cells. In addition, small interfering RNA (siRNA)‐mediated knockdown of Plk‐1 caused cell cycle arrest at G2/M and the reduction of cellular proliferation. More importantly, the treatment of pancreatic cancer cells with Plk‐1 siRNA followed by exposure to gemcitabine dramatically decreased cell viability and increased cellular apoptosis, as compared with treatment with either agent alone. These observations indicate that down‐regulation of Plk‐1 expression by RNAi enhances gemcitabine sensitivity and increases gemcitabine cytotoxicity in pancreatic tumour cells. This is the first demonstration that the combination of Plk‐1 gene therapy and gemcitabine chemotherapy has synergistic anti‐tumour activity against pancreatic carcinoma in vitro. This combination treatment warrants further investigation as an effective therapeutic regimen for patients with resistant pancreatic cancer and other tumours.

HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism

AIMS HSPA12B is a newly discovered and endothelial-cell-specifically expressed heat shock protein. We have reported recently that overexpression of HSPA12B increased endothelial nitric oxide synthase (eNOS) expression in mouse cardiac tissues during endotoxemia. Endothelial NOS has been shown to protect heart from ischaemic injury. We hypothesized that overexpression of HSPA12B will attenuate cardiac dysfunction and remodelling after myocardial infarction (MI) through an eNOS-dependant mechanism. METHODS AND RESULTS MI was induced by permanent ligation of the left anterior descending coronary artery in the transgenic mice (Tg) overexpressing hspa12b gene and its wild-type (WT) littermates. Echocardiographic analysis revealed that Tg mice exhibited improvements in cardiac dysfunction and remodelling at 1 and 4 weeks after MI. These improvements were accompanied by a significant decrease in cardiomyocyte apoptosis and increase in capillary and arteriolar densities. Significant up-regulation of eNOS, VEGF, Ang-1, and Bcl-2 was also observed in Tg hearts compared with WT hearts after MI. However, pharmacological inhibition of eNOS abolished the HSPA12B-induced decrease in cardiomyocyte apoptosis and increase in capillary formation after MI. Most importantly, inhibition of eNOS abrogated the protection of HSPA12B against cardiac dysfunction and remodelling after MI. CONCLUSIONS These data demonstrate for the first time that the overexpression of HSPA12B attenuates cardiac dysfunction and remodelling after MI. This action of HSPA12B was mediated, at least in part, by prevention of cardiomyocyte apoptosis and promotion of myocardial angiogenesis via an eNOS-dependent mechanism. HSPA12B could be a novel target for the management of patients with post-MI cardiac dysfunction and remodelling.

Lithium attenuates bupivacaine-induced neurotoxicity in vitro through phosphatidylinositol-3-kinase/threonine-serine protein kinase B- and extracellular signal-regulated kinase-dependent mechanisms

Local anesthetics (LAs) are necessary for the regional anesthesia, spinal anesthesia, and pain management. However, the application of LAs may cause neurotoxicity and result in postoperative neurological complications. Lithium is a mood stabilizer for the treatment of bipolar disorder and may exert neuroprotective effects. In this study, we evaluated the effects of lithium on bupivacaine (a frequently used LAs)-induced injury in mouse neuroblastoma neuro 2a (N2a) cells. N2a cells were treated with bupivacaine in the presence or absence of lithium. After treatment, the cell injury was evaluated by examination of viability, morphology changes, and nuclear condensation. The levels of mitochondrial transmembrane potential (ΔΨm) and activation of phosphatidylinositol-3-kinase (PI3K)/ threonine-serine protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also examined. In a separate experiment, we investigated the effect of Akt and ERK inhibition on cell injury after bupivacaine and lithium treatment. Pretreatment of N2a cells with lithium significantly attenuated bupivacaine-induced cell injury. Lithium pretreatment completely reversed the suppression of PI3K/Akt and ERK signalings and significantly prevented the decline of ΔΨm in N2a cells after bupivacaine treatment. More importantly, pharmacological inhibition of Akt and ERK diminished the protective effect of lithium against bupivacaine-induced neuronal death. Our data suggest that lithium pretreatment provides a protective effect on bupivacaine-induced neuronal cell injury. This action of lithium is mediated through, at least in part, the activating of PI3K/Akt- and ERK-dependent mechanisms. Because lithium is a clinically proved safety drug for neurons, it is worthwhile to identify whether coadministration of LAs with lithium will decrease the risks of LAs-induced postoperative neurological complications in clinic practice.