Pengfei Liang

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress.

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H2O2-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H2O2-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.

Nucleolin/C23 mediates the antiapoptotic effect of heat shock protein 70 during oxidative stress

Although heat shock protein 70 (Hsp70) has been shown to markedly inhibit H2O2‐induced apoptosis in C2C12 cells, and nucleolin/C23 has also been implicated in apoptosis, the relationship of these two molecules is still largely unknown. The aim of the current study was to investigate the potential involvement of nucleolin/C23 in the antiapoptotic mechanism of Hsp70. We found that primary cultures of neonatal rat cardiomyocytes underwent apoptosis upon H2O2 treatment, and in these cells nucleolin/C23 protein was highly unstable and had a half‐life of less than 4 h. However, transfection with Hsp70 greatly stabilized nucleolin/C23 and also protected the cells from H2O2‐induced apoptosis. When nucleolin/C23 was knocked down with an antisense oligomer, H2O2‐induced apoptosis became more severe, even in Hsp70‐overexpressed cells, demonstrating an essential role of nucleolin/C23 in the antiapoptotic effects of Hsp70. Similar results were obtained by both nuclear morphology observation and caspase‐3 activity assay. Therefore, these data provide evidence that nucleolin/C23 is an essential downstream effecter of Hsp70 in the protection of cardiomyocytes against oxidative stress‐induced apoptosis.

ATP‐binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria‐derived activator of caspase and apoptosis in C2C12 cells

Hydrogen peroxide (H2O2) is a well known oxidative stress inducer causing apoptosis of many cells. Previously, we have shown that heat shock pretreatment blocked the release of the second mitochondria‐derived activator of caspase (Smac) to the cytosol and inhibited apoptosis of C2C12 myoblast cells in response to H2O2. The present study aimed to elucidate the underlying mechanism by over‐expressing a major stress‐inducible protein, heat shock protein (HSP) 70, and characterizing the resulting cellular changes. We demonstrate that HSP70 over‐expression markedly inhibited the release of Smac and prevented the activation of caspases‐9 and ‐3 and apoptosis in C2C12 cells under H2O2 treatment. However, no direct interaction between HSP70 and Smac was observed by co‐immunoprecipitation. Mutational analysis demonstrated that the ATP‐binding domain of HSP70, rather than the peptide‐binding domain, was essential for these observed HSP functions. Taken together, our results provide evidence supporting the role of HSP70 in the protection of C2C12 cells from H2O2‐induced and Smac‐promoted apoptosis by preventing the release of Smac from mitochondria, thereby inhibiting activation of caspases‐9 and ‐3. This mechanism of HSP70 action is dependent on its ATP‐binding domain but independent of its interaction with Smac protein.

Enhancement of PPAR-β activity by repetitive low-grade H2O2 stress protects human umbilical vein endothelial cells from subsequent oxidative stress-induced apoptosis

Repetitive stress has been shown to up-regulate antioxidant defense and increase survival after subsequent oxidative injury. The up-regulation of antioxidant defense has been identified as an underlying cause of the apoptosis-inhibitory effects exerted by repetitive stress. However, it remains unclear what the important signaling mechanisms are by which cells preexposed to low-grade stress deal with apoptosis-inducing stress. In this study, we repetitively stressed human umbilical vein endothelial cells (HUVECs) through multiple exposures to a low dose (30 microM) of H(2)O(2) in culture for 4 weeks. We then examined the effects of repetitive stress on PPAR-beta expression and activity as well as the role of PPAR-beta in the protective potency of repetitive stress. Our results show that repetitive stress enhances PPAR-beta expression and activity, thereby inhibiting oxidative stress-induced apoptosis. Further, PPAR-beta-directed antisense oligonucleotides reduced the PPAR-beta protein content, enhanced the H(2)O(2)-mediated apoptosis, and ablated the protective effect of repetitive low-grade H(2)O(2) stress. The specific PPAR-beta agonist L-165041 significantly potentiated the apoptosis induced by H(2)O(2) (p<0.05) and increased the protective effect of repetitive stress. These findings indicate that repetitive low-grade H(2)O(2) stress protects HUVECs from subsequent oxidative stress-induced apoptosis by enhancing PPAR-beta expression and activity.

Nucleolin/C23 is a negative regulator of hydrogen peroxide-induced apoptosis in HUVECs

Nucleolin plays important roles in chromatin structure, rDNA transcription, rRNA maturation, nucleocytoplasmic transport, and ribosome assembly. Although it has been shown to be anti-apoptotic, the underlying mechanisms remain unclear. In the current study, we first examined endogenous nucleolin expression in response to oxidative stress-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). Flow cytometry and caspase activity assays showed that H2O2 treatment caused apoptosis of the cells; reverse-transcription polymerase chain reaction and Western blotting revealed the downregulation of nucleolin expression and increased protein cleavage during this process. Overexpression of nucleolin protein by transfecting cells with the full-length nucleolin cDNA inhibited apoptosis, but nucleolin deficiency brought about by transfection with antisense oligonucleotide increased apoptosis of HUVECs. Concurrently, the expression of the apoptotic protein gene Bax was also downregulated following nucleolin overexpression. All these results indicate an important negative regulatory role for nucleolin in the apoptosis of endothelial cells, likely involving the Bax pathway.

Role of PPAR-beta in hydrogen peroxide-induced apoptosis in human umbilical vein endothelial cells.

Peroxisome proliferator-activated receptor beta (PPAR-beta) is a ligand activated transcription factor belonging to the nuclear receptor superfamily. Recent evidence suggests that PPAR-beta has clearly defined roles in skin wound healing, inflammation and cell proliferation. However, little is known about the role of PPAR-beta in oxidative stress-induced apoptosis in human umbilical vein endothelial cells (HUVECs). In this study, a specific PPAR-beta ligand, L-165041, and antisense phosphorothioate oligodeoxynucleotides (asODNs) against PPAR-beta were used to reveal the role of PPAR-beta in oxidative stress-induced apoptosis. The results showed that H(2)O(2) at 0.5mM resulted in a marked increase of apoptosis and a significant down-regulation of PPAR-beta expression and activation in HUVECs. Moreover, L-165041 significantly inhibited H(2)O(2)-induced apoptosis (P<0.05) and asODNs against PPAR-beta markedly inhibited the de novo synthesis of PPAR-beta, which was accompanied by enhanced apoptosis induced by H(2)O(2) (P<0.05). These data demonstrated that H(2)O(2) down-regulated the expression and activation of PPAR-beta, which played important roles in H(2)O(2)-induced apoptosis in HUVECs.

An epidemiological analysis of paediatric burns in urban and rural areas in south central China.

OBJECTIVE This study aims to analyse the epidemiology of paediatric burns in south central China, illustrate the differences between rural and urban areas, and discern prevention measures to reduce paediatric burns. METHODS Data were obtained from all paediatric patients admitted to Department of Burns unit of Xiangya Hospital during 2009-2012. A retrospective review was performed, including cause of burn, pre-hospital treatment, place of burn occurrence, anatomical areas involved, extent of burn, date of injury, number of operations, complications, length of hospital stay, hospitalisation cost and cure rate. RESULTS A total of 278 hospitalised paediatric patients were admitted in this study. The majority (56.47%) were 1-3 years old. Rural patients accounted for 67.99% in total; the ratio of boys to girls was 2.05. Scalding with hot fluids was the most common cause of burns in children (62.59%), followed by flame (17.63), fireworks (9.71%), electricity (5.76%) and other factors such as contact and chemical (4.32%). The living room was the location with the highest frequency of burns in children (53.24%). Burns were more likely to happen in winter and the upper extremities were the most involved anatomic site (53.24%). Total burn surface area (TBSA) ranging from 0% to 9% accounted for 55.4% in total. Rural patients underwent more operations and had longer and costlier hospital stays than urban patients. CONCLUSION Compared with treatment in urban areas, rural burn patients received less first-aid treatment, underwent more surgery, had more complications and longer and more costly hospital stays. This finding strongly suggests that it is necessary to make more efforts to prevent burns, especially in rural areas.

Nucleolin protects the heart from ischaemia–reperfusion injury by up-regulating heat shock protein 32

AIMS Nucleolin plays important roles in a variety of cellular processes. In this study, we aimed to investigate the role of nucleolin in cardiac ischaemia-reperfusion (I-R) injury. METHODS AND RESULTS We investigated the expression pattern of nucleolin in hearts subjected to I-R, or neonatal rat cardiomyocytes subjected to hypoxia-re-oxygenation. We found that nucleolin expression was significantly down-regulated and the cleaved protein was present, both in vivo and in vitro. Gene transfection and RNA interference approaches were employed in cardiomyocytes to investigate the function of nucleolin. Over-expression of nucleolin was cytoprotective, whereas nucleolin ablation enhanced both hypoxia- and H₂O₂-induced cardiomyocyte death. Furthermore, transgenic mice with cardiac-specific over-expression of nucleolin were resistant to I-R injury as indicated by decreased cellular necrosis and decreased infarct size. The cardio-protective roles of nucleolin in cardiomyocytes, are attributable to the interaction of nucleolin with the mRNA of heat shock protein 32 (Hsp32), resulting in an increase of Hsp32 mRNA stability, and subsequent up-regulation of Hsp32 expression. The selective Hsp32 inhibitor, zinc protoporphyrin-IX, abrograted the cardiac protection mediated by nucleolin. CONCLUSION This study has demonstrated that nucleolin is involved in the regulation of I-R-induced cardiac injury and dysfunction via the regulation of Hsp32, and may be a novel therapeutic target for ischaemic heart diseases.

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs.

BACKGROUND The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs). METHODS Deep partial thickness burn model in Sprague-Dawley rats and the heat denatured cell model (52°C, 35s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin. RESULTS Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs. CONCLUSIONS The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF. GENERAL SIGNIFICANCE We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis.

Proteomic change of peripheral lymphocytes from scald injury and Pseudomonas aeruginosa sepsis in rabbits

BACKGROUND Increased susceptibility to infection has been related to impairment of lymphocyte-regulated immune responses after severe burn. The aim of this study is to identify the differential expression of proteins in circulating lymphocytes from scald injury and Pseudomonas aeruginosa sepsis in rabbits to provide a basis for pathogenesis of burns and sepsis. METHODS Rabbits were subjected to sham burn (A), 30% scald (B), A+bacterial challenge (C) or B+bacterial challenge (D). Bacterial challenge was inflicted by an injection of 2.0x10(8) CFU P. aeruginosa (ATCC27853) in the auricular vein 22 h after the burn procedure. The animals were sacrificed 24 h later. Lymphocytes were isolated, and the differential proteins in the lymphocytes from the experimental and control animals were identified by two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), two of which were confirmed by Western blotting. RESULTS Nineteen differential protein spots were found by 2-DE and 12 spots (11 proteins) were identified. Differential expression of peroxiredoxin and annexin I was validated by Western blotting. Among the identified proteins, the expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate kinase, glutamate dehydrogenase and annexin I were down-regulated in group B, excessively down-regulated in group D, but mildly in group C, and peroxiredoxin was up-regulated in groups B and D. CONCLUSIONS Proteome changes in lymphocytes from P. aeruginosa sepsis in the scalded rabbits were revealed, which are related to immune suppression and the pathogenesis of sepsis after scald injury.