[The effects of leptin on neuron apoptosis in mice with cerebral ischemia/reperfusion injury].

OBJECTIVE: To study the effect of leptin on neuron apoptosis in mice with cerebral ischemia injury. METHODS: Seventy-five male Kuming mice were randomly divided into 3 groups:sham, model and leptin intervention group, respectively. Focal cerebral ischemia/reperfusion injury model in mice was established by middle cerebral artery occlusion. Leptin intervention group was injected with leptin (1µg/g weight, I. P.) at 0 min of ischemic injury. Neuron apoptosis was detected by TUNEL staining. The mRNA expression of apoptosis relative gene bcl-2 and caspase-3 were detected by RT-PCR. The protein expression of bcl-2 and caspase-3 were detected by immunohistochemistry. RESULTS: In model group, most of the neurons in the central area of cerebral ischemia had necrosis obviously, and the amount of neuron apop-tosis was much higher than that in sham group (P<0.01). Compared with sham group, both expression of pro-apoptosis gene caspase-3 and anti-apoptosis gene bcl-2 increased significantly in model group (P<0.01). Compared with model group, the amount of neuron apoptosis and expression level of caspase-3 were decreased significantly (P<0.01), whereas the mRNA and protein expression of bcl-2 were increased sig-nificantly in leptin intervention group (P<0.01). CONCLUSIONS: Leptin could reduce neuron apoptosis through down-regulation the expression of caspase-3 and up-regulation the expression of bcl-2. It suggests that leptin could play a neuroprotective role in cerebral ischemia injury.

FHL2 inhibits the Id3-promoted proliferation and invasive growth of human MCF-7 breast cancer cells.

BACKGROUND: Id3 plays a key role in the progression of breast cancer. Previously, four and a half LIM protein (FHL2) was identified as a repressor of Id family proteins by interacting with them. This study aimed to investigate the effects of FHL2 on the transcriptional regulation and oncogenic activities of Id3 in human breast cancer cells. METHODS: Cell transfection was performed with SuperFect reagent. Stable transfectants that overexpressed Id3 were obtained by selection on G418. The level of Id3 protein was determined by Western blotting analysis. Dual luciferase assays were used to measure the effect of Id3 and FHL2 on E47-mediated transcriptional activity in MCF-7 human breast cancer cells. The MTT assay was used to measure cell proliferation. The transwell assay was used to measure the invasive capacity of MCF-7 cancer cells. RESULTS: Id3 markedly repressed transcription mediated by the basic helix-loop-helix (bHLH) factor E47 in MCF-7 cells. This Id3-mediated repression was effectively antagonized by FHL2. Overexpression of Id3 markedly promoted the proliferation and invasive capacity of MCF-7 cells; however, these effects were significantly suppressed by the overexpression of FHL2. CONCLUSIONS: FHL2 can inhibit the proliferation and invasive growth of human breast cancer cells by repressing the functional activity of Id3. The functional roles of FHL2-Id3 signaling in the development of human breast cancer need further research.

Leptin administration alleviates ischemic brain injury in mice by reducing oxidative stress and subsequent neuronal apoptosis.

BACKGROUND: Recent research has indicates that leptin plays a protective role in traumatic brain injury. We studied the protective effect of leptin on cerebral ischemia/reperfusion injury by using mice transient focal cerebral ischemia/reperfusion injury model. METHODS: The distribution of 125I-leptin in the mouse brain was assessed by radioimmunoassay method. Mouse models of transient focal cerebral ischemia were established by occlusion of the right middle cerebral artery for two hours followed by 24 hours reperfusion. The neurologic deficits and infarct volume were determined using the Longa's score and 2,3,5-triphenyltetrazolium chloride staining, respectively. Regional cerebral blood flow was monitored by a laser-Doppler blood flowmeter. The levels of malondialdehyde, nitric oxide, nitric oxide synthase, and superoxide dismutase were detected according to respective assay kit. The histologic changes and neuronal apoptosis were observed with hematoxylin and eosin and transferase-mediated dUTP-biotin nick end labeling staining, respectively. The expression of B-cell lymphoma/leukemia-2 (Bcl-2) and cysteineasparateprotease-3 (caspase-3) were investigated by Western blot and real-time polymerase chain reaction assay. RESULTS: Leptin decreased infarct volume and neurologic defects and improved regional cerebral blood flow and microvascular branch blood flow after injury. The malondialdehyde and nitric oxide levels were reduced, and superoxide dismutase level was increased after leptin treatment, which also minimized histologic changes and neuronal apoptosis, led to the upregulation of Bcl-2 and downregulation of caspase-3 expression after injury. CONCLUSIONS: Peripherally administered leptin crossed the blood-brain barrier and was distributed into multiple regions of the brain; in the brain, leptin directly alleviated the injury-evoked damages by reducing oxidative stress and neuronal apoptosis.

[The role of Leptin on neuron apoptosis in mice with cerebral ischemia/reperfusion injury].

OBJECTIVE: To study the effect of Leptin on neuron apoptosis in mice with cerebral ischemia injury and its mechanism. METHODS: Seventy-five mice were randomly divided into three groups. Focal cerebral ischemia/reperfusion injury model in mice was reproduced by middle cerebral artery occlusion for 2 hours followed by reperfusion. In Leptin intervention group mice were given Leptin 1 µg/g during cerebral ischemia by intraperitoneal injection. Mice in the model group were given equal amount of phosphate buffer saline. After reperfusion for 24 hours, the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The mRNA and protein expression of apoptosis relative gene caspase-3 and bcl-2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immuno histochemistry. RESULTS: Most of neuron necrosis was observed in cerebral ischemia center in model group. Compared with sham-operation group, neuron apoptosis rate, mRNA and protein expression of caspase-3 and bcl-2 in model group increased significantly [apoptosis rate: (68.65 ± 0.79)% vs. (4.40 ± 0.00)%, caspase-3 mRNA: 2.563 ± 0.250 vs. 0.153 ± 0.020, bcl-2 mRNA: 0.337 ± 0.100 vs. 0.125 ± 0.030, caspase-3 protein (absorbance value, A value): 0.57 ± 0.05 vs. 0.37 ± 0.03, bcl-2 protein (A value): 0.51 ± 0.04 vs. 0.35 ± 0.01, all P<0.01]. The apoptosis rate of penumbra neurons was reduced in Leptin intervention group significantly compared with model group [(42.30 ± 8.45)% vs. (68.65 ± 0.79)%, P<0.01]. Compared with model group, the mRNA and protein expression of caspase-3 in Leptin intervention group were reduced significantly [caspase-3 mRNA: 2.267 ± 0.040 vs. 2.563 ± 0.250, caspase-3 protein (A value): 0.45 ± 0.04 vs. 0.57 ± 0.05, P>0.05 and P<0.01], and the mRNA and protein expression of bcl-2 in Leptin intervention group upregulated significantly [bcl-2 mRNA: 0.662 ± 0.040 vs. 0.337 ± 0.100, bcl-2 protein (A value): 0.76 ± 0.09 vs. 0.51 ± 0.04, both P<0.01]. CONCLUSION: Leptin could reduce apoptosis of neurons through down-regulation of the expression of caspase-3 and up-regulation of the expression of bcl-2. The results suggest that Leptin plays a neuroprotective role in cerebral ischemia injury.

MSCs: Biological characteristics, clinical applications and their outstanding concerns.

Mesenchymal stem cells (MSCs) are multi-potent adult stem cells harboring multi-lineage differentiation potential and immunosuppressive properties that make MSCs an ideal candidate cell type for immunomodulation and regenerative medicine. Currently, MSC-related researches and clinical trials have evoked exciting promise in a variety of disorders and tissue regeneration. However, it must be recognized that several critical potential problems have also emerged from current clinical trials, for example: (1) the indefinite association between the phenotypic characteristics and the biological functions of MSCs; (2) the lack of clinical data to support the long-term safety of MSCs; (3) the need for further clarification of multiple mechanisms of MSC transplant actions in vivo; and (4) the lack of comparability of MSC transplant efficacy. Therefore, MSC-based therapies could not yet be considered a routine treatment in the clinic. Based on these, we proposed that large-scale and multi-center clinical trials of MSC-based therapies should be initiated under strict supervision. These interventions might help to establish a new clinical paradigm to turn MSC transplantation into a routine therapy for at least some diseases in the near future.

[Effects of leptin on apoptosis of rat cerebral astrocytes with ischemia/hypoxia injury].

OBJECTIVE: To investigate the effect of leptin on apoptosis of rat cerebral astrocytes with ischemia/ hypoxia injury and its mechanism. METHODS: The cerebral astrocytes with ischemic/hypoxia injury were induced in neonatal SD rats. The cellular viability of injury of astrocytes was detected by MTT assay. The apoptosis of astrocyte were detected with Annexin V-FITC kit. The effect of leptin on the expression of apoptosis factor bcl-2, bax, caspase-3 was detected by RT-PCR and Western blot. RESULTS: Compared with the ischemia group, the cellular viability of leptin intervention group increased significantly (P < 0.01), while the astrocytes apoptosis of leptin intervention group decreased significantly (P < 0.01). The mRNA and protein expression level of antiapoptosis factor bcl-2 in leptin intervention group was much higher than that of ischemia group (P < 0.01), whereas the mRNA and protein expression of bax and caspase-3 was much lower than that of ischemia group (P < 0.01). CONCLUSION: Leptin could significantly decrease the apoptosis of astrocytes with ischemia/hypoxia injury, and it i relevant to the increase of bcl-2 expression and the decrease of bax caspase-3 expression level.

[Protective effect of leptin against cerebral ischemia/reperfusion injury in mice].

OBJECTIVE: To investigate the protective effect of leptin against cerebral ischemia/reperfusion injury in mice. METHODS: Mouse models of transient focal cerebral ischemia were established by occlusion of the right middle cerebral artery for 2 h followed by 24 h reperfusion. The infarct volume and neurological deficit scores following leptin treatment were determined using TTC staining and the Longa's score, respectively, to evaluate the protective effect of leptin against ischemic cerebral injury. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA) and nitric oxide (NO) in the brain tissue were measured by colorimetry. The histopathological changes in the brain were observed with HE staining, and the expression of glial fibrillary acidicprotein (GFAP) was detected by immunohistochemistry. RESULTS: Leptin treatment markedly reduced cerebral infarct volume and neurological deficits induced by transient ischemia. The LDH, MDA and NO levels in the brain tissues were significantly decreased after leptin treatment, which also alleviated the histopathological injury, maintained the normal morphology of the astrocytes and increased the expression of GFAP. CONCLUSION: Leptin produces obvious protective effect against cerebral ischemia/reperfusion injury by inhibiting lipid peroxidation, stabilizing the internal environment and adjusting the activity of the astrocytes.

GC-rich promoter elements maximally confers estrogen-induced transactivation of LRP16 gene through ERalpha/Sp1 interaction in MCF-7 cells.

LRP16 gene has been characterized as an estrogen-responsive gene. One 1/2ERE/GC-rich site was previously identified to be indispensable for -676/-214 (region A) fragment within LRP16 regulatory region to confer E2 action. Here, we report that -213/-24 fragment (region B) has higher E2-responsiveness than that of region A in MCF-7 cells, but not in HeLa cells. Deletion and mutation analyses of region B showed that multiple GC-sites are involved in the E2-stimulated response and one 30-bp fragment (-213 to -184 bp) is essential for conferring maximum E2-responsiveness. Results from the cotransfection assays containing Sp1-siRNA revealed that Sp1 is required for the basal transcription activity and E2-responsiveness of both regions A and B. Northern blot analysis demonstrated that inhibition of Sp1 in MCF-7 cells not only decreased the basal expression of LRP16, but markedly impaired its upregulation by E2. Results from gel mobility shift assays exhibited the direct binding of Sp1 protein to the 28-bp fragment (-211 to -184 bp), which was enhanced by the ERalpha titer. Moreover, the functional interaction of ERalpha and Sp1 proteins in the presence of E2 at the GC-rich sites in region B was confirmed by chromatin immunoprecipitation (ChIP) assays. In general, these results demonstrate that GC-rich sites in the proximal promoter of LRP16 gene are sufficient for E2 activation of LRP16 and the -213/-184 fragment containing only one GC site is essential for the maximal induction in MCF-7 cells. We also provide a model for Sp1-dependent regulation of genes by E2 through GC-rich motifs.

Induction of the LRP16 gene by estrogen promotes the invasive growth of Ishikawa human endometrial cancer cells through the downregulation of E-cadherin.

LRP16 was previously identified as an estrogen-induced gene in breast cancer cells. The responsiveness of LRP16 to estrogen and its functional effects in endometrial cancer (EC) cells are still unclear. Here, we show that the mRNA level and promoter activity of the LRP16 gene were significantly increased by 17beta-estradiol (E2) in estrogen receptor alpha (ER alpha)-positive Ishikawa human EC cells. Although the growth rate of Ishikawa cells was not obviously affected by ectopic expression of LRP16, the results of a Transwell assay showed an approximate one-third increase of the invasive capacity of LRP16-overexpressing cells. As a result of molecular screening, we observed that the expression of E-cadherin, an essential adhesion molecule associated with tumor metastasis, was repressed by LRP16. Further promoter analyses demonstrated that LRP16 inhibited E-cadherin transactivation in a dose-dependent manner. However, the inhibition was abolished by estrogen deprivation, indicating that the downregulation of E-cadherin transcription by LRP16 requires ER alpha mediation. Chromatin immunoprecipitation analyses revealed that the binding of ER alpha to the E-cadherin promoter was antagonized by LRP16, suggesting that LRP16 could interfere with ER alpha-mediated transcription. These results suggest that the upregulation of LRP16 by estrogen could be involved in invasive growth by downregulating E-cadherin in human ECs.

[Epidermal growth factor receptor mutation in non-small cell lung cancer and breast cancer].

Somatic mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene is associated with the sensitivity of non-smal cell lung cancer (NSCLC) to TK inhibitor Gefitinib. Mutational analysis for EGFR exons 19 and 21 was performed in 75 NSCLC and 10 breast cancer patients. All patients had not received treatment of Gefitinib. Somatic mutations in TK domain of EGFR were identified in 13 of the 75(13/75, 17.33%) patients, including 7 cases of in-frame deletion in exon 19 (7/75, 9.33%) and 6 cases of amino acid substitution (2573T>G, L858R) in exon 21 (6/75, 8%) . No other mutations were found in 10 breast cancer patients who stained positive for HER2 immunhistochemistry. Adenocarcinoma has a higher rate of mutations than several other types of NSCLC, the mutations occurring more fre-quently in female patients. EGFR mutation rate in Chinese NSCLC patients was higher than that in Caucasians. Our data indicated that Chinese adenocarcinoma patients could benefit from TK inhibitor Gefitinib.

Prevalence of BK virus and JC virus in peripheral blood leukocytes and normal arterial walls in healthy individuals in China.

Several studies have demonstrated that BK virus (BKV) and JC virus (JCV) establish latent infection in peripheral blood leukocytes (PBLs) of healthy individuals; however, the main populations studied are European. In this study, the prevalence of BKV and JCV DNA in PBLs from healthy adult individuals and umbilical cord blood from newborn children in China was detected by semi-nested polymerase chain reaction (snPCR) followed by restriction enzyme analysis. The results suggest that the healthy adult Chinese population harbors BKV and JCV DNA in peripheral leukocytes. Overall, the prevalence of BKV and JCV DNA in PBLs of healthy adult individuals was 42.1% and 7.8%, respectively. The overall prevalence of BKV DNA was significantly higher than that of JCV DNA. None of the umbilical cord blood samples from newborn children were positive for BKV and JCV DNA. To understand further the target tissues involved in establishment of BKV and JCV latency in healthy individuals, the presence of DNA from both viruses was detected in normal arterial wall samples from 20 young trauma victims by the same method used for leukocyte DNA. BKV DNA was detected alone in 20% of samples tested; JCV DNA was not detected alone in any of the samples. DNA from both viruses was found in 5% of samples. This is the first report to show that normal arterial walls of healthy individuals may be another target site of latency for BKV and JCV.