A novel series of tertiary amines
as retinoid-related orphan receptor
gamma-t (RORγt) inverse agonists was discovered through agonist/inverse
agonist conversion. The level of RORγt inhibition can be enhanced
by modulating the conformational disruption of H12 in RORγt
LBD. Linker exploration and rational design led to the discovery of
more potent indole-based RORγt inverse agonists.
RORγt; agonists; inverse agonists; Th17 cell differentiation; cocrystal structure; structure-based design
Background: 5-Fluorouracil (5Fu) chemotherapy is the first treatment of choice for advanced gastric cancer (GC), but its effectiveness is limited by drug resistance. Emerging evidence suggests that the existence of cancer stem cells (CSCs) contributes to chemoresistance. The aim of the present study was to determine whether 5Fu chemotherapy generates residual cells with CSC-like properties in GC. Methods: Human GC cell lines, SGC7901 and AGS, were exposed to increasing 5Fu concentrations. The residual cells were assessed for both chemosensitivity and CSC-like properties. B lymphoma Mo-MLV insertion region 1 (BMI1), a putative CSC protein, was analyzed by immunohistochemical staining and subjected to pairwise comparison in GC tissues treated with or without neoadjuvant 5Fu-based chemotherapy. The correlation between BMI1 expression and recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy was then examined. Results: The residual cells exhibited 5Fu chemoresistance. These 5Fu-resistant cells displayed some CSC features, such as a high percentage of quiescent cells, increased self-renewal ability and tumorigenicity. The 5Fu-resistant cells were also enriched with cells expressing cluster of differentiation (CD)133+, CD326+ and CD44+CD24-. Moreover, the BMI1 gene was overexpressed in 5Fu-resistant cells, and BMI1 knockdown effectively reversed chemoresistance. The BMI1 protein was highly expressed consistently in the remaining GC tissues after 5Fu-based neoadjuvant chemotherapy, and BMI1 levels were correlated positively with recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy. Conclusions: Our data provided molecular evidence illustrating that 5Fu chemotherapy in GC resulted in acquisition of CSC-like properties. Moreover, enhanced BMI1 expression contributed to 5Fu resistance and may serve as a potential therapeutic target to reverse chemoresistance in GC patients.
Gastric cancer; 5-Fluorouracil (5Fu); Chemoresistance; Cancer stem cells
Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks Ach-mediated MSC migration. Interestingly, intracellular Ca2+ ATPase specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca2+ storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca2+, PKC and ERK1/2 signal pathways.
Mesenchymal stem cell; Acetylcholine; Migration; Protein kinase C; Ras mitogen-activated protein kinase; Calcium; Muscarinic receptors; ERK1/2
Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up-regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)-induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG-induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short-hairpin RNA significantly decreased HG-induced cell apoptosis by reducing caspase-3 activation and ratio of Bcl2-associated X protein to B-cell lymphoma/leukemia-2 (bax/bcl-2). Furthermore, HG activated E26 transformation-specific sequence-1 (Ets-1), and HMGB1 inhibition attenuated HG-induced activation of Ets-1 via extracellular signal-regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets-1 significantly decreased HG-induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin-treated diabetic mice. Inhibition of HMGB1 by short-hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets-1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia-induced cardiomyocyte apoptosis by down-regulating ERK-dependent activation of Ets-1.
high glucose; cardiomyocyte; apoptosis; diabetes; HMGB1; Ets-1
Background. The incidence of breast cancer in RA patients remains controversial. Thus we performed a meta-analysis to investigate the impact of RA on breast cancer. Methods. Published literature was available from PubMed, Embase, and Cochrane Library. Pooled standardized incidence rate (SIR) was computed by random-effect model analysis. Results. We identified 16 separate studies in the present study, in which the number of patients ranged from 458 to 84,475. We did not find the increased cancer risk in RA patients (SIR = 0.86, 95% CI = 0.72–1.02). However, subgroup analysis showed that breast cancer risk in RA patients was positively different in Caucasians (SIR = 0.82, 95% CI = 0.73–0.93) and non-Caucasians (SIR = 1.21, 95% CI = 1.19–1.23), respectively. In subgroup analysis by style, a reduced incidence was found in hospital-based case subjects (SIR = 0.82, 95% CI = 0.69–0.97). Similarly, subgroup analysis for adjusted factors indicated that in A3 (age and sex) and A4 (age, sex, and race/ethnicity) the risk was decreased (SIR = 0.87, 95% CI = 0.76–0.99; SIR = 0.63, 95% CI = 0.59–0.67). Conclusions. The meta-analysis revealed no increased breast cancer risk in RA patients. However, in the subgroup analysis, the risk of breast cancer is increased in non-Caucasians patients with RA while it decreased in Caucasian population, hospital-based case subjects, and A3 group. Such relationship may provide preference for risk of breast cancer in different population.
Increased susceptibility to respiratory infections such as influenza is the hallmark of advancing age. The mechanisms underlying the impaired immune response to influenza are not well understood. In the present study, we have investigated the effect of advancing age on dendritic cell (DC) function because they are critical in generating robust antiviral responses. Our results indicate that monocyte derived DCs from the aged are impaired in their capacity to secrete interferon (IFN)-I in response to influenza virus. Additionally, we observed a severe reduction in the production of IFN-III, which plays an important role in defense against viral infections at respiratory mucosal surfaces. This reduction in IFN-I and IFN-III were a result of age-associated modifications in the chromatin structure. Investigations using chromatin immunoprecipitation with H3K4me3 and H3K9me3 antibodies revealed that there is increased association of IFN-I and IFN-III promoters with the repressor histone, H3K9me3 in non-stimulated aged DCs compared to young DCs. This was accompanied by decreased association of these promoters with activator histone, H3K4me3 in aged DCs after activation with influenza. In contrast to interferons, the association of TNF-alpha promoter with both these histones was comparable between aged and young subjects. Investigations at 48 h suggested that these changes are not stable and change with time. In summary, our study demonstrates that myeloid DCs from aged subjects are impaired in their capacity to produce IFNs in response to influenza virus and that age-associated altered histone expression patterns are responsible for the decrease in IFN production.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-012-9477-8) contains supplementary material, which is available to authorized users.
Dendritic cells; Aging; IFN-I; IL-29; Histones; Chromatin Immunoprecipitation
Vascular remodeling due to smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, anti-neointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, due to their non-specific effect on endothelial cells (EC). The objective of this study was to identify a therapeutic target that differentially regulates SMC and EC proliferation.
Approach and Results
By using both rat balloon-injury and mouse wire-injury models, we identified CTP synthase (CTPS) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization due to induction of CTP synthesis salvage pathway enzymes nucleoside diphosphate kinase A and B in ECs. Diphosphate kinase B appeared to preserve EC proliferation via utilization of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelization in vivo.
Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair following injury.
CTP synthase; vascular remodeling; smooth muscle proliferation; re-endothelialization; nucleoside diphosphate kinase
At present, no effective clinical treatment is available for the late effects of radiation myelopathy. The aim of the present study was to assess the therapeutic effects of human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in a rat model of radiation myelopathy.
An irradiated cervical spinal cord rat model was generated. UC-MSCs were injected through the tail vein at 90, 97, 104 and 111 days post-irradiation. Behavioral tests were performed using the forelimb paralysis scoring system, and histological damage was examined using Nissl staining. The microcirculation in the spinal cord was assessed using von Willebrand factor (vWF) immunohistochemical analysis and laser-Doppler flowmetry. The microenvironment in the spinal cord was determined by measuring the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum and the anti-inflammatory cytokines brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in the spinal cord.
Multiple injections of UC-MSCs through the tail veil decreased the forelimb paralysis, decreased spinal cord histological damage, increased the number of neurons in the anterior horn of the spinal cord, increased the endothelial cell density and the microvessel density in the white matter and gray matter of the spinal cord, increased the relative magnitude of spinal cord blood flow, down-regulated pro-inflammatory cytokine expression in the serum, and increased anti-inflammatory cytokine expression in the spinal cord.
Multiple injections of UC-MSCs via the tail vein in a rat model of radiation myelopathy significantly improved the microcirculation and microenvironment through therapeutic paracrine effects.
Radiation myelopathy; Endothelial cell; Spinal cord blood flow; Paracrine system; Human umbilical cord-derived mesenchymal stromal cells
Dendritic cells (DCs) are major antigen-presenting cells that play a key role in initiating and regulating innate and adaptive immune responses. DCs are critical mediators of tolerance and immunity. The functional properties of DCs decline with age. The purpose of this study was to define the age-associated molecular changes in DCs by gene array analysis using Affymatrix GeneChips. The expression levels of a total of 260 genes (1.8%) were significantly different (144 down-regulated and 116 upregulated) in monocyte-derived DCs (MoDCs) from aged compared to young human donors. Of the 260 differentially expressed genes, 24% were down-regulated by more than 3-fold, suggesting that a large reduction in expression occurred for a notable number of genes in the aged. Our results suggest that the genes involved in immune response to pathogens, cell migration and T cell priming display significant age-related changes. Furthermore, downregulated genes involved in cell cycle arrest and DNA replication may play a critical role in aging-associated genetic instability. These changes in gene expression provide molecular based evidence for age-associated functional abnormalities in human DCs that may be responsible for the defects in adaptive immunity observed in the elderly.
BACKGROUND: The study aimed to compare the tolerability and efficacy of gefitinib combined with chemotherapy agents versus chemotherapy alone for the treatment of epidermal growth factor receptor (EGFR)–mutated lung adenocarcinoma in heavily pretreated patients. METHODS: The study was designed as a matched-pair case-control investigation to minimize intergroup heterogeneity. Patients were stratified into gefitinib plus chemotherapy and chemotherapy alone groups with matching for sex, age, ECOG performance status, progress-free survival (PFS) from previous EGFR tyrosine kinase inhibitor treatment, EGFR mutation types, and tumor metastasis status. RESULTS: Sixty-six patients were selected from our database using the matched-pair method. The median age was 61 years (95% confidence interval, 57-65 years). During a follow-up period of 14.5 months on average, the overall response rates of the gefitinib-integrated and chemotherapy alone groups were 9.1% and 6.5%, respectively (P > .05), whereas the corresponding disease-control rates were 39.4% and 30.3%, respectively (P > .05). No statistically significant differences in PFS (median, 4.2 vs 3.3 months; P = .06) and overall survival (median, 10.4 vs 7.9 months; P = .44) were observed between two groups. The 6-month survival rates of the gefitinib-integrated and chemotherapy alone groups were 21.2% and 12.1%, respectively (P < .05). Side effects were mild, and all treatments were well tolerated. CONCLUSIONS: Our results indicated that gefitinib-integrated therapy offered a trend to better PFS and an improved 6-month survival rate in heavily pretreated patients with metastatic EGFR-mutated lung adenocarcinoma. All treatments were well tolerated. Future prospective studies are warranted to confirm our findings.
Mitochondrial dysfunction caused by amyloid β-peptide (Aβ) plays an important role in the pathogenesis of Alzheimer disease (AD). Substantial evidence has indicated that the mitochondrial permeability transition pore (mPTP) opening is involved in Aβ-induced neuronal death and reactive oxygen species (ROS) generation. Astragaloside IV (AS-IV), one of the major active constituents of Astragalus membranaceus, has been reported as an effective anti-oxidant for treating neurodegenerative diseases. However, the molecular mechanisms still need to be clarified. In this study, we investigated whether AS-IV could prevent Aβ1-42-induced neurotoxicity in SK-N-SH cells via inhibiting the mPTP opening. The results showed that pretreatment of AS-IV significantly increased the viability of neuronal cells, reduced apoptosis, decreased the generation of intracellular reactive oxygen species (ROS) and decreased mitochondrial superoxide in the presence of Aβ1-42. In addition, pretreatment of AS-IV inhibited the mPTP opening, rescued mitochondrial membrane potential (ΔΨm), enhanced ATP generation, improved the activity of cytochrome c oxidase and blocked cytochrome c release from mitochondria in Aβ1-42 rich milieu. Moreover, pretreatment of AS-IV reduced the expression of Bax and cleaved caspase-3 and increased the expression of Bcl-2 in an Aβ1-42 rich environment. These data indicate that AS-IV prevents Aβ1-42-induced SK-N-SH cell apoptosis via inhibiting the mPTP opening and ROS generation. These results provide novel insights of AS-IV for the prevention and treatment of neurodegenerative disorders such as AD.
Norovirus (NoV) has been recognized as the most important cause of nonbacterial acute gastroenteritis affecting all age group people in the world. Genetic recombination is a common occurance in RNA viruses and many recombinant NoV strains have been described since it was first reported in 1997. However, the knowledge of recombinant NoV in China is extremely limited.
A total of 685 stool specimens were tested for NoV infection from the acute gastroenteritis patients who visited one general hospital in Beijing from April 2009 to November 2011. The virus recombination was identified by constructing phylogenetic trees of two genes, further SimPlot and the maximum chi-square analysis.
The overall positive rate was 9.6% (66/685). GII.4 New Orleans 2009 and GII.4 2006b variants were the dominant genotype. Four GII.g/GII.12 and one GII.12/GII.3 recombinant strains were confirmed, and all derived from adult outpatients. The predictive recombination point occurred at the open reading frame (ORF)1/ORF2 overlap.
The GII.g ORF1/GII.12ORF2 recombinant has been reported in several countries and it was the first report of this recombinant in China.
Background: In a subset of patients with Hirschsprung's disease (HSCR), gastrointestinal motor dysfunction persisted long after surgical correction. Gastrointestinal motility is achieved through the coordinated activity of the enteric nervous system, interstitial cells of Cajal, and smooth muscle (SMC) cells. Inhibition of four-and-a-half LIM protein-1 (Fhl1) expression by siRNA significantly decreases pulmonary artery SMCs migration and proliferation. Furthermore when up-expressing FHL1 in atrial myocytes, K (+) current density markedly increases, therefore changing myocytes' response to an electrical stimulus. However whether FHL1 in colon SMCs (the final effector organ) influences intestinal motility in HSCR patients has not been clarified. Methods: FHL1 mRNA and protein expressions were analyzed in 32 HSCR colons and 4 normal colons. Results: Smooth muscle layers were thicken and disorganized in HSCR. FHL1 was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the ganglionic colon. FHL1 mRNA relative expression level in aganglionic colons was 1.06±0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83±0.09. FHL1 expression level in ganglionic colon (1.66±0.30) or aganglionic colon (1.81±0.35) was significantly higher than that in normal colons (P=0.045 and P=0.041, respectively). Meanwhile, we found FHL1 expression in aganglionic colon was slightly stronger than that in ganglionic colon (P=0.036). Conclusion: These data suggested that up-regulated FHL1 in smooth muscle in HSCR might be associated with intestinal wall remodeling in HSCR and might be one of the risk factors for gastrointestinal motor dysfunction.
FHL1; Hirschsprung's disease; expression; smooth muscle; prognosis
Objective: To explore the therapeutic effect and safety of target-dose metoprolol in treating chronic heart failure (CHF) patients complicated with diabetes mellitus (DM).
: One hundred and fifty-four elderly patients were randomly divided into an observation group and a control group (n=77), which were treated with target-dose metoprolol and conventional therapy, and routinely treated respectively. The New York Heart Association (NYHA) classification, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), 6-min walking distance and medication safety of the two groups were compared.
Results: Compared with the results before treatment, the NYHA classification, LVESD, LVEDD, LVEF and 6-minutes walking distance of both groups were significantly improved (P<0.05), with significantly better results in the observation group than those in the control group after treatment (P<0.05). In the 6 months of follow-up, the incidence of cardiac events in the observation group (3.90%) was significantly lower than that of the control group (14.29%) (P<0.05). The levels of average fasting blood sugar and glycosylated hemoglobin in the groups showed no significant differences (P>0.05).
Conclusion: Treating CHF patients complicated with DM with target-dose metoprolol can obviously boost the cardiac function and exercise tolerance, leading to satisfactory clinical therapeutic effect, high security and moderate tolerance.
Chronic heart failure; Diabetes mellitus; Metoprolol; Therapeutic effect; Safety
Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.
Methods and Results
We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium.
We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.
Hepatitis B virus (HBV) infection is a global public health problem that causes persistent liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. A large amount of people die annually from HBV infection. However, the pathogenesises of the HBV-related diseases are ill defined and the therapeutic strategies for the diseases are less than optimum. The recently discovered microRNAs (miRNAs) are tiny noncoding RNAs that regulate gene expression primarily at the post-transcriptional level by binding to mRNAs. miRNAs contribute to a variety of physiological and pathological processes. A number of miRNAs have been found to play a pivotal role in the host-virus interaction including host-HBV interaction. Numerous studies have indicated that HBV infection could change the cellular miRNA expression patterns and different stages of HBV associated disease have displayed distinctive miRNA profiles. Furthermore, the differential expressed miRNAs have been found involved in the progression of HBV-related diseases, for instance some miRNAs are involved in liver tumorigenesis and tumor metastasis. Studies have also shown that the circulating miRNA in serum or plasma might be a very useful biomarker for the diagnosis and prognosis of HBV-related diseases. In addition, miRNA-based therapy strategies have attracted increasing attention, indicating a promising future in the treatment of HBV-related diseases.
MicroRNA; Hepatitis B virus; Hepatitis B; Host-virus interaction; Biomarker; Therapy
A large number of studies have confirmed that excessive apoptosis is one of the reasons for deficient neuronal function in neural tube defects (NTDs). A previous study from our laboratory used 2-D gel electrophoresis to demonstrate that 14-3-3ζ expression was low in the spinal cords of rat fetuses with spina bifida aperta at embryonic day (E) 17. As a member of the 14-3-3 protein family, 14-3-3ζ plays a crucial role in the determination of cell fate and anti-apoptotic activity. However, neither the expression of 14-3-3ζ in defective spinal cords, nor the correlation between 14-3-3ζ and excessive apoptosis in NTDs has been fully confirmed.
We used immunoblotting and quantitative real-time PCR (qRT-PCR) to quantify the expression of 14-3-3ζ and double immunofluorescence to visualize 14-3-3ζ and apoptosis. We found that, compared with controls, 14-3-3ζ was down-regulated in spina bifida between E12 and E15. Excessive apoptotic cells and low expression of 14-3-3ζ were observed in the dorsal region of spinal cords with spina bifida during the same time period. To initially explore the molecular mechanisms of apoptosis in NTDs, we investigated the expression of microRNA-7 (miR-7), microRNA-375 (miR-375) and microRNA-451 (miR-451), which are known to down-regulate 14-3-3ζ in several different cell types. We also investigated the expression of p53, a molecule that is downstream of 14-3-3ζ and can be down-regulated by it. We discovered that, in contrast to the reduction of 14-3-3ζ expression, the expression of miR-451, miR-375 and p53 increased in spina bifida rat fetuses.
These data suggest that the reduced expression of 14-3-3ζ plays a role in the excessive apoptosis that occurs in spina bifida and may be partly regulated by the over-expression of miR-451 and miR-375, and the consequent up-regulation of p53 might further promote apoptosis in spina bifida.
The objective of this study was to investigate the effects of varying doses of caffeine on memory impairment and the expression of brain neurotrophic derived factor (BNDF) and TrkB in PS1/APP double transgenic mouse models. PS1/APP double transgenic mice were administered 0.3 ml/day of saline, 1.5 mg/day of caffeine or 0.75 mg/day of caffeine for eight weeks. A water maze test and western blotting were used to determine the memory capability and expression of hippocampal BNDF and TrkB of the mice. The results demonstrated that 0.75 mg/day and 1.5 mg/day doses of caffeine significantly increased memory capability and the expression of hippocampal BDNF and TrkB in PS1/APP mice with a dose-response effect. The results suggested that chronic caffeine treatment may reverse memory impairment in PS1/APP transgenic mice, and BDNF and its receptor TrkB, may be involved in this process.
caffeine; memory; brain neurotrophic derived factor; TrkB; Alzheimer’s disease
Catalase (CAT) breaks down H2O2 into H2O and O2 to protects cells from oxidative damage. However, its translational potential is limited because exogenous CAT cannot enter living cells automatically. This study is aimed to investigate if PEP-1-CAT fusion protein can effectively protect cardiomyocytes from oxidative stress due to hypoxia/reoxygenation (H/R)-induced injury.
H9c2 cardomyocytes were pretreated with catalase (CAT) or PEP-1-CAT fusion protein followed by culturing in a hypoxia and re-oxygenation condition. Cell apoptosis were measured by Annexin V and PI double staining and Flow cytometry. Intracellular superoxide anion level was determined, and mitochondrial membrane potential was measured. Expression of apoptosis-related proteins including Bcl-2, Bax, Caspase-3, PARP, p38 and phospho-p38 was analyzed by western blotting.
PEP-1-CAT protected H9c2 from H/R-induced morphological alteration and reduced the release of lactate dehydrogenase (LDH) and malondialdehyde content. Superoxide anion production was also decreased. In addition, PEP-1-CAT inhibited H9c2 apoptosis and blocked the expression of apoptosis stimulator Bax while increased the expression of Bcl-2, leading to an increased mitochondrial membrane potential. Mechanistically, PEP-1-CAT inhibited p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways, resulting in blockade of Bcl2/Bax/mitochondrial apoptotic pathway.
Our study has revealed a novel mechanism by which PEP-1-CAT protects cardiomyocyte from H/R-induced injury. PEP-1-CAT blocks Bcl2/Bax/mitochondrial apoptotic pathway by inhibiting p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways.
Cell-penetrating peptide; PEP-1; Catalase; Cardiomyocyte; Apoptosis; MAPK
Luteolin (3’,4’,5,7-tetrahydroxyflavone) has powerful anti-apoptotic and antioxidant properties. This study aimed to investigate the effects of luteolin on hyperglycemia-mediated apoptosis in the hippocampi of rats with streptozotocin-induced diabetic encephalopathy after injection into the tail veins, and the molecular mechanisms involved. Biochemistry and terminal deoxynucleotidyl transferase mediated dUTP nick end labelling detection results showed that luteolin treatment (given twice daily for 15 days) significantly inhibited hyperglycemia-mediated apoptosis, decreased malondialdehyde levels and increased glutathione levels in the hippocampi of streptozotocin-induced diabetic rats. Western blot analysis revealed that luteolin also inhibited the expression of apoptosis-related factors and cytochrome c release from mitochondria. Luteolin also improved the learning and memory abilities of rats with diabetic encephalopathy in a water maze test. Further western blot analysis revealed that luteolin treatment facilitated neuronal cell survival through activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, an extracellular signal pathway involved in the suppression of cell apoptosis and promotion of cell survival. These experimental findings indicate that luteolin can inhibit apoptosis of hippocampal nerve cells in rats with diabetic encephalopathy, and that this effect is mediated by an indirect antioxidative effect, the inhibition of activation of apoptosis-related factors and the activation of phosphatidylinositol 3-kinase/Akt signal pathway.
neural regeneration; brain injury; diabetic mellitus; luteolin; apoptosis; learning and memory; spatial recognition memory; nerve cells; hippocampus; anti-oxidation; neuroregeneration
Superoxide anion is elevated during neointima development and is essential for neointimal vascular smooth muscle cell (VSMC) proliferation. However, little is known about the role of manganese superoxide dismutase (MnSOD, SOD2) in the neointima formation following vascular injury. SOD2 in the mitochondria plays an important role in cellular defense against oxidative damage. Because of its subcellular localization, SOD2 is considered the first line of defense against oxidative stress and plays a central role in metabolizing superoxide. Because mitochondria are the most important sources of superoxide anion, we speculated that SOD2 may have therapeutic benefits in preventing vascular remodeling. In this study, we used a rat carotid artery balloon-injury model and an adenoviral gene delivery approach to test the hypothesis that SOD2 suppresses vascular lesion formation. SOD2 was activated along with the progression of neointima formation in balloon-injured rat carotid arteries. Depletion of SOD2 by RNA interference markedly promoted the lesion formation, whereas SOD2 overexpression suppressed the injury-induced neointima formation via attenuation of migration and proliferation of VSMCs. SOD2 exerts its inhibitory effect on VSMC migration induced by angiotensin II by scavenging superoxide anion and suppressing the phosphorylation of Akt. Our data indicate that SOD2 is a negative modulator of vascular lesion formation after injury. Therefore, SOD2 augmentation may be a promising therapeutic strategy for the prevention of lesion formation in proliferative vascular diseases such as restenosis.
Manganese superoxide dismutase; Oxidative stress; Neointima; Migration; Proliferation; Vascular smooth muscle cells; Signal transduction; Free radicals
Poor survival of mesenchymal stem cells (MSC) compromised the efficacy of stem cell therapy for ischemic diseases. The aim of this study is to investigate the role of PEP-1-CAT transduction in MSC survival and its effect on ischemia-induced angiogenesis.
MSC apoptosis was evaluated by DAPI staining and quantified by Annexin V and PI double staining and Flow Cytometry. Malondialdehyde (MDA) content, lactate dehydrogenase (LDH) release, and Superoxide Dismutase (SOD) activities were simultaneously measured. MSC mitochondrial membrane potential was analyzed with JC-1 staining. MSC survival in rat muscles with gender-mismatched transplantation of the MSC after lower limb ischemia was assessed by detecting SRY expression. MSC apoptosis in ischemic area was determined by TUNEL assay. The effect of PEP-1-CAT-transduced MSC on angiogenesis in vivo was determined in the lower limb ischemia model.
PEP-1-CAT transduction decreased MSC apoptosis rate while down-regulating MDA content and blocking LDH release as compared to the treatment with H2O2 or CAT. However, SOD activity was up-regulated in PEP-1-CAT-transduced cells. Consistent with its effect on MSC apoptosis, PEP-1-CAT restored H2O2-attenuated mitochondrial membrane potential. Mechanistically, PEP-1-CAT blocked H2O2-induced down-regulation of PI3K/Akt activity, an essential signaling pathway regulating MSC apoptosis. In vivo, the viability of MSC implanted into ischemic area in lower limb ischemia rat model was increased by four-fold when transduced with PEP-1-CAT. Importantly, PEP-1-CAT-transduced MSC significantly enhanced ischemia-induced angiogenesis by up-regulating VEGF expression.
PEP-1-CAT-transduction was able to increase MSC viability by regulating PI3K/Akt activity, which stimulated ischemia-induced angiogenesis.
Wei-Wei-Kang-Granule(WWKG) is a traditional Chinese medicine (TCM) preparation for the treatment of chronic atrophic gastritis (CAG). We examined the pathologic change and the effects of Wei-Wei-Kang-Granule (WWKG) on the expression of EGFR (epiderminal growth factor receptors) and NF-kB (nuclear transcription factor KappaB) in rats with chronic atrophic gastritis (CAG), and evaluated the possible mechanisms. Ninety rats were randomly divided into control group and four experimental groups. CAG rat models were induced by repeated stimulating experiments in the experimental groups. After modeled rats were intragastrically injected (i.g.) with WWKG at 6000mg/kg (large dose WWKG group), WWKG at 3000mg/kg (small dose WWKG group), San-Jiu-Wei-Tai-Granule(SJWTG) at 1600mg/kg(SJWTG group), and normal saline(0.9%)at 20ml/kg (model group and control group), respectively, once a day for 30 days. After 30 days, all rats were sacrificed and samples were taken from the sinus ventriculi and body of stomach. The gastric specimens were prepared for microscopic view with hematoxylin and eosin (H-E). The immunohistochemistry method was used to observe the expression of protein of EGFR and NF-kB in gastric tissue. The data were analyzed in pre-and post-treatment by computer image automatic analysis system. Immunohistochemistry detection showed that the average optical density of EGFR and NF-kB in antrum was lower in large and small dose WWKG groups than the model group (P<0.01). CAG in rats was related with the damage of barrier in gastric mucosa and the misbalance of cell proliferation and apoptosis. One of the mechanisms is perhaps to reduce the expressing of EGFR and NF-Kb in gastric mucosa.
Chronic atrophic gastritis(CAG); EGFR; NF-kB
The objective of this study was to investigate whether vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSC) improves myocardial survival and the engraftment of implanted MSC in infarcted hearts and promotes recruitment of stem cells through paracrine release of myocardial stromal cell-derived factor-1α (SDF-1α).
Methods and results
VEGF-expressing MSC (VEGFMSC)-conditioned medium enhanced SDF-1α expression in heart slices and H9C2 cardiomyoblast cells via VEGF and the vascular endothelial growth factor receptor (VEGFR). The VEGFMSC-conditioned medium markedly promoted cardiac stem cell (CSC) migration at least in part via the SDF-1α/CXCR4 pathway and involved binding to VEGFR-1 and VEGFR-3. In vivo, VEGFMSC-stimulated SDF-1α expression in infarcted hearts resulted in massive mobilization and homing of bone marrow stem cells and CSC. Moreover, VEGF-induced SDF-1α guided the exogenously introduced CSC in the atrioventricular groove to migrate to the infarcted area, leading to a reduction in infarct size. Functional studies showed that VEGFMSC transplantation stimulated extensive angiomyogenesis in infarcted hearts as indicated by the expression of cardiac troponin T, CD31, and von Willebrand factor and improved the left ventricular performance, whereas blockade of SDF-1α or its receptor by RNAi or antagonist significantly diminished the beneficial effects of VEGFMSC.
Exogenously expressed VEGF promotes myocardial repair at least in part through SDF-1α/CXCR4-mediated recruitment of CSC.
Myocardial infarction; VEGF; SDF-1α; Cardiac stem cell; Migration
The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in vascular lesion formation after experimental angioplasty and to explore the underlying mechanisms.
Methods and Results
Using a rat carotid artery balloon-injury model, we documented for the first time that neointima formation was closely associated with a significantly increased expression of RGC-32 protein. shRNA Knockdown of RGC-32 via adenovirus (Ad)-mediated gene delivery dramatically inhibited the lesion formation by 62% as compared to control groups 14 days after injury. Conversely, RGC-32 overexpression significantly promoted the neointima formation by 33%. Gain and loss of function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that RGC-32 is essential for both the proliferation and migration of RASMCs. RGC-32 induced RASMC proliferation by enhancing p34CDC2 activity. RGC-32 stimulated the migration of RASMC via inducing focal adhesion contact and stress fiber formation. These effects were caused by the enhanced ROKα activity due to RGC-32-induced downregulation of Rad GTPase.
RGC-32 plays an important role in vascular lesion formation following vascular injury. Increased RGC-32 expression in vascular injury appears to be a novel mechanism underlying the migration and proliferation of vascular SMCs. Therefore, targeting RGC-32 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.
Response gene to complement 32; Restenosis; Vascular smooth muscle cells; Migration; Proliferation