Antidementia medication acetylcholinesterase inhibitors have therapeutic benefits on osteoporotic bone by attenuating osteoclastogenesis and bone resorption.

This study was designed to determine whether the use of acetylcholinesterase inhibitors (AChEIs), a group of drugs that stimulate acetylcholine receptors and are used to treat Alzheimer's disease (AD), is associated with osteoporosis protection and inhibition of osteoclast differentiation and function. Firstly, we examined the effects of AChEIs on RANKL-induced osteoclast differentiation and function with osteoclastogenesis and bone resorption assays. Next, we investigated the impacts of AChEIs on RANKL-induced nuclear factor κB and NFATc1 activation and expression of osteoclast marker proteins CA-2, CTSK and NFATc1, and dissected the MAPK signaling in osteoclasts in vitro by using luciferase assay and Western blot. Finally, we assessed the in vivo efficacy of AChEIs using an ovariectomy-induced osteoporosis mouse model, which was analyzed using microcomputed tomography, in vivo osteoclast and osteoblast parameters were assessed using histomorphometry. We found that Donepezil and Rivastigmine inhibited RANKL-induced osteoclastogenesis and impaired osteoclastic bone resorption. Moreover, AChEIs reduced the RANKL-induced transcription of Nfatc1, and expression of osteoclast marker genes to varying degrees (mainly Donepezil and Rivastigmine but not Galantamine). Furthermore, AChEIs variably inhibited RANKL-induced MAPK signaling accompanied by downregulation of AChE transcription. Finally, AChEIs protected against OVX-induced bone loss mainly by inhibiting osteoclast activity. Taken together, AChEIs (mainly Donepezil and Rivastigmine) exerted a positive effect on bone protection by inhibiting osteoclast function through MAPK and NFATc1 signaling pathways through downregulating AChE. Our findings have important clinical implications that elderly patients with dementia who are at risk of developing osteoporosis may potentially benefit from therapy with the AChEI drugs. Our study may influence drug choice in those patients with both AD and osteoporosis.

Elevated Insulin-Like Growth Factor-1-Induced Female Rats Perpetuate the Polycystic Ovary Syndrome Phenotype: Pathological Mechanism of Insulin-Like Growth Factor-1 in Polycystic Ovary Syndrome.

OBJECTIVES: This study was designated to establish a polycystic ovary syndrome (PCOS) rat model with recombinant human insulin-like growth factor-1 (RH-IGF-1). We made assessment on the characteristics of hyperinsulinemia and hyperandrogenism in the rat model. DESIGN: This study performed the characteristics of PCOS upon RH-IGF-1 injection and evaluated the disease process of PCOS syndrome caused by the insulin-resistant pathological condition of IGF-1 based on the comparative study of in vivo test. SETTING: The experiment was conducted in the experimental research center of Yinzhou NO.2 hospital, Ningbo, Zhejiang Province, China. MATERIALS AND METHODS: Thirty-four female Sprague Dawley immature rats aged 21 days were randomly divided into two groups. Those treated with RH-IGF-1 2 mg/100 g daily were in RH-IGF-1 group (n = 20), and those with 0.9% sodium chloride 0.2 mL/100 g daily were in the saline group (n = 14). The experiment was carried out in two stages. In stage I, rats were anesthetized upon the first estrous cycle in the saline group with tissue and blood samples collected (n = 7), and rats in the RH-IGF-1-treated group were anesthetized on the 5th day after vaginal opening (VO) (n = 10). In stage II, rats in the saline group were anesthetized after three complete cycles (n = 7), meanwhile, while on the 15th day after VO (n = 10) for those in the RH-IGF-1 group. RESULTS: We have found that compared with the control group, rats injected with RH-IGF-1 expressed an early VO, disordered estrous cycle, polycystic ovaries, and significantly increased ovarian weight/body weight ratio. And from the perspective of hormone secretion, their androgen increased significantly and the insulin resistance index also elevated distinctly, possessing main characteristics similar to PCOS. LIMITATIONS: In this study, we were limited by the inability to examine IGF-1 in hypothalamus. IGF-1 in hypothalamus and in vitro experiments would be taken into consideration for further study in the future. CONCLUSIONS: These findings suggest that IGF-1 may be a key factor in the pathogenesis of PCOS, and the increase of androgen may be the pathological result, not the cause of PCOS.

LncRNA HCG11 represses ovarian cancer cell growth via AKT signaling pathway.

AIM: Ovarian cancer is a main contributor of cancer-relevant deaths among women worldwide due to high incidence and mortality. Mounting evidence has unveiled that lncRNAs play critical roles in malignancies, including ovarian cancer. Although the tumor suppressor function of HCG11 in prostate cancer and glioma has been proved, investigations on HCG11 role in ovarian cancer are still scarce. METHODS: Gene or protein expression was quantified by RT-qPCR or western blot. HCG11 effects on ovarian cancer were assessed by functional assays. Bioinformatics analysis and mechanism experiments were implemented to identify the association among HCG11, miR-1270, and PTEN. RESULTS: HCG11 was weakly expressed in ovarian cancer and functioned as a tumor suppressor in ovarian cancer by retarding cell proliferation, migration, and EMT. Besides, HCG11 could bind to miR-1270 and PTEN was a target gene of miR-1270. Mechanically, HCG11 competitively bound with miR-1270 to upregulate PTEN. From rescue experiments, HCG11 impeded AKT/mTOR pathway to retard ovarian cancer cell growth by miR-1270/PTEN. CONCLUSIONS: HCG11 was a tumor suppressor in ovarian cancer cells and additionally, HCG11 regulated AKT/mTOR pathway to hinder ovarian cancer cell growth via modulating miR-1270/PTEN, indicating that HCG11 may represent a promising target for effective treatment of ovarian cancer patients.

Detection of HPV E6/E7 mRNA in the diagnosis of cervical cancer and precancerous lesions after kidney transplantation.

OBJECTIVE: To explore the value of human papillomavirus (HPV) E6/E7 mRNA detection in the diagnosis of cervical cancer and its precancerous lesions after kidney transplantation. METHODS: One hundred and sixty-six women who underwent cervical cancer screening after kidney transplantation were selected and received thinprep cytology test (TCT), HPV DNA and HPV E6/E7 mRNA tests. A biopsy under colposcopy was performed for those with suspicious test results. The positive rates of TCT, HPV DNA and HPV E6/E7 mRNA expressions in patients with different biopsy pathological grades, the positive rates of HPV E6/E7 mRNA in TCT and HPV DNA positive patients were compared. Besides, the relationship between the results of the three detections and the pathological results of cervical biopsy as well as the diagnostic efficacy of cervical cancer and its precancerous lesions were compared. RESULTS: Among the 166 women undergoing cervical cancer screening, 87 cases received histopathological biopsy, of which, the positive expression rates of HPV E6/E7 mRNA in the negative, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III and invasive carcinoma (ICC) patients were 51.43%, 54.55%, 66.67%, 81.82% and 100.00%, respectively. The positive expression rates of HPV E6/E7 mRNA in TCT and HPV DNA-positive patients were 47.50% and 51.96%, respectively; those rates for diagnosis of ≥CIN II were significantly greater than that of ≤CIN I (both P<0.01). Receiver operating characteristic curve revealed that the areas under the concentration-time curve of TCT, HPV DNA and HPV E6/E7 mRNA detection for cervical cancer and precancerous lesions were 0.723, 0.833, 0.929, respectively. Their sensibilities were 76.89%, 83.30% and 92.38%, and their specificities were 77.04%, 88.47% and 94.47%, respectively. CONCLUSION: HPV E6/E7 mRNA detection effectively improves the diagnostic sensitivity and specificity of cervical cancer and precancerous lesions, thereby avoiding over-examination and over-treatment.

Multi-Omics Integration Reveals a Competitive Endogenous RNAs Network for the Identification of Progression Biomarkers and the Stratification of Patients Diagnosed With Nephroblastoma.

Specific types of nephroblastoma (Wilms' tumor, WT) are known to associate with poor overall survival. Emerging experimental evidence has demonstrated that competitive endogenous RNA (ceRNA) networks have important roles in regulating cancer occurrence, but the roles of ceRNAs in regulating the WT progression and the patient outcomes remain unclear. Using the multi-omics data of 132 WT patients collected from TARGET database, an integration analysis pipeline was performed to construct a highly reliable ceRNA network. As results, a total of 147 nodes (116 mRNAs, 15 miRNAs, and 16 lncRNAs) were identified and used to explore the underlying mechanism for WT progression. WGCNA analysis further identified several prognostic molecules, including hsa-mir-93, LINC00087 and RP5-1086K13, that significantly associated with the overall survival rate. And, enrichment analysis verified the participation of these molecules in tumor-related pathways, such as those controlling autophagy and cadherin-mediated adhesion. Importantly, the WT patients were classified into three categories according to the ceRNA network, which significantly correlated with the overall survival. In conclusion, the ceRNA network could be a promising tool to further validate the prognostic biomarkers and categories of patients diagnosed with WT.

Expression levels of a gene signature in hiPSC associated with lung adenocarcinoma stem cells and its capability in eliciting specific antitumor immune-response in a humanized mice model.

BACKGROUND: Previous studies have reported that cancer stem cells (CSCs) play a key role in tumorigenesis, metastasis, and recurrence. CSC-based vaccination confers better protection in tumor cells. However, isolation and cultivation of CSCs are difficult. This study aimed to explore the similarities between CSCs and induced pluripotent stem cells (iPSCs). METHODS: ALDH1+ cancer stem cells were isolated from lung adenocarcinoma patients and their gene expression patterns compared with human induced pluripotent stem cells (hiPSCs). In addition, a tumor vaccine was developed using hiPSC and unmethylated cytosine-guanine (CpG). Finally, the antitumor properties of the vaccine were evaluated in a humanized mouse model. RESULTS: Preimmunization of iPSC+CpG elicited stronger antigen presentation and cytotoxic T cell response which suppressed the growth of tumors. Adoptive transfer of spleen T cells from the vaccine preimmunized mice inhibited tumor growth in unvaccinated recipients without any side effects. CONCLUSIONS: This study suggests a universal strategy for tumor therapy which simplifies future clinical procedures. Therefore, the application of hiPSC elicits tumor protective responses.

MiR-182 inhibits kidney fibrosis by regulating transforming growth factor ß1/Smad3 pathway in autosomal dominant polycystic kidney disease.

The aim of the present study was to investigate the molecular mechanism of miR-182 in kidney fibrosis in polycystic kidney disease (PKD). We measured the expression of miR-182 in kidney tissue of autosomal dominant PKD. Additionally, we investigated the relationship between miR-182 and fibrotic protein by transfecting miR-182 mimics and miR-182 inhibitor into polycystic kidney cyst-lined epithelial cells, respectively. Furthermore, we observed the interaction between transforming growth factor ß1 (TGF-ß1) and miR-182 and fibrinogen factors of cyst-lined epithelial cells after TGF-ß1 intervention, and measured the expression of Smad2 and Smad3 protein. Results are presented as follows: (a) MiR-182 was positively correlated with fibrosis of cyst-lined epithelial cells; (b) TGF-ß1 could induce fibrosis of cyst-lined epithelial cells; (c) the expression of miR-182 had a remarkably impact on the fibrosis induced by TGF-ß1, but had little effect on the expression of TGF-ß1; (d) the expression of Smad3 protein in TGF-ß1 induce-cyst-lined epithelial cells was increased. TGF-ß1 and miR-182 promoting the fibrosis of polycystic kidney cyst-lined epithelial cells may be mediated by the TGF-ß1/Smad3 signaling pathway, of which Smad3 was an important regulator.

Histone Deacetylase Inhibitors Reduce Cysts by Activating Autophagy in Polycystic Kidney Disease.

BACKGROUND: Histone deacetylase inhibitors (HDACi) have therapeutic effects on various models of renal diseases including autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanism is unclear. OBJECTIVES: Here, we studied the role of trichostatin A (TSA), a specific HDACi, in regulating cyst growth to test the possibility that HDACi might help manage ADPKD by enhancing autophagy. RESULTS: Autophagy protein expression was higher in cultured Pkd1 knockout (Pkd1-/-) cells, an in vitro model of cystogenesis, compared with control cells. TSA prevented cyst formation in Pkd1-/- cells. We further tested whether TSA could not reduce the size of an already established cyst after inhibition of autophagy by chloroquine in Pkd1-/- cells. In vivo, treatment with TSA significantly slowed cyst growth in Pkd1-/- mice. Moreover, TSA treatment stimulated AMPK and inactivated mTOR during cyst growth in Pkd1-/- cells and kidneys in mice. CONCLUSIONS: Our results suggest that HDACi may prevent cyst formation by activation of the AMPK pathway and autophagy. They also imply that HDACi could have therapeutic potential for ADPKD treatment.

PYNOD reduces microglial inflammation and consequent neurotoxicity upon lipopolysaccharides stimulation.

PYNOD, a nod-like receptors (NLR)-like protein, was indicated to inhibit NF-κB activation, caspase-1-mediated interleukin (IL)-1ß release and cell apoptosis in a dose-dependent manner. Exogenous addition of recombinant PYNOD to mixed glial cultures may suppress caspase-1 activation and IL-1ß secretion induced by Aß. However, to the best of our knowledge, there no study has focused on the immunoregulatory effects of PYNOD specifically in microglia. The present study aimed to explore the roles of PYNOD involved in the lipopolysaccharides (LPS)-induced microglial inflammation and consequent neurotoxicity. Murine microglial BV-2 cells were transfected with pEGFP-C2-PYNOD (0-5.0 µg/ml) for 24 h and incubated with or without LPS (1 µg/ml) for a further 24 h. Cell viability was determined using MTT assay and the secretion of nitric oxide (NO), IL-1ß and caspase-1 was measured using the Griess method or ELISA. Protein expression levels of NF-κB p65 and inducible nitric oxide synthase (iNOS) were detected by immunofluorescent staining and/or western blot analysis. Co-culture of BV-2 cells with human neuroblastoma cell line SK-N-SH was performed in Transwell plates and the cell viability and apoptosis (using flow cytometry) of SK-N-SH cells were determined. Results indicated that PYNOD overexpression inhibited NO secretion and iNOS protein expression induced by LPS in BV-2 cells, with no detectable cytotoxicity. PYNOD overexpression also reduced the secretion of IL-1ß and caspase-1 from BV-2 cells upon LPS stimulation. These effects were dose-dependent. Additionally, PYNOD overexpression prevented LPS-induced nuclear translocation of NF-κB p65 in BV-2 cells. The growth-inhibitory and apoptosis-promoting effects of BV-2 cells towards SK-N-SH cells were alleviated as a result of PYNOD overexpression. In conclusion, PYNOD may mitigate microglial inflammation and consequent neurotoxicity.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm2 and 10 µm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm2 and 10 µm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

α2A-adrenergic blockade attenuates septic cardiomyopathy by increasing cardiac norepinephrine concentration and inhibiting cardiac endothelial activation.

Cardiomyopathy is a common complication associated with increased mortality in sepsis, but lacks specific therapy. Here, using genetic and pharmacological approaches, we explored the therapeutic effect of α2A-adrenergic receptor (AR) blockade on septic cardiomyopathy. CLP-induced septic rats were treated with BRL44408 (α2A-AR antagonist), prazosin (α1-AR antagonist) and/or reserpine. CLP-induced cardiomyopathy, indicated by reduced dP/dt and increased cardiac troponin I phosphorylation, was attenuated by BRL44408, this was associated with reduced cardiac TNF-α and endothelial VCAM-1 expression, cardiomyocyte apoptosis and related signal molecule phosphorylation. BRL44408 increased cardiac norepinephrine (NE) concentration in CLP rats. Pretreatment with reserpine that exhausts cardiac NE without affecting the circulating NE concentration or with prazosin partially abolished the cardioprotection of BRL44408 and reversed its inhibitory effects on myocardial TNF-α, apoptosis and related signal molecule phosphorylation, but not on VCAM-1 expression in septic rats. These effects of BRL44408 were confirmed by α2A-AR gene deletion in septic mice. Furthermore, α2-AR agonist not only enhanced LPS-induced TNF-α and VCAM-1 expression in cardiac endothelial cells that express α2A-AR, but also enhanced LPS-induced cardiac dysfunction in isolated rat hearts. Our data indicate that α2A-AR blockade attenuates septic cardiomyopathy by promoting cardiac NE release that activates myocardial α1-AR and suppressing cardiac endothelial activation.

TRAF3 delays cyst formation induced by NF-κB signaling.

This study aims to investigate the effects of TNF receptors associated factor 3 (TRAF3) on the signaling pathway and expression of downstream products of nuclear factor kappa B (NF-κB) in the epithelial cells of renal ducts in individuals with polycystic kidney disease (PKD). We observe the TRAF3 genic overexpression of the epithelial cells, which form a tubular branch structure, in polycystic kidneys and to explore the protective effect of TRAF3 on the cystogenesis and progression of PKD. Western blotting analysis was conducted to examine the signaling changes of NF-κB in PKD the epithelial cells and TRAF3 transgenic PKD epithelial cells. Changes in the downstream apoptosis factor and cell proliferation in PKD epithelial cells and TRAF3 transgenic PKD epithelial cells were detected. A three-dimensional matrigel culture experiment was performed to examine abnormal tubulomorphogenesis in vitro. The overexpression of TRAF3 significantly inhibited the signaling pathway of NF-κB in the PKD epithelial cells, downregulated the expression of downstream factors Bcl-2 and Bcl-xl, and significantly decreased cystic epithelial cell proliferation. Additional branch structures were observed in the PKD epithelial cells with a three-dimensional culture compared to wildtype cells. TRAF3 may likely induce apoptosis and resistance to proliferation and may be a new target to inhibit the cyst formation in PKD by regulating the NF-κB signaling pathway Bcl-2 and Bcl-xl activity. © 2017 IUBMB Life, 69(3):170-178, 2017.

Ginsenosides from stems and leaves of ginseng prevent ethanol-induced lipid accumulation in human L02 hepatocytes.

OBJECTIVE: To investigate the effect of ginsenosides from stems and leaves of ginseng on ethanol-induced lipid deposition in human L02 hepatocytes. METHODS: L02 cells were exposed to ethanol for 36 h and treated with or without ginsenosides. The viability of L02 cells was evaluated by methylthiazolyldiphenyl-tetrazolium bromide assay and the triglyceride (TG) content was detected. Lipid droplets were determined by oil red O staining. Intracellular reactive oxygen species (ROS) production and the mitochondrial membrane potential were tested by flow cytometry. The ATP level was measured by reverse phase high performance liquid chromatography. The expression of cytochrome p450 2E1 (CYP2E1) and peroxisome proliferator-activated receptor α (PPARα) was detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. RESULTS: Ethanol exposure resulted in the increase of TG level, lipid accumulation and ROS generation, and the decrease of mitochondrial membrane potential and ATP production in the cells. However, ginsenosides significantly reduced TG content (9.69±0.22 µg/mg protein vs. 4.93±0.49 µg/mg protein, P<0.01), and ROS formation (7254.8±385.7 vs. 5825.2±375.9, P<0.01). Meanwhile, improvements in mitochondrial membrane potential (10655.33±331.34 vs. 11129.52±262.35, P<0.05) and ATP level (1.20±0.18 nmol/mg protein vs. 2.53±0.25 nmol/mg protein, P<0.01) were observed by treatment with ginsenosides. Furthermore, ginsenosides could down-regulate CYP2E1 expression (P<0.01) and upregulate PPARα expression (P<0.01) in ethanol-treated cells. CONCLUSIONS: Ginsenosides could prevent ethanol-induced hepatocyte steatosis in vitro related to the inhibition of oxidative stress and the improvement of mitochondrial function. In addition, the modulation of CYP2E1 and PPARα expression may also play an important role in the protective effect of ginsenosides against lipid accumulation.

p38 MAPK signal pathway involved in anti-inflammatory effect of Chaihu-Shugan-San and Shen-ling-bai-zhu-San on hepatocyte in non-alcoholic steatohepatitis rats.

BACKGROUND: Traditional Chinese Medicine (TCM), has over thousands-of-years history of use. Chaihu-Shugan-San (CSS), and Shen-ling-bai-zhu-San (SLBZS), are famous traditional Chinese herbal medicine formulas, which have been used in China, for the treatment of many chronic diseases. MATERIALS AND METHODS: This study investigated the anti-inflammatory effects of CSS and SLBZS on signaling molecules involved in p38 mitogen-activated protein kinase (p38 MAPK), pathway on hepatocytes of non-alcoholic steatohepatitis (NASH), rats induced by high fat diet. SD male rats were randomly divided into 8 groups: negative control group, model control group, high (9.6g/kg/day)/low (3.2g/kg/day)-dose CSS group, high (30g/kg/day)/low (10g/kg/day)-dose SLBZS group, high (39.6g/kg/day)/low (13.2g/kg/day)-dose integrated group. The rats of NASH model were induced by feeding a high-fat diet. After 16, wks, Hepatocytes were isolated from 6, rats in each group by collagenase perfusion. The liver histopathological changes and serum inflammatory cytokines TNF-α, IL-6 were determined. The proteins of TLR4, phosphor-p38 MAPK and p38 MAPK involved in p38 MAPK signal pathway were assayed. RESULTS: The statistical data indicated the NASH model rats reproduced typical histopathological features of NASH in human. CSS and SLBZS ameliorated lipid metabolic disturbance, attenuated NASH progression, decreased the levels of TNF-α and IL-6 in serum, as well as inhibited TLR4 protein expression, p38 MAPK phosphorylation, and activation of p38 MAPK. In conclusion, CSS and SLBZS might work as a significant anti-inflammatory effect on hepatocyte of NASH by inhibiting the activation of TLR4, p-p38 MAPK and p38 MAPK involved in p38 MAPK signal pathway. CONCLUSION: To some extent, CSS and SLBZS may be a potential alternative and complementary medicine to protect against liver injury, alleviate the inflammation reaction, moderate NASH progression.

Berberine inhibits norepinephrine-induced apoptosis in neonatal rat cardiomyocytes via inhibiting ROS-TNF-α-caspase signaling pathway.

OBJECTIVE: To determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes. METHODS: The cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined. RESULTS: NE at a concentration of 50 µ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 µ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 µ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h. CONCLUSION: The present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Mitochondrial mutation impairs cytoplasmic male sterility rice in response to H2O2 stress.

Cytoplasmic male sterility (CMS) is a phenomenon widely observed in various plant species characterized with disrupted anther development caused by mitochondrial mutation. CMS is becoming a model system for the investigations of nucleus-cytoplasmic interaction. To reveal the possible effects of CMS genes on plant growth in adverse environment, plant development and biochemical characters of mitochondria from Honglian (HL)-CMS line Yuetai A and maintainer Yuetai B treated with H(2)O(2) were analyzed. Results showed that 40-60mM H(2)O(2) significantly inhibits rice seedling development and growth. When treated with H(2)O(2), ATP content and mitochondrial membrane potential in Yuetai A decreased significantly faster than those of Yuetai B. These biochemical changes were accompanied by the severe nuclear DNA fragmentation and the release of mitochondrial cytochrome c in the leaf cells of Yuetai A. In addition, the antioxidative enzyme activities and mitochondrial electron transfer chain complexes were significantly down-regulated. Disturbance of the biochemical indexes indicate that HL-CMS line is more susceptible to H(2)O(2) stress than the maintainer line, the deleterious effects caused by the CMS-related ORFH79 peptide compromises the adaptability of HL-CMS line to the adverse environment.

Functional characterization of the p53 binding site in the human PYNOD promoter.

Many members of the NOD-like receptor (NLR) family play important roles in pathogen recognition and inflammation. However, human PYNOD, an NLR-like protein consisting of a pyrin domain and a nucleotidebinding and oligomerization domain (NOD), has been reported to inhibit inflammatory signals. Using bioinformatics, we found a completely preserved canonical p53 binding site in the PYNOD core promoter (-228 to -237 bp) both in humans and in chimpanzees. In this study, we investigated the characterization and biologic function of this binding site in vitro. The results show that either deletion of the p53 binding elements within the PYNOD promoter or treatment with p53 inhibitor (PFT-α) could significantly reduce PYNOD promoter activity and PYNOD expression as detected by the enhanced green fluorescent protein (EGFP) reporter system, reverse transcription-polymerase chain reaction, and Western blot respectively. Furthermore, the chromatin immunoprecipitation (ChIP) method confirmed that p53 could bind to the PYNOD promoter. Our findings suggest that the p53 binding site plays a positive role in regulating PYNOD gene expression, which may maintain an efficient balance between defense and self-inflicted injury in respond to pathogen invasion.

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways.

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.

Pretreatment with berberine and yohimbine protects against LPS-induced myocardial dysfunction via inhibition of cardiac I-[kappa]B[alpha] phosphorylation and apoptosis in mice.

Myocardial dysfunction is a common complication in sepsis and significantly contributes to the mortality of patients with septic shock. Our previous study demonstrated that pretreatment with berberine (Ber) protected against the lethality induced by LPS, which was enhanced by yohimbine, an [alpha]2-adrenergic receptor antagonist, and Ber combined with yohimbine also improved survival in mice subjected to cecal ligation and puncture. However, no studies have examined whether Ber and yohimbine reduce LPS-induced myocardial dysfunction. Here, we report that pretreatment with Ber, Ber combined with yohimbine, or yohimbine significantly reduced LPS-induced cardiac dysfunction in mice. LPS-provoked cardiac apoptosis, I-[kappa]B[alpha] phosphorylation, IL-1[beta], TNF-[alpha], and NO production were attenuated by pretreatment with Ber and/or yohimbine, whereas cardiac Toll-like receptor 4 mRNA expression, malondialdehyde content, and superoxide dismutase activity were not affected. These data demonstrate for the first time that pretreatment with Ber and/or yohimbine prevents LPS-induced myocardial dysfunction in mice through inhibiting myocardial apoptosis, cardiac I-[kappa]B[alpha] phosphorylation, and TNF-[alpha], IL-1[beta], and NO production, suggesting that activation of [alpha]2-adrenergic receptor in vivo may be responsible at least in part for LPS-induced cardiac dysfunction, and Ber in combination with yohimbine may be a potential agent for preventing cardiac dysfunction during sepsis.

Regulation of cell proliferation and apoptosis through fibrocystin-prosaposin interaction.

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). It is widely accepted that cystogenesis is owing to aberrant cell proliferation and apoptosis, increased fluid secretion, and extracellular matrix abnormality. Fibrocystin/polyductin (FPC), the encoded protein product by PKHD1, is a single transmembrane protein and believed to be a novel receptor-like molecule. FPC has been located mainly on the plasma membrane and cilium/basal body. However, its biological functions remain poorly understood. To investigate the roles of FPC in the pathogenesis of ARPKD, we searched for FPC-interacting proteins by yeast two-hybrid assay, and found a novel partner, prosaposin. Prosaposin is a glycoprotein with multiple functions. With GST pull-down assay and co-immunoprecipitation, we confirmed the interaction between FPC and prosaposin. In order to study the effects of FPC-prosaposin interaction on cell proliferation and apoptosis, we have made stable cell lines in which FPC was overexpressed or knocked down alone or in combination with prosaposin overexpression. By MTT assay, we found that FPC knockdown and prosaposin overexpression increased cell proliferation, respectively, while overexpression of FPC C-tail did the opposite. With apoptosis assay, we found that overexpression of FPC C-tail promoted cell apoptosis. However, overexpression of prosaposin significantly enhanced cell survival in FPC knockdown cells. All these findings indicated that FPC and prosaposin may play significant roles in regulation of cell proliferation and apoptosis. Taken together, we have disclosed a novel signaling pathway of FPC, which may be important for the pathogenesis of ARPKD.