Hydrogen Sulfide Promotes Postnatal Cardiomyocyte Proliferation by Upregulating SIRT1 Signaling Pathway.

Hydrogen sulfide (H2S) has been identified as a novel gasotransmitter and a substantial antioxidant that can activate various cellular targets to regulate physiological and pathological processes in mammals. However, under physiological conditions, it remains unclear whether it is involved in regulating cardiomyocyte (CM) proliferation during postnatal development in mice. This study mainly aimed to evaluate the role of H2S in postnatal CM proliferation and its regulating molecular mechanisms. We found that sodium hydrosulfide (NaHS, the most widely used H2S donor, 50-200 µM) increased neonatal mouse primary CM proliferation in a dose-dependent manner in vitro. Consistently, exogenous administration of H2S also promoted CM proliferation and increased the total number of CMs at postnatal 7 and 14 days in vivo. Moreover, we observed that the protein expression of SIRT1 was significantly upregulated after NaHS treatment. Inhibition of SIRT1 with EX-527 or si-SIRT1 decreased CM proliferation, while enhancement of the activation of SIRT1 with SRT1720 promoted CM proliferation. Meanwhile, pharmacological and genetic blocking of SIRT1 repressed the effect of NaHS on CM proliferation. Taken together, these results reveal that H2S plays a promotional role in proliferation of CMs in vivo and in vitro and SIRT1 is required for H2S-mediated CM proliferation, which indicates that H2S may be a potential modulator for heart development in postnatal time window.

Identification of immune-related genes in the prognosis of head and neck cancer using a novel prognostic signature model.

BACKGROUND: Increasing evidence indicates that the immune response plays a critical role in the development of head and neck cancer (HNC). We aimed to develop an immune-related gene signature and evaluate its prognostic value in patients with HNC. METHODS: We retrieved an HNC cohort from The Cancer Genome Atlas database and divided the samples into high-risk and low-risk groups based on the median of the immune and stromal scores. We performed Venn and Cox analyses to identify the immune-related DEGs to use in our prognostic model. We evaluated the correlation between the model and immune-cell infiltration and validated the prognostic value of the model by applying it to 2 external HNC cohorts. RESULTS: We identified 7 DEGs-CCR4, WDFY4, VCAM1, LYZ, VSIG4, XIRP1, and CMKLR1-to use in our prognostic model and validated the model by applying it to 2 external HNC cohorts. We found that risk scores based on the model could reflect the status of the tumor microenvironment and that VSIG4 might be associated with lymph node metastasis in HNC. CONCLUSIONS: We developed a highly accurate immune-related prognostic 7-gene model in HNC predication, indicating that these 7 genes play critical roles in the tumor microenvironment.

Exploration of the effect of Celastrol on protein targets in nasopharyngeal carcinoma: Network pharmacology, molecular docking and experimental evaluations.

Background: Celastrol, an important extract of Tripterygium wilfordii, shows strong antitumor activity in a variety of tumors including nasopharyngeal carcinoma (NPC). However, little is known about its targets in NPC. We aimed to screen the key gene targets of Celastrol in the treatment of NPC by means of in silico analyses (including network pharmacology and molecular docking) and experimental evaluations. Methods: The main target genes of Celastrol and the genes related to NPC were obtained by retrieving the relevant biological databases, and the common targets were screened. Protein-protein interaction analysis was used to screen the hub genes. Then, a "compound-target-disease" network model was created and molecular docking was used to predict the binding of Celastrol to the candidate hub proteins. Afterward, the expression changes of the candidate genes under the administration of Celastrol were verified in vitro and in vivo. Results: Sixty genes common to Celastrol and NPC were screened out, which may be related to numerous biological processes such as cell proliferation, apoptosis, and tube development, and enriched in various pathways such as PI3K- Akt, EGFR tyrosine kinase inhibitor resistance, and Apoptosis. The tight binding ability of the candidate hub proteins (TNF, VEGFA, and IL6) to Celastrol was predicted by molecular docking [Docking energy: TNF, -6.08; VEGFA,-6.76; IL6,-6.91(kcal/mol)]. In vitro experiments showed that the expression of TNF and VEGFA decreased while the expression of IL6 increased in NPC cells (CNE2 and HONE1) treated with Celastrol. In vivo experiments suggested that Celastrol significantly reduced the weight and volume of the transplanted tumors in tumor-bearing mice in vivo. The expression of TNF, VEGFA, and IL6 in the transplanted tumor cells could be regulated by using Celastrol, and the expression trends were consistent with the in vitro model. Conclusion: Several gene targets have been filtered out as the core targets of Celastrol in the treatment of NPC, which might be involved in a variety of signaling pathways. Hence, Celastrol may exert its anti-NPC activity through multiple targets and multiple pathways, which will provide new clues for further research. Future experiments are warranted to validate the findings.

Glucocorticoids rapidly promote YAP phosphorylation via the cAMP-PKA pathway to repress mouse cardiomyocyte proliferative potential.

Adult mammalian cardiomyocytes (CMs) lose their proliferative potential due to cell-cycle withdrawal and polyploidization and fail to mount a proliferative response to regenerate new CMs after cardiac injury. The decline in the proliferative potential of mammalian CMs occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extra-uterine life. There is an increase in circulating glucocorticoid (GC) levels shortly after birth in mammals, and thus, we sought to determine the roles and mechanisms of GCs in regulating CM proliferation. Here, we showed that GCs suppressed CM proliferation in vitro and in vivo, decreased the total number of CMs, and increased the cross-sectional area of CMs. However, the glucocorticoid receptor antagonist had no effect on CM proliferation. Agonists of adenylate cyclase and protein kinase A (PKA) inhibited CM proliferation, while PKA antagonists or knockdown of PKA alleviated the inhibitory effect of GCs on CM proliferation. GCs and the activation of the cyclic adenosine monophosphate (cAMP)/PKA signaling pathway facilitated yes-associated protein (YAP) phosphorylation in mouse CMs and promoted YAP protein translocation from the nucleus to the cytoplasm. Meanwhile, blocking the cAMP/PKA signaling pathway partially blocked the effect of GCs on YAP protein phosphorylation and YAP protein translocation. Thus, our findings suggest that GCs suppress mouse CM proliferation in vitro and in vivo, through a mechanism that involves targeting the cAMP-PKA-YAP signaling pathway.

Brain-derived neurotrophic factor produced long-term synaptic enhancement in the anterior cingulate cortex of adult mice.

Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) is one of the diffusible messengers for enhancing synaptic transmission in the hippocampus. Less information is available about the possible roles of BDNF in the anterior cingulate cortex (ACC). In the present study, we used 64-electrode array field recording system to investigate the effect of BDNF on ACC excitatory transmission. We found that BDNF enhanced synaptic responses in a dose-dependent manner in the ACC in C57/BL6 mice. The enhancement was long-lasting, and persisted for at least 3 h. In addition to the enhancement, BDNF also recruited inactive synaptic responses in the ACC. Bath application of the tropomyosin receptor kinase B (TrkB) receptor antagonist K252a blocked BDNF-induced enhancement. L-type voltage-gated calcium channels (L-VGCC), metabotropic glutamate receptors (mGluRs), but not NMDA receptors were required for BDNF-produced enhancement. Moreover, calcium-stimulated adenylyl cyclase subtype 1 (AC1) but not AC8 was essential for the enhancement. A selective AC1 inhibitor NB001 completely blocked the enhancement. Furthermore, BDNF-produced enhancement occluded theta burst stimulation (TBS) induced long-term potentiation (LTP), suggesting that they may share similar signaling mechanisms. Finally, the expression of BDNF-induced enhancement depends on postsynaptic incorporation of calcium-permeable AMPA receptors (CP-AMPARs) and protein kinase Mζ (PKMζ). Our results demonstrate that cortical BDNF may contribute to synaptic potentiation in the ACC.

Peperomin E Induces Apoptosis and Cytoprotective Autophagy in Human Prostate Cancer DU145 Cells In Vitro and In Vivo.

Peperomin E was first isolated from Peperomia dindygulensis, an anticarcinogenic herb, and exhibited anticancer activity in many cancer cell lines. To date, it is unknown whether peperomin E has an effect on human prostate cancer DU145 cells in vitro and in vivo. In this study, we used MTT to assess the proliferation inhibition activity of peperomin E in DU145 cells in vitro and observed the cell morphological changes by a phase contrast microscope. A DU145 cell xenograft tumor mouse model was used to evaluate the efficacy of peperomin E in vivo. Apoptosis rates were measured by flow cytometry, and protein expression levels were analyzed by western blot. The results showed that peperomin E significantly inhibited the proliferation of DU145 cells in vitro and reduced the weight and volume of tumors in vivo. Peperomin E also significantly induced the apoptosis and autophagic response of DU145 cells. The autophagic inhibitors LY294002 and chloroquine enhanced peperomin E-mediated inhibition of DU145 cell proliferation and induction of DU145 cell apoptosis. The results also showed that the Akt/mTOR pathway participated in peperomin E-induced autophagy in DU145 cells. In summary, our finding showed that peperomin E had an effect on DU145 cells in vitro and in a nude mouse DU145 cell xenograft model in vivo, demonstrated that peperomin E could significantly induce apoptosis and the autophagic response in DU145 cells and that autophagy played a cytoprotective role in peperomin E-treated DU145 cells. These results suggest that the combination of peperomin E treatment and autophagic inhibition has potential for the treatment of prostate cancer.

The Anti-Proliferation, Cycle Arrest and Apoptotic Inducing Activity of Peperomin E on Prostate Cancer PC-3 Cell Line.

Peperomin E is a natural secolignan existing distributed in the plants of the genus Peperomia. Previous investigations demonstrated that peperomin E showed potential antitumor activity in some cancer lines, but it is unclear whether peperomin E has an effect on prostate cancer cell lines. The aim of the present study is to investigate its effects on proliferation inhibition, apoptosis-inducing and cell-cycle arrest activity using a prostate cancer PC-3 cell line. The proliferation inhibition was evaluated by MTT assay, apoptosis was detected by Annexin V/propidium iodide (PI) staining and Hoechst 33258 staining, cell cycle distributions were measured by flow cytometry, and western blot analysis was used to determine specific cellular apoptotic protein expressions of Bcl-2, Bax, caspase-3 and cleaved-caspase-3. According to the results of this study, peperomin E exhibited significant anti-proliferation activity on PC-3 cell lines in vitro in a dose-dependent manner. Peperomin E treatments lead to marked morphological changes. Apoptotic cell count and cell-cycle distribution at G2/M phase significantly increased with increasing concentrations of peperomin E. The down-regulated expression level of Bcl-2 and up-regulated expression level of Bax and cleaved-caspase-3 compared with the controls were also observed after peperomin E treatment. These data suggest that peperomin E exhibited proliferation inhabitation, apoptosis-inducing and cell-cycle arrest activity on PC-3 cell lines. The anti-proliferation effect of peperomin E on PC-3 cells should result partly from its cell-cycle arrest and apoptosis-inducing activity, whereas the increasing of the Bax/Bcl-2 ratio and activation of caspases-3 play an important role in the development of apoptosis.

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell-matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis.

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer.

[Influence of chronic fluorosis on the expression of mitochondrial fission protein dynamin-related 1 in the cortical neurons of rats].

OBJECTIVE: To explore the changes of protein expression of mitochondrial fission gene dynamin-related 1(Drp 1) in the cortical neurons of rats with chronic fluorosis. METHODS: A total of 120 one-month-old SD rats (each weighing approximately 100-120 g at the beginning of the experiment) were randomly divided into three groups, and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride & high-fluoride supplemented with 10 and 50 mg/L fluoride,respectively). After 3 or 6 months exposure, 20 rats from each group were killed. Then the protein expression of mitochondrial fission gene, Drp1, was detected by immunohistochemistry and western-blotting method. RESULTS: Dental fluorosis and urinary fluorosis were obviously found in the rats exposed to fluoride. At the experiment period of 3 months, the numbers of positive cells of Drp1 detected by immunohistochemistry changed. Compared with the control group (36.3 ± 5.8), the changes in low-fluoride group (34.7 ± 4.1) showed no significant difference (t = 1.5, P > 0.05),but the increase in high-fluoride group (45.0 ± 4.7) had statistical significance (t = 8.8, P < 0.05). The western-blotting method had consistent results. Compared with the control group (0.59 ± 0.03), a significant increase of the average topical density in low- fluoride (0.62 ± 0.03) and high-fluoride (0.71 ± 0.02) groups were found (t = 0.02,0.11, P < 0.05). At the experiment period of 6 months, the numbers of positive cells of Drp1 detected by immunohistochemistry significantly changed. Compared with the control group (33.2 ± 4.4), the number in low- fluoride and high-fluoride groups were separately (36.6 ± 3.8) and (39.4 ± 4.2),both increased significantly (t = 3.5,6.3, P < 0.05). Same results could be found in western-blotting method,compared with the control group (0.65 ± 0.06), the average topical density in low- fluoride (0.80 ± 0.09) and high-fluoride (0.76 ± 0.08) groups both increased significantly (t = 0.1,0.1, P < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the changes of expression of Drp1, and the neurons damage from the chronic fluorosis might be associated with the hyperfunction of mitochondrial fusion.

[Changed expression of mito-fusion 1 and mitochondrial fragmentation in the cortical neurons of rats with chronic fluorosis].

OBJECTIVE: To observe the mitochondrial fragmentation and the expression of mito-fusion 1 gene in the cortical neurons of rats with chronic fluorosis, and to reveal their roles in mitochondria damage to neurons due to chronic fluorosis. METHODS: SD rats were divided randomly into three groups of 20 each (a half females and a half males housed individually in stainless-steel cages), and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride and high supplemented with 10 and 50 mg/L fluoride, respectively). After 3 or 6 months exposure, the mitochondrial morphology of the neurons in rat brains were observed by transmission electron microscopy (TEM), then the expression of mitochondrial fusion gene, Mfn1, were detected by immunohistochemistry and western-blotting, respectively. RESULTS: Dental fluorosis was obvious in the rats exposed to excessive fluoride in their drinking water, that is, (16 rats out of 20) numbers of I° detal fluorosis in the low-fluoride group, and (11 rats out of 20) numbers of I° and (9 rats out of 20) numbers of II° detal fluorosis in the high-fluoride group were observed after 3 months exposure. Moreover, (14 rats out of 20) numbers of I° and (6 rats out of 20) numbers of II° detal fluorosis in the low-fluoride group and (6 rats out of 20) numbers of Io, (13 rats out of 20) numbers of II°, and (1 rats out of 20) numbers of III° detal fluorosis in the high-fluoride group were observed after 6 months exposure. And both of untreated controls without detal fluorosis were also observed. The urinary level of fluoride in the low-fluoride group (3.30 ± 1.18) mg/L and in the high-fluoride group (5.10 ± 0.35) were observed after 3 months exposure (F = 3.18, P < 0.05). Moreover, the urinary level of fluoride in the low-fluoride group (4.16 ± 1.39) mg/L and in the high-fluoride group (5.70 ± 1.70) mg/L were also observed after 6 months exposure (F = 3.17, P < 0.05). The normal mitochondrial morphology of neurons in rats without fluorosis was observed after 3 and 6 months, while the abnormal mitochondrial morphology of neurons with fluorosis was shown, presenting mitochondrial fragmentation with swollen cristae and even the fragmented, shortened or stacked punctuate membranes (section observation of three bullous mitochondrial-mitochondrial fission process) by TEM. As compared with controls (53.0 ± 4.54 and 1.21 ± 0.18) at the experiment period of 3 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 51.09 ± 6.25) and western-blotting (1.22 ± 0.26) were no significant difference for low fluoride group (t = 1.7, 1.1, P > 0.05); Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 59.71 ± 5.64) and western-blotting (1.66 ± 0.20) were significantly increasing for high fluoride group (t = 2.1, 2.1, P < 0.05). As compared with controls (36.43 ± 4.04 and 1.00 ± 0.13) at the experiment period of 6 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells 20.05 ± 4.55 and 17.10 ± 3.86) and western-blotting (0.64 ± 0.08 and 0.39 ± 0.06) were significantly decreasing for the two fluoride group (t = 2.1, 2.2; 2.2, 2.2 respectively, all P value were < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the mitochondrial fragmentation for the changed expression of Mfn1, and the neurons damage from the chronic fluorosis might be associated with the dysfunction of mitochondrial fusion.

The influence of chronic fluorosis on mitochondrial dynamics morphology and distribution in cortical neurons of the rat brain.

The present study was designed to evaluate the effects of chronic fluorosis on the dynamics (including fusion and fission proteins), fragmentation, and distribution of mitochondria in the cortical neurons of the rat brain in an attempt to elucidate molecular mechanisms underlying the brain damage associated with excess accumulation of fluoride. Sixty Sprague-Dawley rats were divided randomly into three groups of 20 each, that is, the untreated control group (drinking water naturally containing <0.5 mg fluoride/l, NaF), the low-fluoride group (whose drinking water was supplemented with 10 mg fluoride/l) and the high-fluoride group (50 mg fluoride/l). After 6 months of exposure, the expression of mitofusin-1 (Mfn1), fission-1 (Fis1), and dynamin-related protein-1 (Drp1) at both the protein and mRNA levels were detected by Western blotting, immunohistochemistry, and real-time PCR, respectively. Moreover, mitochondrial morphology and distribution in neurons were observed by transmission electron or fluorescence microscopy. In the cortices of the brains of rats with chronic fluorosis, the level of Mfn1 protein was clearly reduced, whereas the levels of Fis1 and Drp1 were elevated. The alternations of expression of the mRNAs encoding all three of these proteins were almost the same as the corresponding changes at the protein levels. The mitochondria were fragmented and the redistributed away from the axons of the cortical neurons. These findings indicate that chronic fluorosis induces abnormal mitochondrial dynamics, which might in turn result in a high level of oxidative stress.

Contribution of BK(Ca) channels of neurons in rostral ventrolateral medulla to CO-mediated central regulation of respiratory rhythm in medullary slices of neonatal rats.

We recently described that carbon monoxide (CO) participated in the regulation of rhythmic respiration in medullary slices. The present study was undertaken to further assess whether the large-conductance calcium-activated potassium channels (BK(Ca) channels) are involved in the CO-mediated central regulation of respiratory rhythm in medullary slices. The rhythmic discharge of hypoglossal rootlets of medullary slices of neonatal rats was recorded. We observed that blocking BK(Ca) channels could partially abolish the effects of CO on the rhythmic bursts of hypoglossal rootlets. With whole-cell patch-clamp recording technique, we further observed that CO could reversibly augment potassium current density of the neurons in the rostral ventrolateral medulla. The CO-induced increase in potassium current was entirely blocked by the pretreatment of slices with BK(Ca) channels blocker; whereas blockade of CO generation with zinc protoporphyrin-IX produced an opposite response. Altogether, these data indicate that BK(Ca) channels of the neurons in neonatal rostral ventrolateral medulla could be activated by CO and involved in CO-mediated central regulation of respiratory rhythm in medullary slices.

Protective action of endogenously generated H2S on hypoxia-induced respiratory suppression and its relation to antioxidation and down-regulation of c-fos mRNA in medullary slices of neonatal rats.

We previously reported that exogenous H(2)S played roles in protection of respiratory centers against hypoxic injury in medullary slices of neonatal rats. The protective action of endogenous H(2)S and its relation to antioxidation and down-regulation of c-fos mRNA were investigated in the present study. Perfusion of the slices with l-cysteine (Cys), substrate of cystathionine ß-synthase (CBS, H(2)S synthase), could increase frequency of rhythmic respiratory discharge of the hypoglossal rootlets and prevent respiratory suppression induced by hypoxia, whereas perfusion with hydroxylamine (NH(2)OH, inhibitor of CBS) could postpone recovery of respiration from hypoxic inhibition. NH(2)OH also significantly enhanced hypoxia-induced increase in malondialdehyde (MDA) content of the slices. The hypoxia-induced up-regulation of c-fos mRNA could be markedly antagonized by S-adenosyl-l-methionine (SAM, activator of CBS), but greatly increased by NH(2)OH. Neither NH(2)OH, Cys nor SAM had any effect on expression of bcl-2 mRNA in hypoxic medullary slices. These results indicate that endogenously generated H(2)S was involved in protection of the medullary respiratory centers against hypoxic injury partly via antioxidation and down-regulation of c-fos.

Protective effect of hydrogen sulfide on hypoxic respiratory suppression in medullary slice of neonatal rats.

Hydrogen sulfide (H(2)S) has been shown to play a protective role in injury of cells induced by hypoxia. Little is known however about its effect on medullary hypoxia-induced rhythmic respiratory suppression. In the present study, a decrease in frequency of rhythmic discharge of hypoglossal rootlets was observed in medullary slices of neonatal rats perfused with 95% N(2)-5% CO(2) to produce hypoxia. Perfusion with NaHS (H(2)S donor) prevented the inhibitory effect of hypoxia on the burst activity of the rootlets, whereas such action of NaHS was suppressed by pretreatment with glibenclamide, a blocker of K(ATP) channels. In addition, the increase in malondialdehyde content and the up-regulation of c-fos mRNA expression of the slices induced by hypoxia was significantly reduced by NaHS. These results indicate that exogenous H(2)S may protect the medullary respiratory center against hypoxic injury via activation of K(ATP) channels, reduction of lipid peroxidation and down-regulation of c-fos.