Expression Of Active Caspase-3 Research Articles

ETHNOPHARMACOLOGICAL STUDY OF BRAIN OXIDATIVE STRESS IMPROVING POTENTIAL OF CURCUMIN IN INTOXICATED RATS

Objective: The following study aimed to investigate the efficacy of curcumin at preventing amikacin neurotoxicity
 Methods: Twenty-four male Wister albino rats were randomly divided into four groups including-G (1): control group includes six rats, they were administered 0.5 ml of saline orally for 14 consecutive days. G (2): includes six rats; they were administered 200 mg/kg curcumin orally for 14 consecutive days. G (3): includes six rats, they were administered 300 mg/kg body weight/day of amikacin intraperitoneally for 14 consecutive days G (4): includes six rats, they were administered 200 mg/kg curcumin orally concurrently with 300 mg/kg body weight/day of amikacin. All animals were kept in the same conditions from feed, heat and humidity.
 Results: According to the result obtained after sacrification of all animals after the end of 14 d, Results revealed that amikacin at the dose rate of 300 mg/kg b. wt for 14 d induces significant changes in oxidative stress markers compared to the control group, a significant reduction in CAT. SOD. GSH (1.51±0.16, 77.00±0.73 and 84.06±4.42) respectively compared to control (3.63±0.11, 98.48±0.18 and 117.05±0.52) along with a significant increase in MDA activity (219.02±3.34) compared to control group (180.42±0.19), That indicate oxidative stress effect of it. On the beneficial side rats received amikacin 300 mg/kg B. wt I/p concurrently with 200 mg/kg b. wt curcumin for successive 14day result in a significant increase in CAT. SOD. GSH (2.23±0.09,92.00±0.26, 102.25±1.71) and decrease in MDA concentration (139.23±3.89) compared to amikacin treated group levels along with histopathological changes appear in brain tissue in the group treated with amikacin include nuclear pyknosis and degeneration in some neurons in the hippocampus, multiple focal eosinophilic plaque formation in the striatum also this results enhanced by activated caspase-3 expression in the brain tissue following amikacin administration.
 Conclusion: The present study proved that Oral administration of curcumin at the dose of 200 mg/kg for 14 d concurrently with amikacin significantly mitigates its neurotoxic and oxidative stress effects.

Honokiol exerts protective effects on neural myelin sheaths after compressed spinal cord injury by inhibiting oligodendrocyte apoptosis through regulation of ER-mitochondrial interactions

Objective To investigate the effect of honokiol on demyelination after compressed spinal cord injury (CSCI) and it's possible mechanism. Design Animal experiment study. Setting Institute of Neuroscience of Chongqing Medical University. Interventions Total of 69 Sprague–Dawley (SD) rats were randomly divided into 3 groups: sham group (n=15), honokiol group (n=27) and vehicle group (n=27). After established CSCI model by a custom-made compressor successfully, the rats of sham group were subjected to the limited laminectomy without compression; the rats of honokiol group were subjected to CSCI surgery and intraperitoneal injection of 20 mg/kg honokiol; the rats of vehicle group were subjected to CSCI surgery and intraperitoneal injection of an equivalent volume of saline. Outcome measures: The locomotor function of each group was assessed using the Basso, Beattie and Bresnahan (BBB) rating scale. The pathological changes of myelinated nerve fibers of spinal cord in 3 groups were detected by osmic acid staining and transmission electron microcopy (TME). Immunofluorescence and Western blot were used to research the experessions of active caspase-3, caspase-12, cytochrome C and myelin basic protein (MBP) respectively. Results In the vehicle group, the rats became paralyzed and spastic after injury, and the myelin sheath became swollen and broken down along with decreased number of myelinated nerve fibers. Western blot analysis manifested that active caspase-3, caspase-12 and cytochrome C began to increase 1 d after injury while the expression of MBP decreased gradually. After intervened with honokiol for 6 days, compared with the vehicle group, the locomotor function and the pathomorphological changes of myelin sheath of the CSCD rats were improved with obviously decreased expression of active caspase-3, caspase-12 and cytochrome C. Conclusions Honokiol may improve locomotor function and protect neural myelin sheat from demyelination via prevention oligodendrocytes (OLs) apoptosis through mediate endoplasmic reticulum (ER)-mitochondria pathway after CSCI.

Endurance exercise training suppresses Parkinson disease-induced overexpression of apoptotic mediators in the heart.

Thirty randomly selected normal albino mice were equally divided into the following 3 groups: sedentary control (SC), sedentary Parkinson diseased (SPD), and exercised Parkinson diseased (EPD). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p) were used to induce chronic Parkinson disease in the SPD and EPD animals. The expression of p53 and active caspase-3 was investigated, using immunohistochemistry, in the heart in each animal group. Both p53 and active caspase-3 expression was significantly (p value < 0.05) reduced in the PD heart following endurance exercise training. Our present data suggest that chronic exercise training reduced PD-induced upregulation of p53 and active caspase-3 in the heart. Thus, our study suggests that inhibiting p53 and/or active caspase-3 may be considered as a therapeutic approach to ameliorate PD cardiomyopathy.

Renal-targeting peptide-microRNA nanocomplex for near IR imaging and therapy of renal ischemia/reperfusion injury

Renal ischemia-reperfusion injury (RI/R) is one of the main causes of acute renal injury and a common clinical disease with high morbidity and mortality. It is of great significance to deliver microRNAs (miRNAs) to cells and in vivo to realize gene regulation and treatment of related diseases. In this study, we reported that the nanocomplex FMN-17 could realize both therapeutic and functional monitoring simultaneously in vivo and in vitro. The nanocomplex comprised a cationic cell-penetrating peptide nona-arginine, a targeting ligand folic acid, a caspase-3 responsive moiety, and a Cy imaging moiety. The nanocomplex FMN-17 has been shown to deliver miR-17-5p efficiently and selectively into HK-2 cells and tissues. Treatment of HK-2 cells with the nanocomplex significantly increased the miR-17-5p level and inhibited apoptosis, as evident by reducing the expression of active caspase-3 and reactive oxygen species. Uptake of FMN-17 in vivo alleviated renal tissue injury by histological assessment. In summary, we designed and synthesized a new miRNA delivery system with high transfection efficiency, good therapeutic effect, and near-infrared imaging in renal ischemia/reperfusion injury.

The nuclear transporter importin-11 regulates the Wnt/β-catenin pathway and acts as a tumor promoter in glioma

Karyopherins mediate the macromolecular transport between the cytoplasm and the nucleus and participate in cancer progression. However, the role and mechanism of importin-11 (IPO11), a member of the karyopherin family, in glioma progression remain undefined. Effects of IPO11 on glioma progression were detected using CCK-8, colony formation assay, flow cytometry analysis, caspase-3 activity assay, and Transwell invasion assay. Western blot analysis was used to detect the expression of active caspase-3, active caspase-7, active caspase-9, N-cadherin, Vimentin, E-cadherin, β-catenin, and c-Myc. The activity of Wnt/β-catenin pathway was evaluated by the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor reporter assay. Results showed that IPO11 knockdown inhibited proliferation and reduced colony number in glioma cells. IPO11 silencing promoted the apoptotic rate, increased expression levels of active caspase-3, caspase-7, and caspase-9, and enhanced caspase-3 activity. Moreover, IPO11 silencing inhibited glioma cell invasion by suppressing epithelial-to-mesenchymal transition (EMT). Mechanistically, IPO11 knockdown inactivated the Wnt/β-catenin pathway. β-Catenin overexpression abolished the effects of IPO11 silencing on the proliferation, apoptosis, and invasion in glioma cells. Furthermore, IPO11 silencing blocked the malignant phenotypes and repressed the Wnt/β-catenin pathway in vivo. In conclusion, IPO11 knockdown suppressed the malignant phenotypes of glioma cells by inactivating the Wnt/β-catenin pathway.

Visfatin protein may be responsible for suppression of proliferation and apoptosis in the infantile mice ovary

Visfatin is an important adipokines, which are expressed in different tissues including ovary of mammals. The postnatal ovary in rodents undergoes dramatic changes of intra-ovarian factors in relation to proliferation and apoptosis. There are studies which showed that gonadal visfatin changes in postnatal life. However, role of visfatin in the early postnatal period i.e. infantile period has not been studied. Therefore, the present study was aimed to explore the role of visfatin in the early postnatal ovarian functions. Furthermore, to explore the role of visfatin, the endogenous visfatin was inhibited from PND14-PND21 by FK866 with dose of 1.5 mg/kg. Our results showed gain in body weight and ovarian weight after visfatin inhibition. The inhibition of visfatin increased the ovarian proliferation (increase in PCNA, GCNA expression and BrdU incorporation) and apoptosis (increase in BAX and active caspase3 expression). Moreover, visfatin inhibition decreased the expression of antiapoptotic/survival protein, BCL2 in the ovary. These findings suggest that visfatin in the infantile ovary may suppress the proliferation and apoptosis by up-regulating BCL2 expression. An interesting finding has been observed that circulating estrogen and progesterone remain unaffected, although visfatin inhibition up-regulated ER-β and down-regulated ER-α. It may also be suggested that visfatin could regulates proliferation and apoptosis via modulating estrogen signaling. In conclusion, visfatin inhibits the proliferation and apoptosis without modulating the ovarian steroid biosynthesis and visfatin mediated BCL2 expression could also be mechanism to preserve the good quality follicle in early postnatal period.

Z-Guggulsterone Induces Apoptosis in Gastric Cancer Cells through the Intrinsic Mitochondria-Dependent Pathway.

Background To study the effects of z-guggulsterone on gastric cancer cell apoptosis and the mechanism related. Materials and Methods Human gastric tumor SGC-7901 cells and GES-1 normal epithelial cells were treated with z-guggulsterone (0–75 μM) for 24 h. MTT assay was applied to evaluate cell proliferation. Flow cytometry and Hoechst staining were used to assess cell apoptosis. Western blotting was applied to evaluate FXR, small heterodimer partner (SHP), Bcl-2, and Bax protein expression. ELISA was applied to gain the levels of active caspase-3 and the contents of TNF-α, TGF-β1, and VEGF. Results The expression levels of FXR and SHP were higher in tumor cells than in normal epithelial cells. Inhibition of FXR signaling with z-guggulsterone dose-dependently inhibited SGC-7901 cell proliferation and promoted SGC-7901 cell apoptosis. Bcl-2 protein expression was significantly decreased, and active caspase-3 and Bax protein expression was increased in SGC-7901 cells incubated with z-guggulsterone. The content of TNF-α was significantly increased, and the contents of VEGF and TGF-β1 were decreased in SGC-7901 cells incubated with z-guggulsterone. Conclusions Inhibition of FXR signaling with z-guggulsterone induced anticancer effects in SGC-7901 cells by decreasing cell proliferation and promoting apoptosis. Z-guggulsterone induced cell apoptosis through the mitochondria-dependent pathway.

Autophagy Activation by Resveratrol Reduces Severity of Experimental Rheumatoid Arthritis.

Previous work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen-induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response. Animals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule-associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin-1. Also, active caspase-3 expression, as well as poly(ADP-ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol-treated AIA group. Resveratrol also mitigates angiopoietin-1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL-1β, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression. Dietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross-talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.

Abstract 16549: Trimethylamine N-Oxide-Derived Endothelial Microvesicles Induce a Dysfunctional Endothelial Cell Phenotype

Introduction: Gut microbiota-generated increases in trimethylamine N-oxide (TMAO) has been linked to atherosclerosis and major adverse cardiovascular events. Experimental and clinical studies suggest that TMAO impairs endothelial cell function resulting in a proatherogenic endothelial phenotype. Endothelial microvesicles (EMVs) are anucleate vesicles shed constitutively by the endothelium aiding in cell-to-cell communication, activate repair or defense mechanisms, and/or stimulate immune responses. Under pathologic conditions, however, EMVs are released in greater number and their functional phenotype is more likely to evoke dysfunctional cellular effects. The experimental aims of this study were to determine: 1) if TMAO stimulates EMV release from endothelial cells in vitro ; and 2) the effects of TMAO-generated EMVs on endothelial cell inflammation, apoptosis, autophagy and nitric oxide (NO) production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with TMAO (100 μmol) for 24 h. EMVs released into the supernatant from cells treated with TMAO or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were treated with either TMAO-derived or control EMVs for 24 h. Results: EMV release was significantly higher in cells treated with TMAO compared with control (55±3 vs 28±3 EMV/μL). TMAO-generated EMVs induced significantly higher release of interleukin (IL)-6 (33.9±2.6 vs 20.2±1.3 pg/mL) and IL-8 (45.4±2.6 vs 33.1±1.6 pg/mL) and active NF-κB p65 (Ser536) (17.4±1.3 vs 7.7±1.0 AU) expression than control EMVs. TMAO EMVs significantly increased cell expression of apoptotic proteins caspase-9 (198.5±19.4 vs 119.7±11.2 AU) and active caspase-3 (17.7±2.8 vs 11.3±0.7 AU) and markedly depressed eNOS activity (10.6±0.9 vs 6.9±0.6 AU) and NO production (7.7±0.4 vs 5.5±0.6 μmol/L). In addition, cell autophagy was dysregulated by TMAO EMVs; cell expression of Beclin-1 (53.8±6.4 vs 36.6±3.6 AU), p62 (34.0±0.8 vs 16.9±1.2 AU) and LC3BII/LC3BI (15.0±0.1 vs 7.5±0.8 AU) were significantly elevated in cells treated with TMAO vs control EMVs. Conclusions: TMAO-generated EMVs adversely affect major functional characteristics of endothelial cells potentially contributing to the proatherogenic profile of TMAO.

Neuroepithelial cell competition triggers loss of cellular juvenescence

Cell competition is a cell–cell interaction mechanism which maintains tissue homeostasis through selective elimination of unfit cells. During early brain development, cells are eliminated through apoptosis. How cells are selected to undergo elimination remains unclear. Here we aimed to identify a role for cell competition in the elimination of suboptimal cells using an in vitro neuroepithelial model. Cell competition was observed when neural progenitor HypoE-N1 cells expressing RASV12 were surrounded by normal cells in the co-culture. The elimination through apoptosis was observed by cellular changes of RASV12 cells with rounding/fragmented morphology, by SYTOX blue-positivity, and by expression of apoptotic markers active caspase-3 and cleaved PARP. In this model, expression of juvenility-associated genes Srsf7 and Ezh2 were suppressed under cell-competitive conditions. Srsf7 depletion led to loss of cellular juvenescence characterized by suppression of Ezh2, cell growth impairment and enhancement of senescence-associated proteins. The cell bodies of eliminated cells were engulfed by the surrounding cells through phagocytosis. Our data indicates that neuroepithelial cell competition may have an important role for maintaining homeostasis in the neuroepithelium by eliminating suboptimal cells through loss of cellular juvenescence.

Correlation between the granulosa cell layer and active caspase-3 expression in ovarian follicles of Tropidurus hispidus and T. semitaeniatus (Squamata, Tropiduridae): immunohistochemical approach

The greatest threats to terrestrial reptiles are urban development and habitat modification. In this sense, a better understanding of folliculogenesis in these animals would be important to knowledge of reproductive biology. The aim of this study was to analyze the correlation between the thickness of the granulosa cell layer and the expression of the active caspase-3 protein in the previtellogenic and vitellogenic follicles of T. hispidus and T. semitaeniatus . Ovaries were used for histological (morphology and morphometry: thickness of granulosa layer) and immunohistochemical (active caspase-3 expression) analyses. The previtellogenic follicles of T. hispidus and T. semitaeniatus showed a thicker granulosa layer, with pyriform and small cells. The vitellogenic follicles had a monolayer of cuboid cells, and a thicker thecal layer. The thickness of the granulosa layer was significantly higher in the previtellogenic compared to the vitellogenic phase for both species. However, no differences were observed between the species. Active caspase-3 was observed in the pyriform and intermediate cells in previtellogenesis of T. hispidus and T. semitaeniatus . Nevertheless, no immunostaining was observed in the vitellogenic phase in both species. In conclusion, this study shows that the thickness of the granulosa cell layer is higher in the previtellogenic follicles compared to the vitellogenic follicles in the two Tropidurus species. Pyriform and intermediate cells from previtellogenic follicles show high expression of the protein, indicating that remodeling of the epithelium is associated with apoptosis. Finally, our results provide a scientific basis for assisted reproductive techniques and conservation actions to the reptiles in the future.

Isoflurane activates AMP-activated protein kinase to inhibit proliferation, and promote apoptosis and autophagy in cervical carcinoma both in vitro and in vivo

Objective Isoflurane is an extensively used inhalational anesthesia, and its carcinogenic or anti-cancerous effect has been identified recently. However, the specific role of isoflurane in cervical cancer remains unclear. Aim This study aimed to investigate the function of isoflurane in cervical cancer as well as the underlying mechanism. Methods After isoflurane treatment, HeLa cell viability, percentage of apoptotic cells, expression of active caspase-3/9 were examined by CCK-8 assay, Annexin V-FITC/PI double staining, and Western blot analysis, respectively. ROS generation, ratio of NAD+/NADH, and ATP level after isoflurane stimulation were determined using commercial assay kits. Afterwards, activation of AMPK and autophagy was assessed through Western blot analysis and immunofluorescence. Whether AMPK mediated the isoflurane-induced apoptosis and autophagy was explored by adding an AMPK inhibitor (Compound C). The in vivo function of isoflurane was finally investigated on a HeLa cell xenograft model. Results Isoflurane inhibited cell viability and induced apoptosis evidenced by upregulation of active caspase-3/9 in HeLa cells. Oxidative stress was triggered by isoflurane, as isoflurane elevated ROS level, and lowered ratio of NAD+/NADH and ATP level. Further results showed isoflurane activated the AMPK/mTOR pathway and induced autophagy. In addition, inhibition of AMPK led to ameliorated effects of isoflurane on apoptosis and autophagy. In vivo experiments proved isoflurane could repress tumorigenesis, activate AMPK, and induce autophagy in Xenograft mouse. Conclusions Isoflurane activated AMPK to inhibit proliferation and promote apoptosis and autophagy both in vitro and in vivo.

MicroRNA-204-5p mediates sevoflurane-induced cytotoxicity in HT22 cells by targeting brain-derived neurotrophic factor.

Sevoflurane is widely used as an inhalational anesthetic in clinical practice. However, sevoflurane can cause cytotoxicity and induce learning capacity decline in patients. A previous publication indicated that miR-204-5p might have a close relationship with sevoflurane-induced neurotoxicity. When exposed to sevoflurane, the expression of miR-204-5p in neonatal hippocampus of rats was significantly increased. Hence, we aimed to investigate the role of miR-204-5p in sevoflurane-induced neurotoxicity using a mouse hippocampal neuronal cell line (HT22). The levels of miR-204-5p in HT22 cells were detected by RT-qPCR. In addition, the effects of miR-204-5p on cell viability, apoptosis and proliferation were evaluated by CCK-8, flow cytometric, and immunofluorescence assay, respectively. Western blotting was used to detect expressions of Bax, Bcl-2, active caspase 3, BDNF, TrkB, p-TrkB, Akt and p-Akt in HT22 cells. ELISA assay was used to examine the levels of total superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and reactive oxygen species (ROS) in cells. Meanwhile, the dual luciferase reporter system assay was employed to explore the interaction of miR-204-5p and BDNF in cells. The level of miR-204-5p was increased in sevoflurane-treated HT22 cells. Moreover, downregulation of miR-204-5p inhibited sevoflurane-induced apoptosis and promoted cell proliferation by upregulating the proteins of Bcl-2 and downregulating the expressions of Bax and active caspase-3 in HT22 cells. In addition, inhibition of miR-204-5p alleviated sevoflurane-induced oxidative injuries in HT22 cells via decline of ROS and MDA and upregulation of SOD and GSH. Furthermore, bioinformatics and dual luciferase assay demonstrated that miR-204-5p can inhibit the TrkB/Akt pathway by targeting BDNF. Our findings indicated that downregulation of miR-204-5p can decrease oxidative status in HT22 cells and alleviate sevoflurane-induced cytotoxicity through stimulating the BDNF/TrkB/Akt pathway. Therefore, miR-204-5p might be a potential biomarker and therapeutic target for the treatment of sevoflurane-induced neurotoxicity.

Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.

In vivo anti-V-ATPase antibody treatment delays ovarian tumor growth by increasing antitumor immune responses.

Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the invivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. Invitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. Invivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable invivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the invivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.

Rat postnatal prostate development is impaired by in vitro high-glucose environment.

The prostate development has an important postnatal period where cell proliferation begins at the first days after birth and is related to gland growth and ramification. Any metabolic and/or hormonal changes occurring during the postnatal period can interfere with prostate branching. Hyperglycemia is a common condition in low-weight preterm babies at neonatal period and also a disorder found in the offspring of obese mothers. Thus, this study aimed to investigate the in vitro effects of a glucose-rich environment during prostate postnatal development. Wistar rats prostate were removed at birth and cultured for 1, 2 and 3 days in DMEM under normal (5.5 mM) or elevated (7 and 25 mM) glucose concentrations. Samples were processed for morphological analysis, PCNA and smooth muscle α-actin immunohistochemistry, evaluation of active caspase-3, ERK1/2 and Wnt5a gene expression. High glucose concentrations reduced the number of prostatic buds and proliferating cells. The natural increase in smooth muscle cells and collagen deposition observed in control prostates during the first 3 days of development was reduced by elevated glucose concentrations. The amount of active caspase-3 was higher in prostates incubated at 7 mM and TGF-β levels also increased sharply after both glucose concentrations. Additionally, high glucose environment decreased ERK 1/2 activation and increased Wnt5a expression. These data show that high levels of glucose during the first postnatal days affected prostate development by inhibiting cell proliferation which impairs bud branching and this was associated with anti-proliferative signals such as decreased ERK1/2 activation and increased Wnt5a expression.

Polydatin attenuates hepatic stellate cell proliferation and liver fibrosis by suppressing sphingosine kinase 1

Sphingosine kinase 1 (SphK1) plays critical roles in the activation of hepatic stellate cells (HSCs) and liver fibrosis. Our previous study found that polydatin ameliorates chronic liver injury and fibrosis by inhibiting oxidative stress. However, whether polydatin exerts an anti-fibrotic effect on liver fibrosis dependent on SphK1 signaling is unknown. We aimed to investigate the role of polydatin in SphK1, which mediates HSC activation and liver fibrosis. C57BL/6 mice were induced using CCl4 5 μL g−1 i.p. twice a week for 6 weeks and treated with or without polydatin. Human immortalized HSC line (LX-2) was induced using platelet-derived growth factor-BB (PDGF-BB) or adenovirus-SphK1 and treated with polydatin. Hepatic macrophage filtration, collagen deposition, expression of α-smooth muscle, active caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were markedly increased in mice induced by CCl4 for 6 weeks. In contrast, polydatin attenuated collagen synthesis and hepatocyte apoptosis. Furthermore, polydatin exhibited significant anti-proliferative activity against PDGF-BB-induced activated hepatic stellate cells (HSCs). SphK1 was strongly induced in mice exposed to CCl4, whereas its expression and activity were inhibited by polydatin treatment. Finally, SphK1 overexpression in LX-2 cells promoted proliferation of activated HSCs, which could not be reversed by polydatin treatment. These results demonstrate that polydatin attenuates HSC proliferation and activation through inhibition of SphK1 signaling, contributing to the suppression of liver fibrosis.

Deletion of the adenosine A2A receptor increases the survival rate in a mice model of polymicrobial sepsis.

We aim to investigate the role of A2A receptor in peritonitis-related sepsis by injection of a fecal solution (FS) as a model of polymicrobial infection. C57/black J6 wild-type (WT) and A2A-deficient mice (A2AKO) were exposed to sepsis induced by intraperitoneal injection of a FS (FS-induced peritonitis) or instead was injected with saline buffer (Sham). Survival rate and sepsis score were measured up to 48h. The presence of bacteria in tissue homogenates was analyzed. Telemetry and speckle laser Doppler were used for systemic blood pressure and peripheral blood perfusion analysis, respectively. Histological analysis and identification of active caspase 3 were performed in selected organs, including the liver. The survival rate of A2AKO mice exposed to FS-induced peritonitis was significantly higher, and the sepsis score was lower than their respective WT counterpart. Injection of FS increases (50 to 150 folds) the number of colonies forming units in the liver, kidney, blood, and lung in WT mice, while these effects were significantly attenuated in A2AKO mice exposed to FS-induced peritonitis. A significant reduction in both systolic and diastolic blood pressure, as well as in the peripheral perfusion was observed in WT and A2AKO mice exposed to FS-induced peritonitis. Although, these last effects were significantly attenuated in A2AKO mice. Histological analysis showed a large perivascular infiltration of polymorphonuclear in the liver of WT and A2AKO mice exposed to FS-induced peritonitis, but again, this effect was attenuated in A2AKO mice. Finally, high expression of active caspase 3 was found only in the liver of WT mice exposed to FS-induced peritonitis. The absence of the A2A receptor increases the survival rate in mice exposed to polymicrobial sepsis. This outcome was associated with both hemodynamic compensation and enhanced anti-bacterial response.

Dietary methionine restriction attenuates renal ischaemia/reperfusion-induced myocardial injury by activating the CSE/H2S/ERS pathway in diabetic mice.

Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)‐induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine‐restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R‐induced mediated myocardial injury and apoptosis were partially reversed by methionine‐restricted diet (MRD) via the down‐regulation of CSE expression and up‐regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose‐dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R‐induced myocardial injury.

Potent Therapy and Transcriptional Profile of Combined Erythropoietin-Derived Peptide Cyclic Helix B Surface Peptide and Caspase-3 siRNA against Kidney Ischemia/Reperfusion Injury in Mice.

Cause-specific treatment and timely diagnosis are still not available for acute kidney injury (AKI) apart from supportive therapy and serum creatinine measurement. A novel erythropoietin-derived cyclic helix B surface peptide (CHBP) protects kidneys against AKI with different causes, but the underlying mechanism is not fully defined. Herein, we investigated the transcriptional profile of renoprotection induced by CHBP and its potential synergistic effects with siRNA targeting caspase-3, an executing enzyme of apoptosis and inflammation (CASP3siRNA), on ischemia/reperfusion (IR)-induced AKI. Utilizing a mouse model with 30-minute renal bilateral ischemia and 48-hour reperfusion, the renoprotection of CHBP or CASP3siRNA was demonstrated in renal function and structure, active caspase-3 and HMGB1 expression. Combined treatment of CHBP and CASP3siRNA further preserved kidney structure and reduced active caspase-3 and HMGB1. Furthermore, differentially expressed genes (DEGs) were identified with fold change >1.414 and P < 0.05. In IR kidneys, 281 DEGs induced by CHBP were mainly involved in promoting cell division and improving cellular function and metabolism (upregulated signal transducer and activator of transcription 5B and solute carrier family 22 member 7). The additional administration of CASP3siRNA caused 504 and 418 DEGs in IR + CHBP kidneys with or without negative control small-interfering RNA, with 37 genes in common. These DEGs were associated with modulated apoptosis and inflammation (upregulated BCL6, SLPI, and SERPINA3M) as well as immunity, injury, and microvascular homeostasis (upregulated complement factor H and GREM1 and downregulated ANGPTL2). This proof-of-effect study indicated the potent renoprotection of CASP3siRNA upon CHBP at the early stage of IR-induced AKI. Underlying genes, BCL6, SLPI, SERPINA3M, GREM1, and ANGPTL2, might be potential new biomarkers for clinical applications. SIGNIFICANCE STATEMENT: It is imperative to explore new strategies of cause-specific treatment and timely diagnosis for acute kidney injury (AKI). CHBP and CASP3siRNA synergistically protected kidney structure after 48-hour ischemia/reperfusion-induced AKI with reduced injury mediators CASP3 and high mobility group box 1. CHBP upregulated cell division-, function-, and metabolism-related genes, whereas CASP3siRNA further regulated immune response- and tissue homeostasis-associated genes. Combined CHBP and CASP3siRNA might be a potent and specific treatment for AKI, and certain dysregulated genes secretory leukocyte peptidase inhibitor and SERPINA3M could facilitate timely diagnosis.