P53 mRNA Levels Research Articles

Evaluation of Effect of Biologically Synthesized Ethanolic Extract of Propolis-Loaded Poly(-Lactic-co-Glycolic Acid) Nanoparticles on Wound Healing in Diabetic Rats.

Wound healing is interaction of a complex cascade of cellular/biochemical actions leading to restoration of structural and functional integrity with regain of injured tissues strength. This study was aimed at evaluation of application of ethanolic extract of propolis-loaded poly(-lactic-co-glycolic acid) nanoparticles (EEP-PLGA NPs) on wound healing in diabetic rats. Sixty rats were randomized into four groups of 15 rats each: In control group (Control) diabetic wound was treated with normal saline. In Carrier 1 group diabetic wound was treated with PLGA nanoparticles based solution. In Carrier 2 group the diabetic wound was treated with EEP. In Treatment group animals received EEP-PLGA NPs on the wound. Wound size was measured on 7, 14 and 21 days after surgery. The expression of p53, bcl-2, Caspase III, were evaluated using reverse-transcription PCR and Immunohistochemical staining. The Treatment group had significantly reduced the wound size compared to other groups (P = 0.001). histological and morphometric studies, and mean rank of the qualitative studies demonstrated that there was significant difference between Treatment group and other groups (P < .05). Observations demonstrated that ethanolic extract of propolis-loaded PLGA nanoparticles significantly shortened the inflammatory phase and accelerated the cellular proliferation. Accordingly, the animals in Treatment group revealed significantly (P < .05) higher fibroblast distribution/one mm2 of wound area and rapid re epithelialization. The mRNA levels of bcl-2, p53 and caspase III were remarkably (P < .05) higher in Treatment group compared to control and animals. The immunohistochemical analyzes confirmed the RT-PCR findings. EEP-PLGA NPs offered potential advantages in wound healing acceleration and improvement through angiogenesis stimulation, fibroblast proliferation and granulation tissue formation in early days of healing phases, acceleration in diabetic wound repair associated with earlier wound contraction and stability of damaged area by rearrangement of granulation tissue and collagen fibers.

Exposure of Toluene Diisocyanate Induces DUSP6 and p53 through Activation of TRPA1 Receptor.

Toluene diisocyanate (TDI), a major intermediate agent used in the manufacturing industry, causes respiratory symptoms when exposed to the human body. In this study, we aimed to determine the molecular mechanism of TDI toxicity. To investigate the impact of TDI exposure on global gene expression, we performed transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) after TDI treatment. Differentially expressed genes (DEGs) were sorted and used for clustering and network analysis. Among DEGs, dual-specificity phosphatase 6 (DUSP6) was one of the genes significantly changed by TDI exposure. To verify the expression level of DUSP6 and its effect on lung cells, the mRNA and protein levels of DUSP6 were analyzed. Our results showed that DUSP6 was dose-dependently upregulated by TDI treatment. Thereby, the phosphorylation of ERK1/2, one of the direct inhibitory targets of DUSP6, was decreased. TDI exposure also increased the mRNA level of p53 along with its protein and activity which trans-activates DUSP6. Since TRPA1 is known as a signal integrator activated by TDI, we analyzed the relevance of TRPA1 receptor in DUSP6 regulation. Our data revealed that up-regulation of DUSP6 mediated by TDI was blocked by a specific antagonist against TRPA1. TDI exposure attenuated the apoptotic response, which suggests that it promotes the survival of cancerous cells. In conclusion, our results suggest that TDI induces DUSP6 and p53, but attenuates ERK1/2 activity through TRPA1 receptor activation, leading to cytotoxicity.

Safinamide protects against amyloid β (Aβ)-induced oxidative stress and cellular senescence in M17 neuronal cells

ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disorder that is pathologically related to oxidative stress and cellular senescence. Safinamide is one of the clinically prescribed monoamine oxidase B (MAOB) inhibitors. It has been reported to possess therapeutic potential in neurological disorders. However, the therapeutic potential of safinamide in AD is still under investigation. In this study, we explored the effect of safinamide in amyloid (Aβ)1–42 oligomers-stimulated M17 neuronal cells. We established the in vitro model with M17 cells by treating them with 1 μM Aβ1-42 oligomers with or without safinamide (100 or 200 nM). The results show that safinamide ameliorated Aβ1-42 oligomers-induced oxidative stress in M17 cells as revealed by the decreased reactive oxygen species (ROS) production and reduced glutathione (GSH) content. Safinamide treatment significantly ameliorated senescence-associated-β-galactosidase (SA-β-gal)-positive cells and telomerase activity. Further, we show that safinamide treatment resulted in decreased mRNA and protein expressions of p21 and plasminogen activator inhibitor-1 (PAI-1). Moreover, silencing of Sirtuin1 (SIRT1) abolished the effects of safinamide on the mRNA levels of p21 and PAI-1, as well as SA-β-gal-positive cells in Aβ1-42 oligomers-induced M17 cells. In conclusion, we reveal that safinamide exerted a protective function on M17 cells from Aβ1-42 oligomers induction-caused oxidative stress and cellular senescence through SIRT1 signaling. These present results provide meaningful evidence that safinamide may be medically developed for the prevention and therapy of AD.

In vivo assessment of molybdenum and cadmium co-induce nephrotoxicity via causing calcium homeostasis disorder and autophagy in ducks (Anas platyrhyncha)

Excess molybdenum (Mo) and cadmium (Cd) are widespread environmental and industrial metal pollutants. To evaluate the combined effects of Mo and Cd on calcium homeostasis and autophagy in duck kidneys. 160 healthy 7-day-old ducks (Anas platyrhyncha) were randomized into 4 groups and given to a basic diet, adding various doses of Mo or/and Cd for 16 weeks. On the 4th, 8th, 12th and 16th weeks, kidney tissues were collected. The study exhibited that Mo or/and Cd caused histological abnormality, reduced the activities of Ca2+ ATPase, Mg2+ ATPase, Na+-K+ ATPase and Ca2+-Mg2+ ATPase, K and Mg contents, and increased Na and Ca contents, upregulated CaMKKβ, CaMKIIɑ, CaN, IP3R, GRP78, GRP94, CRT mRNA levels and CaMKIIɑ, CaN, IP3R protein levels. Moreover, exposure to Mo or/and Cd notably promoted the amount of autophagosomes and LC3II immunofluorescence, upregulated AMPKα1, ATG5, Beclin-1, LC3A, LC3B mRNA levels and Beclin-1, LC3II/LC3I protein levels, downregulated mTOR, Dynein, P62 mRNA levels and P62 protein level. The changes of above indicators in combined group were more obvious. Overall, the results suggest that Mo and Cd co-exposure may can synergistically induce nephrotoxicity via causing calcium homeostasis disorder and autophagy in ducks.

Sanguinarine Reverses Pulmonary Vascular Remolding of Hypoxia-Induced PH via Survivin/HIF1α-Attenuating Kv Channels.

Background: Similarities in the biology of pulmonary hypertension and cancer suggest that anticancer therapies, such as sanguinarine, may also be effective in treating pulmonary hypertension. This, along with underlying biochemical pathways, is investigated in this study. Methods: Rats were subjected to 4-week hypoxia (or control) with or without sanguinarine treatment. In addition, pulmonary artery smooth muscle cells (PASMCs) were examined after 24–48 h hypoxia (with normoxic controls) and with or without sanguinirine. Pulmonary artery pressures and plasma survivin levels were measured in vivo. Ex vivo tissues were examined histologically with appropriate staining. mRNA and protein levels of survivin, HIF-1α, TGFb1, BMPR2, Smad3, P53, and Kv 1.2, 1.5, 2.1 were determined by real-time PCR and Western blot in PASMCs and distal PAs tissue. PASMC proliferation and changes of TGFb1 and pSmad3 induced by sanguinarine were studied using MTT and Western blot. Electrophysiology for Kv functions was measured by patch-clamp experiments. Results: Four-week hypoxia resulted in an increase in serum survivin and HIF-1α, pulmonary artery pressures, and pulmonary vascular remodeling with hypertrophy. These changes were all decreased by treatment with sanguinarine. Hypoxia induced a rise of proliferation in PASMCs which was prevented by sanguinarine treatment. Hypoxic PASMCs had elevated TGFb1, pSmad3, BMPR2, and HIF1α. These increases were all ameliorated by sanguinarine treatment. Hypoxia treatment resulted in reduced expression and function of Kv 1.2, 1.5, 2.1 channels, and these changes were also modulated by sanguinarine. Conclusion: Sanguinarine is effective in modulating hypoxic pulmonary vascular hypertrophy via the survivin pathway and Kv channels.

Sidt2 is a key protein in the autophagy-lysosomal degradation pathway and is essential for the maintenance of kidney structure and filtration function

The regulation and homeostasis of autophagy are essential for maintaining organ morphology and function. As a lysosomal membrane protein, the effect of Sidt2 on kidney structure and renal autophagy is still unknown. In this study, we found that the kidneys of Sidt2−/− mice showed changes in basement membrane thickening, foot process fusion, and mitochondrial swelling, suggesting that the structure of the kidney was damaged. Increased urine protein at 24 h indicated that the kidney function was also damaged. At the same time, the absence of Sidt2 caused a decrease in the number of acidic lysosomes, a decrease in acid hydrolase activity and expression in the lysosome, and an increase of pH in the lysosome, suggesting that lysosomal function was impaired after Sidt2 deletion. The accumulation of autophagolysosomes, increased LC3-II and P62 protein levels, and decreased P62 mRNA levels indicated that the absence of the Sidt2 gene caused abnormal autophagy pathway flow. Chloroquine experiment, immunofluorescence autophagosome, and lysosome fusion assay, and Ad-mcherry-GFP-LC3B further indicated that, after Sidt2 deletion, the production of autophagosomes did not increase, but the fusion of autophagosomes and lysosomes and the degradation of autophagolysosomes were impaired. When incubating Sidt2−/− cells with the autophagy activator rapamycin, we found that it could activate autophagy, which manifested as an increase in autophagosomes, but it could not improve autophagolysosome degradation. Meanwhile, it further illustrated that the Sidt2 gene plays an important role in the smooth progress of autophagolysosome processes. In summary, the absence of the Sidt2 gene caused impaired lysosome function and a decreased number of acidic lysosomes, leading to formation and degradation disorders of the autophagolysosomes, which eventually manifested as abnormal kidney structure and function. Sidt2 is essential in maintaining the normal function of the lysosomes and the physiological stability of the kidneys.

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5'-Terminal Region of p53 mRNA.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3′ end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5′ terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

Effects of K-ras gene silence on the expression of oncogenes in HBE cells treated with PM(2.5)

Objective: To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial (HBE) cells which were exposed to PM(2.5). Methods: According to the mRNA sequence of K-ras gene provided by GenBank in September 2019, interference sequences were designed and synthesized, and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells. HBE cells and K-ras gene-silenced cells were exposed to 10 μg/ml, 50 μg/ml PM(2.5) suspension and 10 μmol/L Cr(6+). Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc, c-fos, N-ras, cyclin-D1, p16 and p53 genes, the expression levels of p53 and c-myc proteins were detected by Western blot. Results: In K-ras silenced cell group, K-ras mRNA expression level decreased (80.5%±3.6%) and K-ras protein level decreased (58.9%±4.7%) when compared with the control group (P<0.01) . Compared with the correspoding cell control group without exposure, the mRNA expression levels of c-myc, c-fos, N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM(2.5), K-ras silenced cell group exposed to different concentrations of PM(2.5), HBE cell group exposed to 10 μmol/L Cr(6+) and K-ras silenced cell group exposed to 10 μmol/L Cr(6+) were increased, the mRNA expressions of p16 and p53 genes were decreased (P<0.01) . Compared with HBE cell group exposed to 10 μg/ml PM(2.5), the mRNA expressions of c-myc, c-fos and p16 genes in K-ras silenced cells exposed to 10 μg/ml PM(2.5) were decreased, and the p53 mRNA level was increased (P<0.01) . Compared with HBE cell group exposed to 50 μg/ml PM(2.5), the mRNA expression levels of c-fos, N-ras, cyclin-D1, p16 and p53 genes in K-ras silenced cell group exposed to 50 μg/ml PM(2.5) were decreased (P<0.01) . Compared with the HBE cell group without exposure, c-myc protein increased and p53 protein decreased in HBE cells exposed to 50 μg/ml PM(2.5) (P<0.05) . Compared with the K-ras silenced cell group without exposure, c-myc protein increased in K-ras silenced cells exposed to 50 μg/ml PM(2.5) (P<0.05) . Conclusion: PM(2.5) can increase the expression levels of oncogenes in HBE cells, and K-ras gene silencing can inhibit the expression levels of oncogenes in HBE cells treated with PM(2.5).

Isoliensinine induces cervical cancer cell cycle arrest and apoptosis by inhibiting the AKT/GSK3α pathway.

Isoliensinine is a bis-benzylisoquinoline alkaloid that can be isolated from the lotus Nelumbo nucifera Gaertn. It has been reported to exert a variety of anti-cancer properties. In the present study, the potential effects of isoliensinine on cervical cancer Siha, HeLa, Caski and C33A cell lines were investigated by using Cell Counting Kit-8 (CCK-8), flow cytometry, western blotting and reverse transcription-PCR (RT-PCR) to measure cell proliferation, the cell cycle and apoptosis, in addition to elucidating the underlying molecular mechanism. Protein levels of p21, CDK2, Cyclin E, Mcl-1, cleaved Caspase-9, AKT, phosphorylated-AKT, glycogen synthase kinase (Gsk)3α, PTEN, and mRNA levels of p21, p15, p27, CDK2, CDK4, Cyclin E, Cyclin D, Gsk3α, Gsk3β and PTEN were measured. Molecular docking assays were used to calculate the strength of binding of isoliensinine to AKT using AutoDock 4.0. Isoliensinine was found to induce cell cycle arrest at the G0/G1 phase by upregulating p21 expression and downregulating CDK2 and cyclin E in cervical cancer cells. In addition, in previous research, isoliensinine promoted cell apoptosis by downregulating myeloid-cell leukemia 1 expression and activating caspase-9. Upstream, isoliensinine significantly downregulated AKT (S473) phosphorylation and GSK3α expression in a dose- and time-dependent manner. The AKT inhibitor AKTi-1/2 enhanced the function of isoliensinine on cell cycle arrest and apoptosis through the AKT/GSK3α pathway. AutoDock analysis showed that isoliensinine can bind to the AKT protein. These findings suggest that isoliensinine can induce cervical cancer cell cycle arrest and apoptosis by inhibiting the AKT/GSK3α pathway, which represents a novel strategy for the treatment of cervical cancer.

Fluoride induced down-regulation of IKBKG Gene expression inhibits hepatocytes senescence

BackgroundAccumulating evidences have confirmed that liver is one of the more severely damaged organs during chronic fluorosis. However, the detail mechanism is unclear to data. At present, the objective of this study was to investigate the relationship between down-regulation of IKBKG gene expression and hepatocyte senescence induced by sodium fluoride (NaF). MethodsChronic fluorosis rats and NaF-exposure human liver L02 cells were reproduced the model of hepatocyte senescence in vivo and in vitro. The mRNA and protein levels of p16, p21 and IKBKG, the IL-8 level were determined. The role of IKBKG in fluoride-induced senescence of hepatocytes was explored by knock down in hepatocytes in vivo and in vitro. ResultsThe number of senescence-positive cells in rat liver tissues was increased as well as the level of IL-8 and the expression levels of p16, p21 and IKBKG in fluoride exposure to rat depending on the fluoride concentration. The similar results were obtained in NaF treated liver L02 cells, and the number of cells that stagnated in the G2 phase increased significantly. Further, our results confirmed that decreasing the expression of IKBKG in hepatocytes could reduce fluoride-induced hepatocyte senescence and the changes of senescence-related indicators both in vivo and in vitro. ConclusionThese results indicated that the elevated expression of IKBKG was positive relation with the fluoride-induced senescence in hepatocytes, suggesting the hepatocyte senescence might have a special relationship with fluoride-caused liver damage. Because of the present results limitation, the mechanism of fluoride induced senescence in hepatocytes should be concentrated in the future in detail, especially the novel targets for fluoride induced liver injury.

Protective effect of small peptides from Periplaneta americana on cyclophosphamide-induced premature ovarian failure.

To investigate the protective effect of small peptides from Periplaneta americana (SPPA) on cyclophosphamide (CP)-induced premature ovarian failure (POF) in mice. Silent mating type information regulation 2 homolog 1 (SIRT1) /tumor-associated protein 53 (p53) signaling pathway plays an important role in delaying POF. Hematopoietic progenitor cell antigen (CD34) reflects ovarian aging from the side. However, whether SPPA inhibits POF in mice by influencing the SIRT1/p53 pathway and CD34 expression remains to be studied. Forty female Kun Ming (KM) mice were divided into four groups: a control group (normal saline, n=10), POF model group (160 mg/kg CP, n=10), SPPA low-dosage group (160 mg/kg CP + 100 mg/kg SPPA, n=10), and SPPA high-dosage group (160 mg/kg CP + 200 mg/kg SPPA, n=10). CP administration route is intraperitoneal injection, and SPPA administration route is intragastric. Eyeball enucleation blood samples and the ovaries of mice were collected by midline laparatomy and oopherectomy, and the malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) concentrations were tested. Immunohistochemical tests for the expressions of SIRT1, p53, and CD34 were carried out. Finally, ovarian mRNA levels of SIRT1 and p53 were detected with real-time fluorescence quantification PCR (qRT-PCR). A mouse model of POF was generated using 160 mg/kg of CP. Compared with POF group, we found that plasma NO, MDA, and FSH decreased, while AMH and SOD increased in the SPPA low-dose group. Compared with the POF group, the SPPA low- and high-dosage groups achieved significant growth in the number of primordial, primary, and total number of healthy follicles at all levels, but sharp reductions in the number of atretic follicles. In addition, we found downregulated protein and mRNA expression of SIRT1, and upregulated that of p53 were observed in ovarian tissues of treated mice with POF, in immunohistochemistry experiments and qPCR experiments. In contrast, high protein and mRNA expression of SIRT1, and low that of p53 were observed in SPPA treatment groups. And the results of CD34 protein expression were consistent with that of SIRT1. In total, SPPA significantly inhibited POF caused by CP in mice via activation of the SIRT1/p53 signaling pathway in the mouse ovary.

Targeted Nanotherapeutics Using LACTB Gene Therapy Against Melanoma.

Introductionβ-lactamase (LACTB) is a tumor suppressor gene in various tumors including melanoma. However, it remains challenging to efficiently deliver the LACTB gene into melanoma. Recently, we designed a nonviral nanocarrier iRGD/DOTAP/MPEG-PDLLA (iDPP) that could deliver gene targetedly to melanoma efficiently without obvious adverse effects.MethodsIn this study, the tumor-targeted nanoparticle iDPP was prepared to deliver LACTB gene to treat melanoma in vitro and in vivo. First, the expression level of LACTB in 6 clinical specimens of melanoma patients was evaluated. Subsequently, the characteristics of iDPP/LACTB nanocomplexes were studied. Afterwards, the in vitro and in vivo anti-tumor efficacy of the iDPP/LACTB nanocomplexes were explored utilizing the B16-F10 mouse melanoma cell line and the B16-F10 subcutaneous melanoma model.ResultsCompared with the normal epithelium, the expression level of LACTB in melanoma tissues was significantly downregulated. In vitro B16-F10 cell tests showed iDPP/LACTB nanocomplexes could increase the mRNA levels of P21, Bid, Bax, Pidd1, and Sival genes and up-regulate the p53 signaling pathway of melanoma cells, thus promoting cell apoptosis and blocking the cell cycle. Injected intravenously, iDPP nanoparticles could deliver DNA to the subcutaneous melanoma targetedly. Based on in vivo mouse xenograft model, iDPP/LACTB nanocomplexes could effectively inhibit tumor proliferation and induce tumor apoptosis, thus significantly inhibiting melanoma growth (tumor inhibition rate is about 68%) in the subcutaneous B16-F10 melanoma model.ConclusionThe downregulated LACTB might be a potential target for melanoma therapy. The iDPP/LACTB nanocomplexes could inhibit the growth of the mouse melanoma without obvious side effects, which provide a new option for melanoma gene therapy research.

Research on Mechanism of miRNA-22 Related with Hepatocellular Carcinoma Metastasis for Regulating Process of Tumor Protein P53

The action of miRNA-22 related with HCC metastasis was analyzed in our study and the mechanism of miRNA-22 related with HCC metastasis was discussed. The HCC hep2 cell was transfected with miRNA-22 mimics and miRNA-22 NC instantaneously followed by analysis of cell migration by Transwell assay, cell viability by MTT and clone formation and cell apoptosis by flow cytometry. The action of miRNA-22 mimics and miRNA-22 on the expression of P53 mRNA in HCC Hep2 cell was detected by RT-PCR. The cell activity in miRNA-22 mimics group was significantly elevated compared with miRNA-22 NC group (P &lt; 0.01). Meanwhile, the apoptotic rate, migrated and invaded capacity of HCC cell was significantly elevated (P &lt; 0.01). The expression level of P53 mRNA was reduced (P &lt; 0.01). In conclusion, overexpression of miRNA-22 could restrain the apoptosis of HCC hep2 cell and down-regulated the expression of P53 so as to prompt cell invasion capacity.

Anti-Proliferative and Pro-Apoptotic Activities of Synthesized 3,4,5 Tri-Methoxy Ciprofloxacin Chalcone Hybrid, through p53 Up-Regulation in HepG2 and MCF7 Cell Lines.

Background:Cancer is a significant health problem around the world and one of the leading causes of human death. The need for novel, selective and non-toxic anti-cancer agents is still urging. Aim of the work: to investigate the anti-proliferative and pro-apoptotic effects of the synthesized ciprofloxacin 3,4,5 tri-methoxy chalcone hybrid (CCH) on the HepG2 hepatocellular carcinoma and MCF7 breast carcinoma cell lines. Materials and Methods:HepG2 and MCF7cell lines were treated with CCH. Cell viability and cell cycle analysis were performed. Protein and mRNA expression levels of P53, COX-2 and TNF-α were analyzed by western blotting and RT-PCR respectively. Results:CCH caused concentration and time-dependent reduction in the viability of human HepG2 and MCF7 cells, pre-G1 apoptosis and cell cycle arrest at G2/M stage, significantly higher P53 and TNF-α mRNA and protein expression levels but significantly lower COX2 mRNA and protein expression levels. Conclusion:CCH showed obvious anti-proliferative and apoptosis-inducing activities in both cell lines.

Nobiletin, a hexamethoxyflavonoid from citrus pomace, attenuates G1 cell cycle arrest and apoptosis in hypoxia-induced human trophoblast cells of JEG-3 and BeWo via regulating the p53 signaling pathway.

BackgroundHypoxia is associated with abnormal cell apoptosis in trophoblast cells, which causes fetal growth restriction and related placental pathologies. Few effective methods for the prevention and treatment of placenta-related diseases exist. Natural products and functional foods have always been a rich source of potential anti-apoptotic drugs. Nobiletin (NOB), a hexamethoxyflavonoid derived from the citrus pomace, shows an anti-apoptotic activity, which is a non-toxic constituent of dietary phytochemicals approved by the Food and Drug Administration. However, their effects on hypoxia-induced human trophoblast cells have not been fully studied.ObjectiveThe aim of this study was to investigate the protective effects of NOB on hypoxia-induced apoptosis of human trophoblast JEG-3 and BeWo cells, and their underlying mechanisms.DesignFirst, the protective effect of NOB on hypoxia-induced apoptosis of JEG-3 and BeWo cells was studied. Cell viability and membrane integrity were determined by CCK-8 assay and lactate dehydrogenase activity, respectively. Real Time Quantitative PCR (RT-qPCR) and Western blot analysis were used to detect the mRNA and protein levels of HIF1α. Propidium iodide (PI)-labeled flow cytometry was used to detect cell cycle distribution. Cell apoptosis was detected by flow cytometry with Annexin V-FITC and PI double staining, and the expression of apoptosis marker protein cl-PARP was detected by Western blot analysis. Then, the molecular mechanism of NOB against apoptosis was investigated. Computer molecular docking and dynamics were used to simulate the interaction between NOB and p53 protein, and this interaction was verified in vitro by Ultraviolet and visible spectrum (UV-visible spectroscopy), fluorescence spectroscopy and circular dichroism. Furthermore, the changes in the expression of p53 signaling pathway genes and proteins were detected by RT-qPCR and Western blot analysis, respectively.ResultsHypoxia treatment resulted in a decreased cell viability and cell membrane integrity in JEG-3 and BeWo cell lines, and an increased expression of HIF1α, cell cycle arrest in the G1 phase, and massive cell apoptosis, which were alleviated after NOB treatment. Molecular docking and dynamics simulations found that NOB spontaneously bonded to human p53 protein, leading to the change of protein conformation. The intermolecular interaction between NOB and human p53 protein was further confirmed by UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism. After the treatment of 100 μM NOB, a down-regulation of mRNA and protein levels of p53 and p21 and an up-regulation of BCL2/BAX mRNA and protein ratio were observed in JEG-3 cells; however, there was also a down-regulation of mRNA and protein levels observed for p53 and p21 in BeWo cells after the treatment of NOB. The BCL2/BAX ratio of BeWo cells did not change after the treatment of 100 μM NOB.ConclusionNOB attenuated hypoxia-induced apoptosis in JEG-3 and BeWo cell lines and might be a potential functional ingredient to prevent pregnancy-related diseases caused by hypoxia-induced apoptosis. These findings would also suggest the exploration and utilization of citrus resources, and the development of citrus industry.

Influence of miRNA-30a-5p on Pulmonary Fibrosis in Mice with Streptococcus pneumoniae Infection through Regulation of Autophagy by Beclin-1.

The study is aimed at observing the influence of microribonucleic acid- (miRNA-) 30a-50p on the pulmonary fibrosis in mice with Streptococcus pneumoniae infection through the regulation of autophagy by Beclin-1. Specific pathogen-free mice were instilled with Streptococcus pneumoniae through the trachea to establish the pulmonary fibrosis model. Then, they were divided into the miRNA-30a-50p mimics group (mimics group, n = 10) and miRNA-30a-5p inhibitors group (inhibitors group, n = 10), with the control group (n = 10) also set. Pulmonary tissue wet weight/dry weight (W/D) was detected. The content of tumor necrosis factor-α (TNF-α), interleukin- (IL-) 6, and myeloperoxidase (MPO) was determined using enzyme-linked immunosorbent assay (ELISA). Besides, the changes in the pulmonary function index dynamic lung compliance (Cdyn), plateau pressure (Pplat), and peak airway pressure (Ppeak) were monitored, and the gene and protein expression levels were measured via quantitative PCR (qPCR) and Western blotting. The expression level of miRNA-30a-5p was substantially raised in the mimics group (p < 0.05), but extremely low in the inhibitors group (p < 0.05). The mimics group had obviously raised levels of serum aminotransferase (AST), glutamic-pyruvic transaminase (GPT), alkaline phosphatase (ALP), and pulmonary tissue W/D (p < 0.05). Additionally, the expression levels of TNF-α, IL-6, and MPO were notably elevated in the mimics group, while their expression levels showed the opposite conditions in the inhibitors group (p < 0.05). According to the HE staining results, the inhibitors group had arranged orderly cells, while the mimics group exhibited lung injury, pulmonary edema, severe inflammatory response, and alveolar congestion. In the inhibitors group, Cdyn was remarkably elevated, but Pplat and Ppeak declined considerably (p < 0.05). Besides, the inhibitors group exhibited elevated messenger RNA (mRNA) levels of Beclin-1 and LC3, lowered mRNA levels of α-SMA and p62, a raised protein level of Beclin-1, and a markedly decreased protein level of p62 (p < 0.05). Silencing miRNA-30a-5p expression can promote the expression of Beclin-1 to accelerate the occurrence of autophagy, thereby treating pulmonary fibrosis in mice with Streptococcus pneumoniae infection.

Fluoride regulates chondrocyte proliferation and autophagy via PI3K/AKT/mTOR signaling pathway

Fluorine is an essential trace element for human health. However, excessive fluoride intake causes skeletal fluorosis which affects cartilage development. Fluoride inhibited chondrocyte proliferation which is the initial and critical step of endochondral ossification, but the underlying mechanism has not been clearly illustrated. Mammalian target of rapamycin (mTOR) is an important protein kinase which modulates various cellular processes and is believed to be a central regulator of chondrocyte proliferation and autophagy. In this study, we explored the effect of fluoride on the proliferation and autophagy of chondrocytes and the regulatory role of mTOR signaling pathway. Our results suggested that NaF inhibited the protein expressions of proliferating cell nuclear antigen (PCNA) and pS6 in cultured fetal rat tibias. Furthermore, NaF significantly downregulated the expressions of mTOR signaling pathway-related genes, including PI3K, AKT, mTOR, 4EBP1 and S6K1 in mouse ATDC5 chondrogenic cell line. We also found that NaF increased autophagy in ATDC5 cells. The mRNA and protein levels of autophagy-related genes LC3, Beclin1 and p62 were significantly changed after NaF treatment. Further studies demonstrated that MHY1485, a small-molecular mTOR activator, totally reversed fluoride-induced promotion of autophagy. MHY1485 also recovered the downregulation of proliferative chondrocytes markers Sox9 and Type Ⅱ Collagen (Col2a1) induced by fluoride in ATDC5 cells. Taken together, our result demonstrate that fluoride suppressed proliferation and facilitated autophagy via PI3K/AKT/mTOR signaling pathway in chondrogenesis.

Synthesis, characterization, and biological evaluation of doxorubicin containing silk fibroin micro- and nanoparticles

The aim of this study was biological evaluation of doxorubicin containing silk fibroin micro- and nanoparticles (Dox-MF and Dox-NF). Dox-MF and Dox-NF were synthesized. Cell toxicity on MCF-7, Saos2, and HFF cells was assessed using MTT assay. Induced apoptosis was assessed using flow cytometry and staining with PI/annexin V. Production of reactive oxygen species (ROS) was measured. Gene expression of p53 was evaluated by real-time PCR. FTIR, SEM, and DLS confirmed the accurate synthesis. Cytotoxicity of Dox-MF and Dox-NF showed significant inhibition of cell growth compared with the controls. Regarding Dox-NF, a significant increase was seen in mRNA level of P53 in MCF-7 and SAOS-2 ​cells and a significant decrease in HFF cells compared to the controls. There was a significantly higher expression of P53 gene in MCF-7 and HFF cells treated by Dox-MF. However, a significant decrease in P53 gene expression was detected in SAOS-2 ​cells. Significant apoptotic induction of cell lines by Dox-MF and Dox-NF was observed in both early and late stages. Dox-MF and Dox-NF acted in the direction of cell death through the apoptotic pathway and changing p53 gene expression. So, Dox-MF and Dox-NF can be considered as a candidate for new anticancer agents.

Long-term ketamine administration induces bladder damage and upregulates autophagy-associated proteins in bladder smooth muscle tissue.

Long-term ketamine abuse can cause significant lower urinary tract symptoms in humans, termed ketamine-associated cystitis (KC). Here, we established a model of long-term (6 months) ketamine administration in wild-type (C57BL/6) mice. We elucidated the pathological effects of ketamine in the bladder and investigated changes in autophagy-associated protein expression (i.e., LC3, Beclin-1, and P62) and inflammatory cytokines (i.e., IL-6 and IL-1β) in the bladder smooth muscle tissue. Long-term ketamine administration reduced the number of layers in the bladder mucosal epithelial cells (4-5 layers in the saline group vs. 2-3 layers in the ketamine groups), but increased the number of mast cells and collagen fibers. LC3-II/LC3-I, Beclin-1, IL-6, and IL-1β protein expression in the bladder smooth muscle tissues of ketamine-treated mice was significantly increased. The mRNA and protein levels of P62 in the Ket-60 mg/kg group were also significantly increased, but not the Ket-30 mg/kg group. Our results reveal that long-term ketamine administration can cause cystitis-like pathological changes in mice, and the disordered autophagy in the bladder tissue may be involved in the persistent bladder damage following long-term administration of ketamine at 60 mg/kg.

Abstract P426: Inactivating Grk5 Impairs Basal Cardiac Function And Survival Via P53 Modulation

Introduction: G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent. Hypothesis: The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress. Methods: We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies. Results: Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels. Conclusions: Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.