Search

Abstract Background Hepatic progenitor cells serve not only as the origin of combined hepatocellular cholangiocarcinoma (cHCC-CCA) but are also responsible for malignancy recurrence after surgical resection. Nucleophosmin 1 (NPM1) has been implicated in cancer metastasis and poor prognosis. This study aimed to determine the expression of NPM1 by hepatic progenitor cells in cHCC-CCA and the effects of targeting NPM1 on hepatic progenitor cells and BEL-7402 cells with characteristics of both progenitor cells and cHCC-CCA. Methods First, NPM1 was detected by RT‒PCR, western blotting, and double-immunofluorescence staining in cHCC-CCA tissues. NPM1 expression was subsequently analysed in rat hepatic progenitor cells cultured in vitro and in interleukin 6 (IL6)-treated cells. The effects and mechanism of NPM1 on hepatic progenitor cells were determined by knocking down NPM1 and performing RNA sequencing analysis. Finally, NSC348884, a small-molecule inhibitor that disrupts NPM1 dimer formation, was used to confirm the function of NPM1 in BEL-7402 cells. Results Both human hepatic progenitor cells in cHCC-CCA tissues and rat in vitro cultured hepatic progenitor cells highly expressed NPM1. IL6, a cytokine involved in the malignant transformation of hepatic progenitor cells, dose-dependently increased NPM1 and PCNA expression. Knocking down NPM1 reduced IL6R transcription (P

Inflammatory macrophages play a crucial role in atherosclerosis development. The long non-coding RNA growth arrest-specific 5 (GAS5) regulates THP-1 macrophage inflammation by sponging microRNAs. The purpose of this study was to investigate the regulatory mechanism of GAS5 in atherosclerosis development. GSE40231, GSE21545, and GSE28829 datasets from the Gene Expression Omnibus database were integrated after adjusting for batch effect. Differential analysis was performed on the integrated dataset and validated using the Genotype-Tissue Expression and GSE57691 datasets. Potential biological functions of GAS5 and annexin A2 (ANXA2) were identified using gene set enrichment analysis (GSEA). ssGSEA, CIBERSORTx, and ImmuCellAI algorithms were used to identify immune infiltration in plaque samples. GAS5 and ANXA2 expression levels in RAW264.7 cells treated with oxidized low-density lipoprotein (ox-LDL) were measured by qRT-PCR and Western blot. Small interfering and short hairpin RNA were used to silence GAS5 expression. Plasmids of ANXA2 were used to establish ANXA2 overexpression. Apoptosis and inflammatory markers in macrophages were detected by Western blot. Aortic samples from APOE−/− mice were collected to validate the expression of GAS5 and ANXA2. GAS5 expression was significantly increased during atherosclerosis. GAS5 expression was positively correlated with macrophage activation and ANXA2 expression in plaques. Furthermore, ANXA2 upregulation was also related to the activation of macrophage. GSEA indicated similar biological functions for GAS5 and ANXA2 in plaques. Moreover, in vitro experiments showed that both GAS5 and ANXA2 contributed to macrophage apoptosis and inflammation. Rescue assays revealed that the inflammatory effects of GAS5 on macrophages were ANXA2-dependent. In vivo experiments confirmed the highly expression of Gas5 and Anxa2 in the plaque group. We identified the atherogenic roles of GAS5 and ANXA2 in the inflammatory response of macrophages. The inflammatory response in ox-LDL-treated macrophages was found to be mediated by GAS5-ANXA2 regulation, opening new avenues for atherosclerosis therapy.

Reproductive disorders and declining fertility rates are significant public health concerns affecting birth rates and future populations. Male infertility, often due to spermatogenesis defects, may be linked to environmental pollutants like nickel nanoparticles (Ni NPs). Ni NPs are extensively utilized across different industries. Nevertheless, their potential adverse effects cannot be overlooked. Previous studies have linked the reproductive toxicity induced by Ni NPs with disturbances in mitochondrial function. Mitochondrial division/fusion dynamics are crucial to their proper function, yet little is known about how Ni NPs perturb these dynamics and whether such perturbation contributes to the impairment of the male reproductive system. Herein, we demonstrated that the exposure of Ni NPs to the mouse-derived spermatogonia cell line (GC-1 cells) triggered DRP1-mediated mitochondrial division and the enhanced impairment of mitochondria, consequently promoting mitochondria-dependent cell apoptosis. Notably, both the mitochondrial division inhibitor (Mdivi-1) and lentiviral-transfected cells with low expression of Dnm1l-DK in these cells could mitigate the toxic effects induced by Ni NPs, pointing to the potential role of mitochondrial dynamics in Ni NP-induced reproductive toxicity. Collectively, our work contributes to the understanding of the mechanisms by which Ni NPs can impact male reproductive function and identifies mitochondrial division as a potential target for intervention.

Abstract Background Cardiac‐resident or ‐enriched microRNAs (miRNAs) could be released into the bloodstream becoming circulating cardiac miRNAs, which are increasingly recognized as non‐invasive and accessible biomarkers of multiple heart diseases. However, dilated cardiomyopathy (DCM)‐associated circulating miRNAs (DACMs) and their roles in DCM pathogenesis remain largely unexplored. Methods Two human cohorts, consisting of healthy individuals and DCM patients, were enrolled for serum miRNA sequencing (10 vs. 10) and quantitative polymerase chain reaction validation (46 vs. 54), respectively. Rigorous screening strategy was enacted to define DACMs and their potentials for diagnosis. DCM mouse model, different sources of cardiomyocytes, adeno‐associated virus 9 (AAV9), gene knockout, RNAscope miRNA in situ hybridization, mRFP‐GFP‐LC3B reporter, echocardiography and transmission electron microscopy were adopted for mechanistic explorations. Results Serum miRNA sequencing revealed a unique expression pattern for DCM circulating miRNAs. DACMs miR‐26a‐5p, miR‐30c‐5p, miR‐126‐5p and miR‐126‐3p were found to be depleted in DCM circulation as well as heart tissues. Their expressions in circulation and heart tissues were proven to be correlated significantly, and a combination of these miRNAs was suggested potential values for DCM diagnosis. FOXO3, a predicted common target, was experimentally demonstrated to be co‐repressed within cardiomyocytes by these DACMs except miR‐26a‐5p. Delivery of a combination of miR‐30c‐5p, miR‐126‐5p and miR‐126‐3p into the murine myocardium via AAV9 carrying an expression cassette driven by cTnT promoter, or cardiac‐specific knockout of FOXO3 (Myh6‐CreERT2, FOXO3 flox+/+) dramatically attenuated cardiac apoptosis and autophagy involved in DCM progression. Moreover, competitively disrupting the interplay between DACMs and FOXO3 mRNA by specifically introducing their interacting regions into murine myocardium crippled the cardioprotection of DACMs against DCM. Conclusions Circulating cardiac miRNA‐FOXO3 axis plays a pivotal role in safeguarding against myocardial apoptosis and excessive autophagy in DCM development, which may provide serological cues for DCM non‐invasive diagnosis and shed light on DCM pathogenesis and therapeutic targets.

Objective(s): Vitexin, a natural flavonoid, is commonly found in many foods and traditional herbal medicines and has clear health benefits. However, the role of vitexin in cholestasis is presently unclear. This study investigated whether vitexin mitigated glycochenodeoxycholate (GCDC)-induced hepatocyte injury and further elucidated the underlying mechanisms.Materials and Methods: A cell counting kit-8 (CCK-8) assay was conducted to evaluate cell viability. The mitochondrial membrane potential (MMP, Δψm), reactive oxygen species (ROS) levels, and apoptosis rate of hepatocytes exposed to GCDC were detected by flow cytometry (FCM). We then measured the cytoprotective effects of vitexin against oxidative stress. The molecular signaling pathway was further investigated by using Western blotting and signaling pathway inhibitors.Results: Here, we showed that vitexin increased cell viability and reduced cell apoptosis, necroptosis, and oxidative stress in a dose-dependent manner in GCDC-treated hepatocytes. In addition, by using selective inhibitors, we further confirmed that inhibition of the JAK2/STAT3 pathway by vitexin was mediated by prolonged activation of Sirtuin 6 (SIRT6).Conclusion: Vitexin attenuated GCDC-induced hepatocyte injury via SIRT6 and the JAK2/STAT3 pathways.

Abstract Background Synovial fibroblasts (SFs) with the abnormal expressions of miRNAs are the key regulator in rheumatoid arthritis (RA). Low-expressed miR-140-3p was found in RA tissues. Therefore, we attempted to investigate the effect of miR-140-3p on SFs of RA. Methods RA and normal synovial fibrous tissue were gathered. The targets of miR-140-3p were found by bioinformatics and luciferase analysis. Correlation between the expressions of miR-140-3p with sirtuin 3 (SIRT3) was analyzed by Pearson correlation analysis. After transfection, cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The expressions of miR-140-3p, SIRT3, Ki67, Bcl-2, Bax, and cleaved Caspase-3 were detected by RT-qPCR or western blot. Results Low expression of miR-140-3p and high expression of SIRT3 were found in RA synovial fibrous tissues. SIRT3 was a target of miR-140-3p. SIRT3 expression was negatively correlated to the expression of miR-140-3p. MiR-140-3p mimic inhibited the MH7A cell viability and the expressions of SIRT3, Ki67, and Bcl-2 and promoted the cell apoptosis and the expressions of Bax and cleaved Caspase-3; miR-140-3p inhibitor showed an opposite effect to miR-140-3p mimic on MH7A cells. SIRT3 overexpression not only promoted the cell viability and inhibited cell apoptosis of MH7A cells but also reversed the effect of miR-140-3p mimic had on MH7A cells. Conclusions The results in this study revealed that miR-140-3p could inhibit cell viability and promote apoptosis of SFs in RA through targeting SIRT3.

Abstract Background The present study aimed to investigate the underlying role of interferon-regulatory factor 2 (IRF2)–inositol polyphosphate-4-phosphatase, type-II (INPP4B) axis in the regulation of autophagy in acute myeloid leukemia (AML) cells. Methods Quantitative real time PCR (QRT-PCR) and western blot were performed to determine the expression levels of IRF2, INPP4B and autophagy-related markers in AML cell lines. Autophagy was assessed by elevated Beclin-1 expression, the conversion of light chain 3 (LC3)-I to LC3-II, downregulated p62 expression and green fluorescent protein (GFP)-LC3 puncta formation. The colony formation and apoptosis assays were performed to determine the effects of IRF2 and INPP4B on the growth of AML cells. Results IRF2 and INPP4B were highly expressed in AML cell lines, and were positively correlated with autophagy-related proteins. Overexpression of IRF2 or INPP4B stimulated autophagy of AML cells, whereas inhibition of IRF2 or INPP4B resulted in the attenuation of autophagy. More importantly, IRF2 or INPP4B overexpression reversed autophagy inhibitor, 3-methyladenine (3-MA)-induced proliferation-inhibitory and pro-apoptotic effects, while IRF2 or INPP4B silencing overturned the proliferation-promoting and anti-apoptotic effects of autophagy activator rapamycin. Conclusion IRF2–INPP4B signaling axis attenuated apoptosis through induction of autophagy in AML cells.

Stem cell apoptosis exists widely in embryonic development, tissue regeneration, repair, aging and pathophysiology of disease. The molecular mechanism of stem cell apoptosis has been extensively investigated. However, alterations in biomechanics and nanomorphology have rarely been studied. Therefore, an apoptosis model was established for bone marrow mesenchymal stem cells (BMSCs) and the reconstruction of the mechanical properties and nanomorphology of the cells were investigated in detail. Atomic force microscopy (AFM), scanning electron microscopy (SEM), laser scanning confocal microscopy (LSCM), flow cytometry and Cell Counting Kit-8 analysis were applied to assess the cellular elasticity modulus, geometry, nanomorphology, cell surface ultrastructure, biological viability and early apoptotic signals (phosphatidylserine, PS). The results indicated that the cellular elastic modulus and volume significantly decreased, whereas the cell surface roughness obviously increased during the first 3 h of cytochalasin B (CB) treatment. Moreover, these alterations preceded the exposure of biological apoptotic signal PS. These findings suggested that cellular mechanical damage is connected with the apoptosis of BMSCs, and the alterations in mechanics and nanomorphology may be a sensitive index to detect alterations in cell viability during apoptosis. The results contribute to further understanding of apoptosis from the perspective of cell mechanics.

Background: Lung cancer has been the most common cancer worldwide. Microsomal glutathione S-transferase 1 (MGST1) has been reported to play vital roles in oxidative stress, tumor occurrence and drug resistance. However, the biological function and molecular mechanism of MGST1 in lung adenocarcinoma (LUAD) has not yet been elucidated. Methods: The expression of MGST1 in LUAD tissues and cell lines was evaluated by immunohistochemistry and western blotting, respectively. MGST1 was knocked down by shRNA lentivirus. Cell proliferation was evaluated by MTS, colony formation and EdU assays. Apoptosis was detected by flow cytometry. The potential molecules involved in cell proliferation and apoptosis were examined by western blotting. Finally, the effect of MGST1 on tumor growth in vivo was evaluated in a nude mouse xenograft model. Results: TCGA database analysis and immunohistochemistry demonstrated that MGST1 was highly expressed in LUAD tissues. MGST1 expression in LUAD was correlated with AJCC stage and poor overall survival of patients. MGST1 knockdown significantly inhibited LUAD cell proliferation and induced apoptosis. Mechanistic analyses revealed that MGST1 knockdown might inhibit cell proliferation by inactivating the AKT/GSK-3β pathway signaling and promote cell apoptosis by regulating the mitochondrial apoptosis pathway related proteins. Moreover, knockdown of MGST1 suppressed tumor growth in vivo. Conclusions: MGST1 plays an important role in LUAD tumorigenesis and might serve as a potential prognostic factor and therapeutic target in LUAD.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-β1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl4-induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-β1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-β1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-β1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis.

The intestinal protozoan parasite, Giardia duodenalis, infects a large number of people in the world annually. Giardia infection has been considered a negative effect on intestinal epithelial cell growth, while the underlying mechanisms remain to be explored. Here we evaluated reactive oxygen species (ROS) production and apoptotic events in Giardia trophozoites-stimulated Caco-2 cells via fluorescence microscopy, transmission electron microscopy, flow cytometry, western blot, and cell counting kit-8 analyses. The results showed that Giardia trophozoite treatment could induce lactate dehydrogenase release and Caco-2 cell apoptosis. The ROS levels were increased post treatment. The observed typical characteristics of mitochondria damage include significant swelling and degeneration of matrix and cristae. After trophozoite treatment, the level of Bax protein expression was increased, while Bcl-2 protein decreased. Trophozoite stimulation also led to reduction of mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytoplasm, and this process was accompanied by activation of caspase-9 and caspase-3 and poly (ADP-ribose) polymerase 1 cleavage. Pretreatment with N-acetyl-L-cysteine, a ROS inhibitor, reversed G. duodenalis-induced Caco-2 cell apoptosis. Taken together, we indicated that G. duodenalis could induce Caco-2 cell apoptosis through a ROS- and mitochondria-mediated caspase-dependent pathway. This study furthers our understanding of the cellular mechanism of the interaction between Giardia trophozoites and host cells.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Objective: To study the effect and mechanism of Tip60 regulating DNA on mouse embryo development, and to provide theoretical basis for the study of embryo development. Methods: mice embryos were randomly divided into two groups: control group and experimental group. The embryos of experimental group were given Tip60 inhibitor, the mice of control group were given the same dose of normal saline. The level of Tip60 in the two groups of mice embryos, the effect of Tip60 on p53-p21 pathway, the expression of DNA repair factor 53BP1 protein, the content of active oxygen, the effect of autophagy and apoptosis, and the development of embryos were analyzed. Results: the level of Tip60 in the experimental group was significantly lower than that in the control group; the p53-p21 pathway in the experimental group was activated, the DNA damage of the experimental group was greater, the expression of DNA repair factor was lower, the content of ROS in the experimental group was significantly higher than that in the control group; the autophagy and apoptosis of the experimental group were enhanced; the capsule of the control group was enhanced. The embryogenesis rate of the experimental group was (65.13 ± 4.85)%, and that of the experimental group was (29.36 ± 1.75)%, which was significantly lower than that of the control group. Conclusion: Tip60 can regulate DNA damage and repair by mediating p53-p21 pathway in early mouse embryo development. Inhibition of Tip60 can increase the active oxygen, induce autophagy and over expression of apoptosis in mouse embryo cells, and inhibit the development of embryo to a certain extent. It has important guiding significance to measure the level of Tip60 during embryo culture. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the molecular mechanisms and effect of Beclin-1 on autophagy, proliferation and apoptosis in the colorectal cancer (CRC) HCT116 and SW620 cells. Beclin-1 was silenced by RNA interference (RNAi) in HTC116 and SW620 cells. Reverse transcription-polymerase chain reaction and western blot were used to measure the expression of Beclin-1. The percentage of apoptotic cells was analyzed by cell counting kit-8 (CCK-8) and flow cytometry (FCM). Cell cycle and cell proliferation were analyzed by FCM and the MTT assay. The present study created 3 groups in the two cell lines, consisting of the targeting siRNA (TS) group, in which Beclin-1 was partially silenced, non-specific siRNA (NS) group and control group (CG; without transfection). By siRNA transfection, the mRNA and protein level of Beclin-1 in the TS group were significantly inhibited compared with the NS group and CG (P<0.05). After 0, 24, 48 and 72 h, the survival rate of the cells in the TS group was significantly decreased compared with the survival rate of the cells in the NS group and CG, as detected by CCK-8 methods (P<0.05). FCM and MTT results showed the apoptotic rate of the cells in the TS group was significantly decreased compared with the rate in the NS group and CG (P<0.05), and the proliferation of the cells in the NS group was evidently increased compared with the CG. In conclusion, Beclin-1 played an important role in regulating autophagy, proliferation and apoptosis in HCT116 and SW620 cells. The inhibition of Beclin-1 by RNAi suppressed the autophagic activity and proliferation, but promoted apoptosis in CRC cells. Beclin-1 was the new target of gene therapy for CRC. [ABSTRACT FROM AUTHOR]

Sesquiterpenoids are a major type of compound found in Solanum lyratum (S. lyratum). The present study aimed to investigate whether sesquiterpenoids from S. lyratum demonstrated cytotoxicity against the MCF-7, HCT-8, A-549, SGC-7901 and BEL-7402 cell lines, and the mechanism of solajiangxin H and lyratol D, which exhibited high cytotoxicity against SGC-7901 cells (half maximal inhibitory concentration, IC50=4.8 and 5.9 µg/ml), was associated with mitochondria-mediated apoptosis. The results of the Cell Counting Kit-8 assay indicated that 15 sesquiterpenoids had cytotoxicity against the aforementioned cultured cells. The results of DAPI staining and western blot analysis, used to study the anticancer mechanisms of solajiangxin H and lyratol D in SGC-7901 cells, suggested that solajiangxin H and lyratol D induced the apoptosis of SGC-7901 cells significantly (P<0.01), downregulated the expression of the antiapoptotic proteins B-cell lymphoma (Bcl)-2 and survivin, and upregulated the expression of the proapoptotic proteins Bcl-2-like protein 4, second mitochondria-derived activator of caspase, cleaved (c)-caspase-3 and c-caspase-9. The present study therefore demonstrated that 15 sesquiterpenoids from S. lyratum exhibited anticancer activity in MCF-7, HCT-8, A-549, SGC-7901 and BEL-7402 cells, and that the anticancer mechanisms of solajiangxin H and lyratol D may be associated with mitochondria-mediated apoptosis. Additionally, the present study provides evidence in support of the hypothesis that S. lyratum may be a promising candidate for the development of novel cancer therapies. [ABSTRACT FROM AUTHOR]

Background: Regulating cardiac differentiation to maintain normal heart development and function is very important. At present, biological functions of H19 in cardiac differentiation is not completely clear. Methods: To explore the functional effect of H19 during cardiac differentiation. Expression levels of early cardiac-specific markers Nkx-2.5 and GATA4, cardiac contractile protein genes α-MHC and MLC-2v were determined by qRT-PCR and western lot. The levels of lncRNA H19 and miR-19b were detected by qRT-PCR. We further predicted the binding sequence of H19 and miR-19b by online softwares starBase v2.0 and TargetScan. The biological functions of H19 and Sox6 were evaluated by CCK-8 kit, cell cycle and apoptosis assay and caspase-3 activity. Results: The expression levels of α-MHC, MLC-2v and H19 were upregulated, and miR-19b was downregulated significantly in mouse P19CL6 cells at the late stage of cardiac differentiation. Biological function analysis showed that knockdown of H19 promoted cell proliferation and inhibits cell apoptosis. H19 suppressed miR-19b expression and miR-19b targeted Sox6, which inhibited cell proliferation and promoted apoptosis in P19CL6 cells during late-stage cardiac differentiation. Importantly, Sox6 overexpression could reverse the positive effects of H19 knockdown on P19CL6 cells. Conclusion: Downregulation of H19 promoted cell proliferation and inhibited cell apoptosis during late-stage cardiac differentiation by regulating the negative role of miR-19b in Sox6 expression, which suggested that the manipulation of H19 expression could serve as a potential strategy for heart disease. [ABSTRACT FROM AUTHOR]

Background: Developmental dysplasia of the hip (DDH) is an important factor leading to early adult osteoarthritis. Chondrocyte apoptosis has been proven to be an important factor causing osteoarthritis. Aims: The current study aims to explore whether a rabbit model of developmental dysplasia of the hip through cast immobilization in the legs results in chondrocyte apoptosis. Study Design: Animal experimentation. Methods: Thirty-two New Zealand white rabbits were divided in three groups with cast plaster-induced dislocation at 2, 4 and 6 weeks. The contralateral hip joint was utilized as a control group. Ten rabbits in each group were sacrificed, and hip specimens were obtained. Bcl-2/Bax, cleaved caspase-3 and cleaved caspase-8 expression were examined by western blot analysis. Chondrocyte apoptosis was analyzed through transmission electron microscopy (TEM) and TUNEL analysis. All experiments were repeated at least three times. Results: In the experimental group, Bcl-2/Bax, cleaved caspase-3 and cleaved caspase-8 expression were significantly altered. The Bcl-2/Bax ratio decreased with time (all p<0.01), whereas levels of cleaved caspase-3 (p<0.01 and p<0.05) and cleaved caspase-8 (all p<0.05) gradually increased. Chondrocyte apoptosis was observed through transmission electron microscopy (TEM) and TUNEL analysis (p<0.05 at 4 weeks and p<0.01 at 6 weeks). Conclusion: Prolonged immobilization of rabbit hip caused chondrocyte apoptosis. Reduction of the hip joint may protect chondrocytes from apoptosis, thus preventing secondary osteoarthritis. [ABSTRACT FROM AUTHOR]

Background: Previous studies have reported that eEF-2 kinase is associated with tumour cell sensitivity to certain therapies. In the present study, we investigated the relationship between eEF-2 kinase and lapatinib, a dual inhibitor of EGFR and HER-2, in nasopharyngeal carcinoma (NPC) cells. Methods: The effect of treatment on the growth and proliferation of NPC cells was measured by three methods: cell counting, crystal violet staining and colony counting. Apoptosis was evaluated by flow cytometry to determine Annexin V-APC/7-AAD and cleaved PARP levels, and the results were further confirmed by Western blot analysis. The expression of eEF-2 kinase and the impacts of different treatments on different signalling pathways were analysed by Western blot analysis. Results: The expression of eEF-2 kinase was significantly associated with NPC cell sensitivity to lapatinib. Therefore, suppression of this kinase could increase the cytocidal effect of lapatinib, as well as reduce cell viability and colony formation. Furthermore, inhibition of eEF-2 kinase, by either RNA interference (eEF-2 kinase siRNA or shRNA) or pharmacological inhibition (NH125), enhanced lapatinib-induced apoptosis of NPC cells. The results also showed that lapatinib combined with NH125 had a synergistic effect in NPC cells. In addition, mechanistic analyses revealed that downregulation of the ERK1/2 and Src pathways, but not the AKT pathway, was involved in this sensitizing effect. Conclusions: The results of this study suggest that targeting eEF-2 kinase may improve the efficacy of therapeutic interventions such as lapatinib in NPC cells. [ABSTRACT FROM AUTHOR]

The medicinal value of hydrogen (H2) was ignored prior to research illustrating that inhalation of 2% H2 can significantly decrease the damage of cerebral ischemia/reperfusion caused by oxidative stress via selective elimination of hydroxyl freebase (OH) and peroxynitrite anion (ONOO-). Subsequently, there have been numerous experiments on H2. Most research and trials involving the mechanisms underlying H2 therapy show the effects of antioxygenation, anti-inflammation, and anti-apoptosis. Among quantities of diseases related with H2 therapy, the brain disease is a hotspot as brain tissue and cell damage are easier to be induced by oxidative stress and other stimulations. In this review, emphasis is on stroke, traumatic brain injuries, and degenerative diseases, such as Alzheimer's disease and Parkinson's disease. Taking into account the blood-brain barrier, penetrability, possible side effects, and the molecular properties of H2 within a single comprehensive review should contribute to advancing both clinical and non-clinical research and therapies. A systematic introduction of H2 therapy with regards to mechanisms and cerebral diseases both in animal and human subjects can make it easier to comprehend H2 therapy and therefore provide the basis for further clinical strategy. [ABSTRACT FROM AUTHOR]

Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D.galactose (D.gal), and revealed that chronic injection of D.gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D.gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D.gal for 8 weeks. In addition, the levels of 8.hydroxy.2.deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase.mediated deoxyuridine triphosphate nick.end.labelling.positive cells were increased in the cochleae of D.gal.treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3.associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3.dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age.associated hearing loss, also termed presbycusis. [ABSTRACT FROM AUTHOR]

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs. [ABSTRACT FROM AUTHOR]

Background: Studies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells.Methods: Cell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques.Results: We explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence.Conclusions: Taken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC. [ABSTRACT FROM AUTHOR]

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let- 7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death. [ABSTRACT FROM AUTHOR]

Renal ischemia and reperfusion (I/R) injury, which commonly occurs in kidney transplantation, is the leading cause of acute kidney injury. Picroside II possesses a wide range of pharmacological effects, including anti-apoptosis effects. In the present study, the ability of picroside II to attenuate apoptosis in a rat model of renal I/R injury was investigated. Sprague-Dawley rats were subjected to 45 min of ischemia followed by 24 h of reperfusion. Prior to reperfusion, the rats were treated with picroside II or an equal volume of phosphate-buffered saline. It was observed that renal function was significantly improved by the treatment with picroside II. Morphological analysis indicated that picroside II markedly reduced tissue damage and the expression of cleaved caspase-3. Reverse transcription-quantitative polymerase chain reaction and western blotting revealed that the expression levels of Bax and poly(ADP-ribose) polymerase-1 (PARP-1) were upregulated in the I/R group, whereas those of Bcl-2 were downregulated. However, the treatment with picroside II inhibited these changes induced by renal I/R injury. In conclusion, picroside II has potent anti-apoptotic activity against renal I/R injury. [ABSTRACT FROM AUTHOR]

Guan Chang Fu Fang (GCFF) is a natural compound, which is extracted from three medicinal plants, Agrimonia pilosa Ledeb., Patrinia scabiosaefolia and Solanum nigrum L. GCFF has demonstrated clinical efficacy in the treatment of colon cancer. At present, 5-fluorouracil (5-FU) is the primary active chemotherapeutic agent used for treating colon cancer. Using median-effect and apoptosis analyses, fluorescence microscopy and western blotting, the present study analyzed the association between GCFF and 5-FU in the human colon adenocarcinoma LoVo cell line. The effect of GCFF on the expression of chemotherapeutic agent-associated genes was also investigated. The results of the synergistic analysis revealed that GCFF exhibited a significant effect upon 5-FU-associated cytotoxicity within the LoVo cell line. This effect was observed over a broad dose-inhibition range (5-95%), but was particularly significant in the lower concentrations. The flow cytometry results revealed that low doses of GCFF or 5-FU induced S -phase arrest, as did a low-dose combination of the two drugs. After 48 h, GCFF significantly suppressed the expression levels of the chemotherapeutic agent resistance-associated genes within the colon cancer cells. The western blot analysis revealed that the combined effects of 5-FU and GCFF were due to a regulation of the B-cell lymphoma-2 family of proteins. The findings of the present study suggested that GCFF, when combined with 5-FU, has the potential to be a novel, chemotherapeutic compound for the treatment of colon cancer. [ABSTRACT FROM AUTHOR]

Bone loss is a major issue for patients with osteoporosis, arthritis, periodontitis, and bone metastasis; however, anti-resorption drugs used to treat bone loss have been linked to a variety of adverse effects. Helminthostachys zeylanica (L.) Hook, belonging to the family Ophioglossaceae, is commonly used in traditional Chinese medicine to treat inflammation and liver problems. In the current study, ugonin L extracted from H. zeylanica was shown to reduce the receptor activator of nuclear factor kappa beta ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells in a concentration-dependent manner. Ugonin L treatment also inhibited the mRNA expression of osteoclast markers. Ugonin L was also shown to promote cell apoptosis in mature osteoclasts and suppress RANKL-induced ERK, p38, JNK, and NF-κB activation. Taken together, ugonin L appears to be a promising candidate for the development of novel anti-resorption therapies.

Abstract Preeclampsia (PE) is a multisystemic and placental inflammatory disease that causes maternal and infant health issues. As one of the active components in peony root extract, paeonol (Pae) exerts anti‐apoptosis and anti‐inflammatory effects. Nonetheless, the protective role of Pae in PE has not yet been characterized. A mouse model of PE was constructed through tail vein injection of 1 mg/d phosphatidylserine/dioleoyl‐phosphatidycholine suspension. The levels of inflammatory cytokines in the placenta were examined via enzyme‐linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines (TNF‐α, IL‐6, IFN‐γ, and IL‐4) and apoptosis markers (Bax, Bcl‐2, and caspase‐3) were tested using quantitative reverse transcription‐polymerase chain reaction (qRT–PCR). Western blot analysis was performed to detect the protein levels of apoptosis markers and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway‐related molecules. Here, Pae repressed the inflammatory response in the placenta of PE‐like mouse models, as demonstrated by the decreased concentrations and mRNA levels of TNF‐α, IL‐6, and IFN‐γ and the increased concentrations and mRNA levels of IL‐4. Apoptosis in the placentas of PE‐like mouse models was attenuated by Pae, as manifested by the downregulated mRNA and protein levels of Bax and cleaved‐caspase‐3 and the upregulated Bcl‐2. Administration of Pae inhibited the phosphorylation of JAK2 and STAT3 in the placental tissues of PE mice. The JAK2/STAT3 pathway agonist (SC‐39100) reversed Pae treatment‐mediated suppression of placental inflammation and apoptosis in PE mice. Overall, Pae inhibits the JAK2/STAT3 signaling pathway to attenuate placental inflammation and apoptosis in PE mice.

Abstract Background Pazopanib is an approved multitarget anticancer agent for soft tissue sarcoma (STS) and renal cell carcinoma (RCC), which is also under clinical investigation for other malignancies, including small cell lung cancer (SCLC). However, the potential anti‐SCLC mechanisms of pazopanib remain unclear. Methods Cell viability was evaluated by CCK‐8, apoptotic cell detection was conducted using annexin V/PI staining followed by flow cytometry, and Western blot analysis was used to detect the apoptotic‐related molecules and ER‐stress pathway effectors. The intracellular reactive oxygen species (ROS) level was determined by DCFH‐HA staining followed by flow cytometry. An NCI‐H446 xenograft model was established to evaluate pazopanib on tumor suppression in vivo. Immunohistochemistry (IHC) was used to assess the proliferative activity of xenograft in NCI‐H446 cell‐bearing NOD‐SCID mice. Results Pazopanib dose‐ and time‐dependently inhibited SCLC cell proliferation induced significant apoptosis in SCLC cell lines, increased cleaved‐caspase3 and Bax, and decreased Bcl‐2. Moreover, the PERK‐related ER‐stress pathway was potently activated by pazopanib treatment, inhibiting ER‐stress by salubrinal significantly reversing pazopanib‐mediated apoptosis in SCLC cell lines. Furthermore, pazopanib‐induced intracellular ROS levels increased, while inhibiting ROS by NAC significantly reversed pazopanib‐induced apoptosis in SCLC cells. In addition, pazopanib significantly suppressed NCI‐H446 xenograft growth and decreased Ki67 positive cells in the tumor. Conclusion Our findings indicate that pazopanib induces SCLC cell apoptosis through the ER‐stress process via upregulation of ROS levels. Further investigation of relevant biomarkers to accurately select patients for benefit from pazopanib should be further investigated.

Heart failure is a type of heart disease that causes increased pressure in the heart or a decrease in cardiac output. Unfortunately, despite seriousness of the condition there is a lack of effective early diagnostic markers or effective pharmacotherapy. Atractylodes is known as a tonic herb in ethno-medical systems in East Asia, especially in China, for the treatment of gastrointestinal dysfunction, cancer, osteoporosis, obesity, and fetal irritability. Atractylodes Lactone III is the main active component of Atractylodes rhizome. Atractylodes Lactone III increases the survival and inhibits apoptosis of H9C2 cells induced by angiotensin II. Angiotensin II is closely involved in apoptosis, hypertrophy, and fibrosis of cardiomyocytes, and may be an important cause of the deterioration of cardiovascular disease. Although, Atractylodes Lactone III reduces angiotensin II-induced H9C2 cell hypertrophy by activating the phosphatidylinositol 3-kinase/protein kinase B pathway, the detailed mechanism remains poorly understood. Additionally, inhibition of phosphatidylinositol 3-kinase/protein kinase B pathway inhibits the myocardial protective effect of Atractylodes Lactone III. We therefore hypothesized that Atractylodes Lactone III could serve as a promising therapeutic drug for the treatment of heart failure. [ABSTRACT FROM AUTHOR]

Thyroid cancer is the most common type of endocrine malignancy. Although the general prognosis is good, the treatment of advanced disease is still challenging. Exosomes are vesicle units containing specific components that transmit information between cells. In order to explore its role in papillary thyroid cancer (PTC), our study screened exosome enriched lncRNA SNHG9 by lncRNA chip and explored its biological function. We used lncRNA chips combined with bioinformatics analysis to screen lncRNA SNHG9 enriched in exosomes. GO analysis suggested its relationship with autophagy and apoptosis. Quantitative PCR showed SNHG9 was highly expressed in PTC cells and exosomes and its correlation with PTC tumor size was analyzed by clinical characteristics. SNHG9 could inhibit the protective cell autophagy induced by starvation of human normal thyroid epithelial cell line Nthy-ori-3 and promote its apoptosis through PTC cell exosomes. RNA-pull down combined with protein spectrum showed that SNHG9 could interact with YBOX3. Western blot and RNA immunoprecipitation further confirmed their interaction. Western blot showed that SNHG9 could induce degradation of YBOX3, thus interfering with the stability of P21 mRNA and inducing cell apoptosis. In conclusion, our study identified SNHG9 as a PTC cell exosome-enriched lncRNA. SNHG9 could inhibit cell autophagy and promote apoptosis of Nthy-ori-3 cell through YBOX3/P21 pathway.

Podophyllotoxin (PPT) is a kind of lignans extracted from the roots and stems of the genus Podophyllum from the tiller family, and it has been widely used in the treatment of condyloma acuminatum, multiple superficial epithelioma in the clinics. However, PPT has been reported to be toxic and can cause liver defects and other organ poisoning. In addition, emerging evidences also indicate that PPT has reproductive toxicity and causes female reproduction disorders. In this study, we used fertilized oocytes and tried to explore the effects of PPT on the early embryonic development with the mouse model. The results showed that exposure to PPT had negative effects on the cleavage of zygotes. Further analysis indicated that PPT could disrupt the organization of spindle and chromosome arrangement at the metaphase of first cleavage. We also found that PPT exposure to the zygotes induced excessive reactive oxygen species (ROS), suggesting the occurrence of oxidative stress. Moreover, in the PPT-exposed embryos, there was positive γH2A.X and Annexin-V signals, indicating that PPT induced embryonic DNA damage and early apoptosis. In conclusion, our results suggested that PPT could affect spindle formation and chromosome alignment during the first cleavage of mouse embryos, and its exposure induced DNA damage-mediated oxidative stress which eventually led to embryonic apoptosis, indicating the toxic effects of PPT on the early embryo development.

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (N-acetyl-5-methoxytryptamine, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O2) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MTNR1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MTNR1B, we further confirmed the involvement of MT–MTNR1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.

Panax notoginseng possesses therapeutic potential for cardiovascular disorders. Endoplasmic reticulum (ER) stress plays a critical role in cardiovascular disease. Here we show that a rare ginsenoside-standardized extract (RGSE) from notoginseng exerts cardioprotection via attenuating ER stress-associated apoptosis. A total of 18 ginsenosides with a concentration of 816.75 mg/g were characterized and quantified in RGSE. Ginsenoside Rh4, Rg5, Rk3, and Rk1 were the most abundant. RGSE at 50 and 100 mg/kg significantly attenuated myocardial infarction and improved cardiac function in acute myocardial infarction (AMI). RGSE also prevented myocardial ischemia-induced oxidative stress and apoptosis in vivo and in vitro. In addition, RGSE decreased the overexpression of GRP78, GRP94 and CHOP, and inhibited the phosphorylation of PERK and IRE1α in ER stress. Further investigation revealed that RGSE effectually inactivated TXNIP/NLRP3 signaling pathway in AMI hearts. These observations suggest a novel role for notoginseng in regulating AMI.

Toxicological studies have shown that phthalates, a class of widely used chemicals, can impair male reproductive function, but epidemiological evidence is in consistent This study aimed to investigate the associations of semen phthalate metabolites with sperm apoptosis and DNA damage in a Chinese population. We assessed sperm apoptosis markers with Annexin V/PI analysis and sperm DNA integrity with comet assay before measuring eight phthalate metabolites in semen by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS ) among 463 men from Wuhan, China. We found a suggestive do se-response relationship between semen mono-(2-ethylhexyl) phthalate (MEHP) and an increased percentage of Annexin V7PF sperm (p for trend of <0.10). We also observed that semen monomethyl phthalate (MMP) and monoethyl phthalate (MEP) were associated with significant dose-related increases in tail length of the comet (both p for trend of < 0.01). In conclusion, our data indicate that semen MEHP is associated with increased sperm apoptosis and that semen MMP and MEP are associated with increased sperm DNA damage in a Chinese population. [ABSTRACT FROM AUTHOR]

Developmental abnormalities, neuronal apoptosis and associated cognitive impairment following isoflurane exposure in neonatal rodents have been reported. The study was undertaken to investigate the effect of vitamin C supplementation against isoflurane-induced neurotoxicity. Seven day old rats were exposed to 1.1% isoflurane, or air for 6 hours. Treatment groups were administered with vitamin C (30 mg/kg, orally) from postnatal day 1 (P1) to P10 and were exposed to isoflurane on P7. Isoflurane exposure induced apoptosis was determined by Fluoro-Jade C and terminal deoxynucleotidyltransferase- mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling assay. Vitamin C considerably improved memory and learning impairments, modulated neuroapoptosis and improved expressions of brain-derived neurotrophic factor, nerve growth factor, Bcl-xL and decreased activated caspase-3 expressions. Thus, vitamin C effectively offered protection against isoflurane-induced neuronal apoptosis, learning and memory disturbances. [ABSTRACT FROM AUTHOR]