Expression Levels Of B-cell Lymphoma Research Articles

Hepatoprotective Effect of Allium ochotense Extracts on Chronic Alcohol-Induced Fatty Liver and Hepatic Inflammation in C57BL/6 Mice.

This study was performed to investigate the protective effects of Allium ochotense on fatty liver and hepatitis in chronic alcohol-induced hepatotoxicity. The physiological compounds of a mixture of aqueous and 60% ethanol (2:8, w/w) extracts of A. ochotense (EA) were identified as kestose, raffinose, kaempferol and quercetin glucoside, and kaempferol di-glucoside by UPLC Q-TOF MSE. The EA regulated the levels of lipid metabolism-related biomarkers such as total cholesterol, triglyceride, low-density lipoprotein (LDL), and high-density lipoprotein (HDL)-cholesterol in serum. Also, EA ameliorated the levels of liver toxicity-related biomarkers such as glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and total bilirubin in serum. EA improved the antioxidant system by reducing malondialdehyde contents and increasing superoxide dismutase (SOD) levels and reduced glutathione content. EA improved the alcohol metabolizing enzymes such as alcohol dehydrogenase, acetaldehyde dehydrogenase, and cytochrome P450 2E1 (CYP2E1). Treatment with EA alleviated lipid accumulation-related protein expression by improving phosphorylation of AMP-activated protein kinase (p-AMPK) expression levels. Especially, EA reduced inflammatory response by regulating the toll-like receptor-4/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR-4/NF-κB) signaling pathway. EA showed an anti-apoptotic effect by regulating the expression levels of B-cell lymphoma 2 (BCl-2), BCl-2-associated X protein (BAX), and caspase 3. Treatment with EA also ameliorated liver fibrosis via inhibition of transforming growth factor-beta 1/suppressor of mothers against decapentaplegic (TGF-β1/Smad) pathway and alpha-smooth muscle actin (α-SMA). Therefore, these results suggest that EA might be a potential prophylactic agent for the treatment of alcoholic liver disease.

Hyperforin modulates MAPK/CCL11 signaling to reduce the inflammatory response of nasal mucosal epithelial cells caused by allergic rhinitis by targeting BCL6.

Hyperforin is a type of bicyclic tetraketone with four isoprenoid chains extracted from Hypericum perforatum L. that has multiple biological activities such as anti-diabetes, antitumor and anti-inflammation. However, the role and potential mechanism of hyperforin in allergic rhinitis (AR) remains to be clarified. In the present study, cell viability was analyzed using Cell Counting Kit-8 assay, while inflammation was detected using ELISA and reverse transcription-quantitative PCR. Epithelial cell barrier damage was measured using western blotting and immunofluorescence staining. The expression levels of B-cell lymphoma 6 (BCL6) and the p38 MAPK/C-C motif chemokine 11 (CCL11) pathway were detected using western blotting. In addition, the association between hyperforin and BCL6 was analyzed by SWISS TargetPrediction, DisGeNET, Gene Ontology and Pathway databases. Molecular docking was performed using AutoDockTools 1.5.6 and Discovery Studio 4.5 software. The data demonstrated that there were 16 interlinking target genes of hyperforin with AR, in which BCL6 was the most relevant one with hyperforin in AR. The binding between hyperforin and BCL6 was verified, and molecular docking was modeled. The results revealed that hyperforin inhibited IL-13-induced nasal epithelial inflammatory cytokine release and repressed the damage to the cellular barrier from IL-13 stimulation. In addition, hyperforin activated BCL6 expression and significantly suppressed the expression of p38 MAPK/CCL11. Silencing of BCL6 reversed the effects of hyperforin on IL-13-induced inflammation and barrier damage. In summary, the present results revealed that hyperforin suppressed IL-13-induced nasal epithelial cell inflammation and barrier damage by targeting BCL6/p38 MAPK/CCL11, which may provide promising therapeutic targets for AR.

Mechanism of albiflorin in improvement of Alzheimer's disease based on network pharmacology and in vitro experiments

This study aimed to explore the mechanism of albiflorin in the treatment of Alzheimer's disease(AD) based on network pharmacology, molecular docking, and in vitro experiments. Network pharmacology was used to predict the potential targets and pathways of albiflorin against AD, and molecular docking technology was used to verify the binding affinity of albiflorin to key target proteins. Finally, the AD cell model was induced by Aβ_(25-35) in rat pheochromocytoma(PC12) cells and intervened by albiflorin to validate core targets and pathways. The results of network pharmacological analysis showed that albiflorin acted on key targets such as mitogen-activated protein kinase-1(MAPK1 or ERK2), albumin(ALB), epidermal growth factor receptor(EGFR), caspase-3(CASP3), and sodium-dependent serotonin transporter(SLC6A4), and signaling pathways such as MAPK, cAMP, and cGMP-PKG. The results of molecular docking showed that albiflorin had strong binding affinity to MAPK1(ERK2). In vitro experiments showed that compared with the blank group, the model group showed decreased cell viability, decreased expression level of B-cell lymphoma 2(Bcl-2), increased Bcl-2-associated X protein(Bax), and reduced phosphorylation level of extracellular signal-regulated kinase 1/2(ERK1/2) and the relative expression ratio of p-ERK1/2 to ERK1/2. Compared with the model group, the albiflorin group showed potentiated cell viability, up-regulated expression of Bcl-2, down-regulated Bax, and increased phosphorylation level of ERK1/2 and the relative expression ratio of p-ERK1/2 to ERK1/2. These results suggest that the mechanism of albiflorin against AD may be related to its activation of the MAPK/ERK signaling pathway and its inhibition of neuronal apoptosis.

Enhanced Apoptosis in Choroidal Tissues in Lens-Induced Myopia Guinea Pigs by Activating the RASA1 Signaling Pathway.

PurposeThis study aimed to explore the role of the RAS p21 protein activator 1 (RASA1) signaling pathway in apoptosis in choroid tissues from guinea pigs with negative lens-induced myopia (LIM).MethodsBiometric measurements were performed to examine refractive status, ocular parameters, and choroidal thickness (ChT) after myopia induction. The choroidal morphology was observed by hematoxylin and eosin (H&E) staining and TUNEL assay. The expression of the RASA1 signaling pathway at the mRNA and protein levels in choroidal tissues was measured by real-time quantitative PCR (qPCR) and western blot assays.ResultsCompared with the normal control (NC) group, the ocular length of the guinea pigs in LIM increased remarkably, as did the myopic refraction. ChT decreased after myopia induction. H&E staining showed that the thickness and laxity of the choroidal tissues in LIM were strikingly reduced. The number of apoptotic cells in the LIM eyes was increased. Moreover, qPCR and western blot assays showed that the expression levels of both RASA1 and BCL-2-associated agonist of cell death (BAD) were higher in the LIM group than in the NC group, whereas the expression level of B-cell lymphoma 2 (BCL-2) was decreased after 2 weeks of experimental myopia. However, the trend of RASA1, BAD, and BCL-2 expression was reversed after 4 weeks of experimental myopia compared with levels after 2 weeks of experimental myopia.ConclusionsResults showed that the RASA1 signaling pathway is activated in choroid tissues in myopic guinea pigs. Activated RASA1 signaling induces high BAD expression and low BCL-2 expression, which in turn promotes apoptosis and ultimately causes ChT thinning in myopic guinea pigs.

Antitumor Potential of Withanolide Glycosides from Ashwagandha (Withania somnifera) on Apoptosis of Human Hepatocellular Carcinoma Cells and Tube Formation in Human Umbilical Vein Endothelial Cells.

Hepatocellular carcinoma (HCC) is the fastest-growing tumor capable of spreading to other organs via blood vessels formed by endothelial cells. Apoptosis and angiogenesis-targeting therapies are attractive for cancer treatment. In this study, we aimed to study the in vitro cytotoxicity of Withania somnifera against human HCC (HepG2) cells, identify potential antitumoral withanolide glycosides from the active fraction, and elucidate cytotoxic molecular mechanisms of identified bioactive compounds. W. somnifera (Solanaceae), well-known as ‘ashwagandha’, is an Ayurvedic medicinal plant used to promote health and longevity, and the MeOH extract of W. somnifera root exhibited cytotoxicity against HepG2 cells during initial screening. Bioactivity-guided fractionation of the MeOH extract and subsequent phytochemical investigation of the active n-BuOH-soluble fraction resulted in the isolation of five withanolide glycosides (1–5), including one new metabolite, withanoside XIII (1), aided by liquid chromatography–mass spectrometry-based analysis. The new compound structure was determined by 1D and 2D nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectroscopy, electronic circular dichroism, and enzymatic hydrolysis. In addition, withanoside XIIIa (1a) was identified as the new aglycone (1a) of 1. Isolated withanolide glycosides 1–5 and 1a were cytotoxic toward HepG2 cells; withagenin A diglucoside (WAD) (3) exhibited the most potent cytotoxicity against HepG2 cells, with cell viability less than 50% at 100 μM. WAD cytotoxicity was mediated by both extrinsic and intrinsic apoptosis pathways. Treatment with WAD increased protein expression levels of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Bcl-2-associated X protein (Bax), and cleaved poly(ADP-ribose) polymerase (cleaved PARP) but decreased expression levels of B-cell lymphoma 2 (Bcl-2). Moreover, WAD inhibited tubular structure formation in human umbilical vein endothelial cells (HUVECs) by inhibiting the protein expression of vascular endothelial growth factor receptor 2 and its downstream pathways, including extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). These effects were also enhanced by co-treatment with ERK and PI3K inhibitors. Overall, these results indicate that WAD (3) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers.

Cardiac Protection of a Novel Lupane-Type Triterpenoid from Injuries Induced by Hypoxia-Reperfusion.

Myocardial ischemia–reperfusion injury (MIRI) leads to cardiac remodeling and heart failure associated with acute myocardial infarction, which is one of the leading causes of death worldwide. Betulinic acid (BA), a widely distributed lupane-type triterpenoid, has been reported to possess antioxidative activity and inhibit apoptosis in MIRI. Due to the low bioavailability and water insolubility of BA, a previous study found a series of BA-derivative compounds by microbial transformation. In this study, we observe whether there are anti-MIRI effects of BTA07, a BA derivative, on cardiac injuries induced by hypoxia/reoxygenation (H/R) in adult rat cardiomyocytes in vitro and in Langendorff-perfused hearts ex vivo, and further explore its mechanism of cardioprotection to find more efficient BA derivatives. The hemodynamic parameters of isolated hearts were monitored and recorded by a Lab Chart system. The markers of oxidative stress and apoptosis in isolated hearts and adult rat cardiomyocytes (ARCMs) were evaluated. The expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), protein kinase B (Akt) and phospho-Akt (pAkt, Ser473) induced by H/R were detected via Western blot. The Langendorff experiments showed that BTA07 improves hemodynamic parameters, reduces myocardium damage and infarct size, inhibits levels of myocardial tissue enzymes lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary outflow and reduces oxidative stress and the activation of caspase-3 in the myocardium. In vitro, BTA07 reduced cell death and caspase-3 activation and inhibited reactive oxygen species (ROS) generation. Furthermore, the protective effects of BTA07 were attenuated by inhibition of the PI3K/Akt signaling pathway with LY294002 in ARCMs. BTA07 protects ARCMs and isolated hearts from hypoxia–reperfusion partly by inhibiting oxidative stress and cardiomyocyte apoptosis.

Human umbilical vein endothelial cells-derived exosomes enhance cardiac function after acute myocardial infarction by activating the PI3K/AKT signaling pathway

ABSTRACT Currently, acute myocardial infarction (AMI) is one of the leading causes of human health issues worldwide. The sudden and continuous occlusion of the coronary artery results in myocardial hypoxic-ischemic necrosis, which is accompanied by inflammatory infiltration and fibrosis, leading to pathological cardiac remodeling. Exosome-based therapy is a promising cell-free approach for repairing the ischemic myocardium. This study aimed to explore the effects and mechanism of human umbilical vein endothelial cells (HUVECs)-derived exosomes on AMI. The results indicated that the localized injection of HUVECs-derived exosomes in the infarcted area could significantly improve cardiac function in AMI mouse models. It could also ameliorate myocardial fibrosis and decrease infarct size after AMI. Additionally, HUVECs-derived exosomes had cardioprotective effects on the H9C2 cells in hypoxic culture conditions, including increased cell viability and decreased lactate dehydrogenase (LDH) release. In both the in-vivo and in-vitro experiments, HUVECs-derived exosomes could effectively inhibit cardiomyocyte apoptosis. The low expression levels of Bcl-2–associated X protein (Bax) and cleaved caspase-3, high expression levels of B-cell lymphoma 2 (Bcl-2), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) were detected in AMI mouse models treated with HUVECs-derived exosomes in-vivo. In conclusion, HUVECs-derived exosomes effectively enhanced cardiac function after AMI and inhibited cardiomyocyte apoptosis, which might be regulated through the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) signaling pathway.

Combination of sodium butyrate and decitabine promotes transgene expression in CHO cells via apoptosis inhibition

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for production of recombinant therapeutic proteins (RTPs). Small-molecule additives related to cell cycle apoptosis and autophagy regulation have been used to promote RTP production. By combining two small-molecule additives, positive synergistic effects on transgene expression were observed in CHO cells. In the present study, six small-molecule additives were used, including hydrocinnamic acid (HCA), sodium butyrate (NaB), lithium acetate (LiAc), sodium succinate dibasic hexahydrate (SDH), decitabine (DAC), and sodium propionate (SP). Experiments to test the effects of their pairwise combinations on two different recombinant CHO cell lines (rCHO) were designed using Design-Expert 12.0. Different effects of various pairs of small molecules on apoptosis- and autophagy-related protein expression were observed in the rCHOs. The results showed that compared to the control culture, NaB alone increased the volumetric yield and specific productivity (Qp) by 166% and 143%, respectively. The volumetric yield and Qp of NaB combined with DAC (Cg1)-treated cells increased by 178% and 212%, respectively. Cg1 selectively blocked the cells in the G0/G1 cell cycle stage. The relative expression levels of B-cell lymphoma 2 (Bcl-2), Beclin 1, and microtubule-associated protein light chain 3 (LC3B) in Cg1-treated CHO cells were significantly increased, while relative levels of cleaved caspase-3 expression were significantly decreased. In conclusion, Cg1 had the most obvious effect on RTP production and Qp in CHO cells, suggesting the Cg1 combination of small molecules may be used to improve the expression of recombinant protein in CHO cells.

Long non-coding RNA ncRuPAR regulates gastric cancer cell proliferation and apoptosis via phosphoinositide 3-kinase/protein kinase B signaling.

Objective: To determine the effect and mechanism of the long non-coding RNA (lncRNA) ncRuPAR (non-protein coding RNA, upstream of coagulation factor II thrombin receptor [F2R]/protease-activated receptor-1 [PAR-1]) in human gastric cancer. Methods: HGC-27-ncRuPAR overexpression and MGC-803-ncRuPAR-RNAi knockdown gastric cancer cell lines were established. We assessed the effect of ncRuPAR on cell proliferation, apoptosis, migration, and invasion using Cell Counting Kit 8, flow cytometry, scratch and transwell assays, respectively. Differentially expressed genes in HGC-27-ncRuPAR overexpression and HGC-27-empty vector cell lines were identified using Affymetrix GeneChip microarray analysis. Ingenuity Pathway Analysis (IPA) of the microarray results was subsequently conducted to identify ncRuPAR-enriched pathways, followed by validation using real time-quantitative PCR (RT-qPCR). As one of the top enriched pathways, phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was further examined by western blotting to determine its role in ncRuPAR-mediated regulation of gastric cancer pathogenesis. Results: ncRuPAR inhibited human gastric cancer cell proliferation and induced G1/S phase arrest and apoptosis, but did not affect migration or invasion in vitro. Overexpression of ncRuPAR in vitro was found to inhibit its known target PAR-1, as well as PI3K/Akt signaling. The downstream targets of PI3K/Akt, cyclin D1 was downregulated, but there was no change in expression level of B-cell lymphoma 2 (Bcl-2). Conclusions: We showed that lncRNA-ncRuPAR could inhibit tumor cell proliferation and promote apoptosis of human gastric cancer cells, potentially by inhibiting PAR-1, PI3K/Akt signaling, and cyclin D1. The results suggest a potential role for lncRNAs as key regulatory hubs in GC progression.

Transferrin-conjugated liposomes loaded with carnosic acid inhibit liver cancer growth by inducing mitochondria-mediated apoptosis

Our previous studies have proven that carnosic acid (CA) induces apoptosis of liver cancer cells. However, the poor chemical properties of CA limit its in vivo anti-cancer effects. In this study, CA was loaded into liposomes (LP-CA), and LP-CA was further conjugated with transferrin (Tf-LP-CA) to overcome the shortcomings of poor solubility and absorption at the lesion site. In HepG2 and SMMC-7721 cells, compared with CA and LP-CA, more Tf-LP-CA was absorbed by liver cancer cells, which induced higher levels of apoptosis and reduced the mitochondrial membrane potential more effectively. In HepG2- and SMMC-7721-xenotransplanted mice, Tf-LP-CA inhibited tumor growth with no cytotoxicity to the liver, spleen, or kidney. Furthermore, compared with CA and LP-CA, Tf-LP-CA targeted the tumor site more effectively, enhanced the expressions of cleaved poly(ADP-ribose) polymerase, and Caspase-3 and -9, and regulated the expression levels of B-cell lymphoma 2 (Bcl2) family members in the tumor tissues. Tf-LP-CA was taken up by tumor cells and targeted at tumor tissues, ensuring the precise delivery of CA, which further promoted mitochondria-mediated intrinsic apoptosis in the liver cancer cells. These results provide evidence for the clinical application of the Tf-LP-based CA drug delivery system for liver cancer.

Effect of MiR-210 on the Chemosensitivity of Breast Cancer by Regulating JAK-STAT Signaling Pathway.

The study is aimed at exploring the effect of microribonucleic acid- (miR-) 210 on the chemosensitivity of breast cancer and its potential molecular mechanism. Cell Counting Kit-8 (CCK-8) was applied to detect the half maximal inhibitory concentration (IC50) of cisplatin (DDP) on cell, and quantitative polymerase chain reaction (qPCR) was carried out to measure the relative expression level of miR-210. The IC50 value of DDP on cells was detected via CCK-8 after downregulating the expression of miR-210 in MCF-7/DDP cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) confirmed the effect of themiR-210 downregulation on the apoptosis of drug-resistant MCF-7/DDP cells. Besides, the impacts of the miR-210 downregulation on apoptosis-related proteins and Janus-activated kinase- (JAK-) signal transducer and activator of transcription (STAT) signaling pathway-related proteins were examined by Western blotting. The interaction between miR-210 and the target protein was detected through luciferase activity assay, qPCR, and Western blotting. Drug-resistant MCF-7/DDP cells had significantly stronger resistance to DDP and a remarkably higher expression level of miR-210 than control parental MCF-7 cells (p < 0.05). After the downregulation of the miR-210 expression, MCF-7/DDP cells had markedly reduced resistance but obviously increased sensitivity to DDP (p < 0.05). MiR-210 downregulation increased the apoptosis of MCF-7/DDP cells (p < 0.05). In addition, after miR-210 was knocked down, the expression level of b-cell lymphoma 2 (Bcl-2) was decreased, while the expression levels of Bcl-2-associated X protein (Bax) and cysteinyl aspartate-specific proteinase-3 (caspase-3) were increased. Besides, miR-210 was able to suppress the expression of protein inhibitor of the activated STAT 4 (PIAS4) gene by directly targeting its 3′ untranslated region (3′UTR). The expression of miR-210 has a correlation with chemoresistance of breast cancer MCF-7 cells. MiR-210 regulates the JAK-STAT signal transduction pathway by targeting PIAS4, thus exerting an effect on breast cancer chemosensitivity.

Low temperature exerts protective effects by inhibiting mitochondria-mediated apoptosis pathway following pressure injury to rat muscle.

We aimed to determine the effect of different low-temperature range interventions at different time-points in a rat model of pressure injury (PI) produced by Ischemia/Reperfusion (I/R) injury. Sprague-Dawley rats were randomly assigned to blank control, injury control, and temperature intervention groups. Rats in the injury control and temperature intervention groups (involving exposure to different temperature range at different time-points) were subjected to three cycles of I/R injury with 2-h ischemia and 0.5-h reperfusion to induce PI. The muscle tissues exhibited degenerative changes after compression. Low temperature intervention of 16-18°C in the ischemia period resulted in the lowest degree of tissue damage and significantly decreased levels of Bcl-2-associated X protein (Bax), caspase-9, and caspase-3. Moreover, it resulted in the highest expression level of B-cell lymphoma 2 (Bcl-2) and lowest expression levels of Bax, caspase-9, and caspase-3 in muscle tissues among all intervention groups. Low-temperature intervention at 16-18°C during the ischemia period showed optimal effects on the expressions of apoptotic factors during the development of PI with I/R-induced tissue damage.

Interleukin-33 promotes proliferation and inhibits apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis

The aim of our study was to determine the effect of interleukin (IL)-33 on the proliferation, apoptosis, and secretion of inflammatory cytokines by fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) and to investigate the underlying mechanisms. Cultured RA FLSs and osteoarthritis (OA) FLSs were cocultured with different concentrations of IL-33. TUNEL assay and flow cytometry were used to detect apoptosis. Western blotting and Real-time (RT)-PCR were used to detect the expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), while the Cell Counting Kit-8 was used to determine cell proliferation in each cocultured group. Enzyme-linked immunosorbent assay was used to detect the expression levels of tumour necrosis factor (TNF)-α and IL-6 in the supernatant from each cell culture. Western blot analysis was used to determine the phosphorylated expression levels of the nuclear factor-kappa light chain enhancer of the activated B cells (NF-κB) pathway in each group. IL-33 inhibited RA FLS apoptosis, promoted FLS proliferation, increased Bcl-2 protein expression levels, and decreased Bax protein expression levels. It also increased the expression levels of inflammatory cytokines TNF-α and IL-6 and increased the expression levels of P-NF-κ B in FLSs. IL-33 inhibited apoptosis and promoted proliferation of FLSs; in addition, IL-33 increased the serum levels of inflammatory cytokines. The effect of IL-33 on RA FLSs was likely mediated via the NF-κB pathway.

Photodynamic Effects of Vitamin K3 on Cervical Carcinoma Cells Activating Mitochondrial Apoptosis Pathways.

Photodynamic Therapy (PDT) is a photoactivation or photosensitization process, wherein vitamin K3 (Vit K3) serves as a photosensitizer to produce Reactive Oxygen Species (ROS) against bacteria at appropriate wavelengths. In this study, we used Vit K3 treatment combined with Ultraviolet radiation A (UVA) to produce photodynamic effects on cervical cancer. The dose-concentration relationship between Vit K3 treatment and UVA on tumor cells was analyzed through the Cell Counting Kit-8 method. Then, the morphological characteristics of apoptosis cells were observed through fluorescent staining and fluorescence microscopy. Apoptosis after treatment with Vit K3 treatment, UVA, and Vit K3 treatment plus UVA was further observed through Western blot analysis, flow cytometry, and TUNEL assay. The xenograft models from HeLa cells were established for the exploration of the photodynamic effect of Vit K3 treatment on cervical cancer in vivo. Vit K3 treatment plus UVA reduced tumor cell viability in a dose-dependent manner. Further studies indicated that Vit K3 treatment plus UVA can inhibit tumor growth and enhance the apoptosis of cervical cancer cells. In the combination group, the expression levels of cleaved caspase-3, cleaved caspase-9, B-cell lymphoma- extra large (Bcl-xl), and cytochrome c (cyt-c) increased obviously, whereas the expression level of Bcell lymphoma 2 (Bcl-2) decreased relative to the expression levels of UVA- or Vit K3-treated cells. In the in vivo experiments, tumor growth was inhibited significantly in the VitK3 treatment plus UVA group. Additionally, we demonstrated that the combination therapy mediated an increase in cleaved caspase-3 and cleaved caspase-9 expression and decrease in Bcl-2 expression in vivo. Our results showed that Vit K3 treatment combined with UVA exerted photodynamic effects on cervical cancer cells by activating mitochondrial apoptosis pathways.

MiR-219a-2 relieves myocardial ischemia-reperfusion injury by reducing calcium overload and cell apoptosis through HIF1α/ NMDAR pathway

ObjectiveDuring the process of myocardial ischemia-reperfusion injury (MIRI), the intracellular Ca2+ concentration ([Ca2+]i) continues to increase, leads to the cardiomyocyte apoptosis and eventually causes myocardial damage, while the upstream regulation mechanism of calcium overload is still unknown. This study focuses on the role of miR-219a-2 in MIRI and aims to elaborate its regulatory mechanism on calcium overload that occurs during MIRI. MethodsThe expression of miR-219a-2 was determined in the heart tissues of MIRI mice by qRT-PCR. The [Ca2+]i was measured by fluo-3 using a fluorescence microplate reader. The expression of hypoxiainducible factor 1α (HIF1α) and NR1, the obligatory subunit of N-methyl-d-aspartate receptor 1 (NMDAR), were measured by qRT-PCR and western blot. The luciferase reporter assay was used to confirm the interplay between miR-219a-2 and HIF1α and the interplay between HIF1α and NR1. The cell apoptosis was measured by the expression level of B-cell lymphoma 2 interacting mediator of cell death (Bim) and the number of TUNEL-positive cells. The myocardial infarct size of mice was measured by TTC/Evans Blue staining. ResultsMiR-219a-2 was down-regulated in the heart tissues of MIRI mice. miR-219a-2 overexpression decreased [Ca2+]i and the expression of HIF1α and NR1 in hypoxia/reoxygenation (H/R)-treated HL-1 cells. Then, the luciferase reporter assay showed that miR-219a-2 inhibited the transcription of HIF1α and HIF1α promoted the transcription of NR1. Both HIF1α overexpression and NMDAR function enhancement removed the inhibitory effect of miR-219a-2 on calcium overload and cell apoptosis in H/R-treated HL-1 cells. Finally, the overexpression of miR-219a-2 decreased Ca2+ concentration, cell apoptosis, and myocardial infarction size in MIRI mice, while the NMDAR function enhancer reversed the therapeutic effect of miR-219a-2. ConclusionMiR-219a-2 reducing NMDAR-mediated calcium overload via HIF1α/NR1 axis, thus alleviating cell apoptosis in MIRI.

Kaempferide Induces G0/G1 Phase Arrest and Apoptosis via ROS-Mediated Signaling Pathways in A549 Human Lung Cancer Cells

Kaempferide is an O-methylated flavonol that has received much attention due to its various biological activities. In this study, we explored the underlying mechanisms of kaempferide in human lung cancer A549 cells. The Cell Counting Kit-8 (CCK-8) assay, Hoechst 33342/propidium iodide double staining, flow cytometry, scratch wound healing assay, and Western blot analysis were used to measure cell apoptosis, the cell cycle, reactive oxygen species (ROS) levels, and cell migration of human lung cancer cells. Kaempferide significantly inhibited human lung cancer cell proliferation, and its toxic effects on normal cells were significantly lower than those of 5-fluorouracil. Kaempferide induced A549 cell apoptosis by decreasing the mitochondrial membrane potential and the expression level of B-cell lymphoma 2, and by increasing the expression levels of Bcl-2-associated X protein and caspase-3. It also regulated mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) signaling pathways by increasing the expression levels of phosphorylated c-Jun N-terminal kinase, p-p38, I kappa B, and by decreasing the expression levels of phosphorylated extracellular signal-regulated kinase, p-STAT3, and NF-κB. Kaempferide induced cell cycle arrest in the G0/G1 phase in A549 cells by downregulating the expression levels of p-AKT, cyclin D1, and cyclin-dependent kinase 2. Furthermore, kaempferide blocked A549 cell migration by downregulating the expression levels of transforming growth factor beta 1 (TGF-β1), p-β-catenin, p-glycogen synthase kinase 3 beta, N-cadherin, and vimentin, and by upregulating the expression level of E-cadherin. Kaempferide enhanced the accumulation of ROS, and N-acetyl-l-cysteine (a ROS inhibitor) decreased the regulation of MAPK, NF-κB, AKT, and TGF-β signaling pathways by kaempferide, inhibited cell apoptosis, and reversed cell cycle arrest. Our results showed that kaempferide induced apoptosis via ROS-mediated MAPK, NF-κB, AKT, and TGF-β signaling pathways in A549 cells. Thus, kaempferide may be a novel drug candidate for lung cancer.

Dexmedetomidine pretreatment protects the heart against apoptosis in ischemia/reperfusion injury in diabetic rats by activating PI3K/Akt signaling in vivo and in vitro

Dexmedetomidine (DEX) exerts cardioprotection against ischemia/reperfusion injury. However, the precise mechanisms underlying this cardioprotective effect in diabetic rats are still not fully understood. The aim of the present study was to investigate the cardioprotective mechanism of DEX pretreatment on myocardial ischemia/reperfusion (I/R) injury in diabetic rats. A total of 25 streptozotocin-induced diabetic rats were equally randomized into five groups: i) Sham, ii) DEX (100 μg/kg); iii) myocardial I/R; iv) myocardial I/R+DEX (10 μg/kg); and v) myocardial I/R+DEX (100 μg/kg) groups. Primary cardiomyocytes were cultured in DEX for 1 h, and then oxygen and glucose deprivation (OGD)/R for 36 h. These results showed that pretreatment with DEX significantly decreased the I/R-induced size of the myocardial infarction, structural damage, morphological changes and apoptosis in myocardial cells, as well as levels of creatinine kinase, malondialdehyde and cardiac troponin I, and increased the I/R-induced superoxide dismutase activity in vivo and in vitro. Furthermore, immunohistochemical staining and western blot analysis revealed that DEX pretreatment significantly increased the I/R-induced expression levels of B-cell lymphoma 2 (Bcl-2), phosphorylated phosphoinositide 3-kinase (pPI3K) and pAkt, and significantly decreased those of pBcl-2 associated agonist of cell death, Bcl-2-associated X protein and cleaved caspase 3 in vivo and in vitro. In addition, all of these cardioprotective effects of DEX were reversed by yohimbine and LY294002 pretreatment. These results suggested that DEX pretreatment may activate the PI3K/Akt signaling pathway in an α2 adrenoceptor-dependent manner. DEX pretreatment may exert cardioprotective effects against myocardial ischemia/reperfusion injury in diabetic rats through the I/R-induced inhibition of cell apoptosis by activating the PI3K/Akt signaling pathway.

Ulinastatin improves myocardial ischemia-reperfusion injury in rats through endoplasmic reticulum stress-induced apoptosis pathway.

To investigate the protective role of ulinastatin (UTI) on myocardial ischemia-reperfusion (I/R) injury in rats via endoplasmic reticulum stress (ERS)-induced apoptosis pathway. A total of 60 rats were randomly divided into normal group (n=20), myocardial I/R model group (model group, n=20), and myocardial I/R model+UTI treatment group (treatment group, n=20). The myocardial function indicators [creatinine (Scr) and creatine kinase (CK)] were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to measure serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and matrix metalloproteinase-9 (MMP-9). Meanwhile, the contents of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) in rat left ventricular tissues were determined by ELISA as well. The cardiac function indexes were determined via magnetic resonance imaging (MRI) and echocardiography (ECG). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect the apoptosis of myocardial tissues. Additionally, the expression levels of endoplasmic reticulum stress and apoptosis genes were measured through quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay and Western blotting analysis, respectively. Serum levels of alanine aminotransferase (ALT), CK, and Scr in model group were significantly higher than those in normal group (p<0.05). Besides, rats in model group had significantly lowered SOD, ejection fraction (EF, %), and fractional shortening (FS, %) than those in normal group (p<0.05). In addition, remarkably increased contents of TNF-α, IL-6, MMP-9, MDA, and ROS, as well as higher left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd) were observed in model group in comparison with normal group (p<0.05). TUNEL staining results revealed that there were more apoptotic cells in model group than that in the other two groups (p<0.05). Expression levels of cysteine aspartic acid-specific protease 12 (Caspase-12) and glucose-regulated protein 78 (GRP78) were evidently higher in model group than those in normal group (p<0.05), while the expression level of B-cell lymphoma 2 (Bcl-2) was clearly lower in model group than that in normal group (p<0.05). UTI treatment partially reversed the above expression changes (p<0.05). UTI has a protective effect against myocardial I/R injury in rats by repressing the occurrence of ERS-induced apoptosis.

M1 macrophage-derived exosome-encapsulated cisplatin can enhance its anti-lung cancer effect.

The aim of this study was to study the efficacy of cisplatin-loaded M1 macrophage secreted-exosomes (DDP-M1-Exos) in enhancing DDP antitumor effect. M1-Exos were first extracted using density gradient centrifugation, and the DDP-M1-Exos system was established via electroporation. Then, the morphology, particle size and maker proteins of the DDP-M1-Exos system were assessed using transmission electron microscope, DLS and Western blotting (WB), respectively. The uptake of Exos by mouse Lewis lung cancer (LLC) cells was observed under a confocal laser scanning microscope, and the influence of the DDP-M1-Exos system on the viability of Lewis cells was determined using methyl thiazolyl tetrazolium assay and 4',6-diamidino-2-phenylindole staining. Besides, its impacts on the messenger ribonucleic acid (mRNA) levels and protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and Caspase-3 in LLC cells were examined using quantitative real-time PCR (qRT-PCR) and WB, respectively. Finally, with the LLC-bearing mouse model as the object of study, the efficacy was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and hematoxylin-eosin staining staining in vivo. Saucer-shaped Exos with a bilayer membrane, uniform particle size and highly expressed CD81, Alix and HSP70 were successfully isolated from M1 macrophages via density gradient centrifugation, and they were absorbed by LLC cells. Meanwhile, the DDP-M1-Exos drug delivery system was successfully prepared using electroporation technique. Compared with Control group, DDP and DDP-M1-Exos groups exhibited a decrease in the proliferation rate of mouse LLC cells and an increase in their apoptosis rate (*P<0.05), and the apoptosis rate in DDP-M1-Exos group was substantially higher than that in DDP group (#P<0.01). According to the qRT-PCR and WB results, DDP-M1-Exos up-regulated Bax and Caspase-3 in the apoptosis signaling pathway to induce tumor cell apoptosis, thereby resisting tumors. Moreover, through in-vivo experiments, it was found that M1-Exos alone had a potential to suppress tumor growth, and that the carrier of M1-Exos could not only kill tumor cells, but also encapsulate DDP to enhance its anti-lung cancer effect. The DDP-M1-Exos drug delivery system is prepared using Exos extracted via density gradient centrifugation by electroporation, and the drug-loaded Exos are able to effectively inhibit the proliferation of tumor cells and induce their apoptosis, exerting an antitumor effect.

Effect of TGF-β1 on myocardial cell apoptosis in rats with acute myocardial infarction via MAPK signaling pathway.

Transforming growth factor beta 1 (TGF-β1) can promote myocyte hypertrophy, thus playing an important role in ventricular remodeling after myocardial infarction (MI). In this study, the model of MI was established in rats through ligating the left anterior descending coronary artery. Subsequently, the messenger ribonucleic acid (mRNA) and protein expression levels of TGF-β1 in myocardial cells in both model group and sham operation group were determined. The effects of TGF-β1 treatment on myocardial cell apoptosis in MI rats were explored. Moreover, the changes of mitogen-activated protein kinase (MAPK) signaling pathway in rats with acute MI were verified. In addition, the protein expressions of phosphorylated-MAPK kinases 3/6 (p-MKK3/6) and MKK3/6 in myocardial cells of the two groups were analyzed. The mRNA and protein expression levels of TGF-β1 in myocardial cells of acute MI rats were significantly higher than those in the sham operation group (p<0.01). After treatment with TGF-β1, the expression level of B-cell lymphoma 2 (Bcl-2) associated X protein (Bax) was obviously down-regulated. The Bax/Bcl-2 ratio was notably lower than that in control group (p<0.01). Meanwhile, the proportion of apoptotic cells decreased remarkably (p<0.01). In the model group, no evident change was observed in the protein expression level of MKK3/6, whereas the levels of p-MKK3/6 were prominently up-regulated (p<0.01). TGF-β1 can activate MKK3/6 in the MAPK signaling pathway to resist the apoptosis of myocardial cells in acute MI rats.