Protein Expression Levels Of Caspase-3 Research Articles

IL-39 increases ROS production and promotes the phosphorylation of p38 MAPK in the apoptotic cardiomyocytes.

The cytokine interleukin (IL)-39 is a novel member of the IL-12 family. Our previous study found that the serum level of IL-39 significantly increased in patients with acute myocardial infarction. However, the role of IL-39 in cardiomyocyte apoptosis remains unclear. In this study, the cultured mouse HL-1 cardiomyocytes were incubated with PBS, 0-100 ng/mL IL-39, 200 μM H2O2 or 20 μM Trolox. IL-39 promoted the production of intracellular reactive oxygen species (ROS) in a concentration dependent manner in HL-1 cardiomyocytes. IL-39 and H2O2 both significantly promoted the production of intracellular ROS, increased the level of intracellular CCL2, stimulated the apoptotic progress of cardiomyocytes, increased the mRNA and protein expression levels of Bax, caspase-3, and p-p38 MAPK, and decreased the mRNA and protein expression levels of Bcl-2. ROS production, CCL2 level, cardiomyocyte apoptosis, and expression of Bax, caspase-3, and p-p38 MAPK were significantly amplified by the administration of IL-39 combined with H2O2, and these processes were significantly alleviated by an antioxidant Trolox. This study was novel in revealing that IL-39 promoted apoptosis by stimulating the phosphorylation of p38 MAPK in mouse HL-1 cardiomyocytes.

Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines

IntroductionDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. MethodsThe cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. ResultsTreatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. ConclusionDHA might be a potential anti-cancer therapeutic for CLL.

The inducing of caspase and Bcl-2 pathway with royal jelly decreases the muscle tissue damage exposed with fluoride in rats.

In this study, 42 Wistar albino male rats (n = 42, 8 weeks old) were used. Rats were divided into 6 groups and 7 rats included each group. Groups: (i) Control group: Standard diet; (ii) RJ (royal jelly) group: Standard diet + royal jelly; (iii) F50 group: Standard diet + 50 mg/kg fluoride; (iv): F100 group: Standard diet + 100 mg/kg fluoride; (v) F50+RJ group: Standard diet + 50 mg/kg fluoride + royal jelly; (vi): F100+RJ group: Standard diet + 100 mg/kg fluoride + royal jelly. After 8 weeks, the rats were decapitated, and their muscle tissues were removed. Expression levels of Caspase-3, Caspase-6, Bax, tumor necrosis factor-α (TNF-α), interleukin 1 alpha (IL1-α) and Bcl-2 proteins in muscle tissue were determined by western blotting method. Histopathological analyses were also performed on the muscle tissue. Malondialdehyde (MDA), glutathione (GSH) and catalase (CAT) analyses were determined by a spectrophotometer. According to the obtained results, Bcl-2, TNF-α and IL1-α protein expression was increased in damage groups compared to the control and royal jelly groups, while Caspase-3, Caspase-6 and Bax protein expression levels decreased in damage groups. MDA level increased in damage groups compared to the control and royal jelly groups, while CAT and GSH levels increased with royal jelly application in royal jelly-given group in comparison to the flouride-exposed group. According to histopathological analysis results, edema and inflammatory cell formations were found in the injury groups, a tendency to decrease in these injuries was observed in the treatment groups. Based on these results, we can say that royal jelly has protective effects on muscle tissue against fluoride damage.Graphical abstract

Study on anti-inflammatory activity and mechanism of indolealkylamines in toad skin on LPS-activated neutrophils

Indolealkylamines(IAAs) are the main hydrophilic substances in toad skin, mainly including free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine. In this study, the LPS-activated neutrophils were used to investigate the structure-activity relationship and anti-inflammatory mechanism of the above-mentioned five monomers from the toad skin in vitro. The neutrophils were divided into the control group, model group(1 μg·mL~(-1) LPS), positive drug group(100 μg·mL~(-1) indometacin), as well as the low-(50 μg·mL~(-1)), medium-(100 μg·mL~(-1)) and high-dose(200 μg·mL~(-1)) free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine groups. The levels of IL-6, TNF-α and IL-1β in the neutrophil supernatant of each group was measured by enzyme-linked immunosorbent assay(ELISA) after LPS stimulation, followed by the detection of apoptosis in each group after Annexin V/PI staining. The protein expression levels of caspase-3, Bax, Bcl-2, beclin1, LC3-I, and LC3-Ⅱ were assayed by Western blot. The results showed that IAAs reduced the excessive secretion of inflammatory cytokines caused by LPS compared with the model group. Besides, the activity of each free IAAs(N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine and dehydrobufotenine), especially bufotenine, was stronger than that of the binding bufothionine. As revealed by Annexin V/PI staining, LPS delayed the early apoptosis of neutrophils compared with the control group, while bufotenine promoted the apoptosis of neutrophils in a dose-dependent manner, which might be related to the elevated expression of apoptosis-related protein Bax/Bcl-2. In addition, LPS activated the autophagy pathways in neutrophils. This study confirmed the efficacy of IAAs in reducing the secretion of inflammatory cytokines in neutrophils induced by LPS for the first time. For instance, bufotenine exerts the anti-inflammatory effect possibly by inducing the apoptosis of neutrophils.

Innate immunity receptors in depression and suicide: upregulated NOD-like receptors containing pyrin (NLRPs) and hyperactive inflammasomes in the postmortem brains of people who were depressed and died by suicide.

Background:Abnormalities of inflammation have been implicated in the pathophysiology of depression and suicide, based on observations of increased levels of proinflammatory cytokines in the serum of people who were depressed and died by suicide. More recently, abnormalities in cytokines and innate immunity receptors such as toll-like receptors have also been observed in the postmortem brains of people who were depressed and died by suicide. In addition to toll-like receptors, another subfamily of innate immunity receptors known as NOD-like receptors containing pyrin (NLRPs) are the most widely present NOD-like receptors in the central nervous system. NLRPs also form inflammasomes that play an important role in brain function. We studied the role of NLRPs in depression and suicide.Methods:We determined the protein and mRNA expression of NLRP1, NLRP3 and NLRP6 and the components of their inflammasomes (i.e., adaptor molecule apoptosis-associated speck-like protein [ASC], caspase1, caspase3, interleukin [IL]-1β and IL-18) postmortem in the prefrontal cortex of people who were depressed and died by suicide, and in healthy controls. We determined mRNA levels using quantitative polymerase chain reaction, and we determined protein expression using Western blot immunolabelling.Results:We found that the protein and mRNA expression levels of NLRP1, NLRP3, NLRP6, caspase3 and ASC were significantly increased in people who were depressed and died by suicide compared to healthy controls.Limitations:Some people who were depressed and died by suicide were taking antidepressant medication at the time of their death.Conclusion:Similar to toll-like receptors, NLRP and its inflammasomes were upregulated in people who were depressed and died by suicide compared to healthy controls. Innate immunity receptors in general — and NLRPs and inflammasomes in particular — may play an important role in the pathophysiology of depression and suicide.

Profiles of messenger RNAs and MicroRNAs in hypoxia-induced hepatic stellate cells.

BackgroundMicroRNA (miRNA) plays an important role in hepatic stellate cell (HSCs) activation and liver fibrosis. The purpose of this study is to explore the effect of hypoxia on the differential expression of mRNAs and miRNAs in rat HSCs.MethodsHSC-T6 cells were treated with cobalt chloride (CoCl2), and the activity of HSC-T6 cells was measured by the CCK-8 assay. The mRNA expression levels of hypoxia inducible factor-1α (HIF-1α), collagen type I, transforming growth factor-β1 (TGF-β1), and Smad7 were measured by RT-qPCR. The protein expression levels of HIF-1α, Bax, Bcl-2, and caspase-3 were assayed by western blot. We used basal medium and 400 µmol/L CoCl2 medium to treat HSC-T6 cells for 48 h. Cells were harvested after 48 h to extract RNA. Transcriptome sequencing was performed to investigate differentially expressed miRNAs and mRNAs (fold change >2; P<0.05). Bioinformatics analysis was performed to predict the functions of differentially expressed miRNAs and mRNAs. Further, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ResultsWith the increase of CoCl2 concentration, the activity of HSC-T6 cells decreased (P<0.05). The mRNA expression levels of HIF-1α, collagen I, TGF-β1, and Smad7, and the protein expressions levels of HIF-1α, Bax, caspase-3, and the Bcl-2/Bax ratio were increased compared with the control group (P<0.05), while the expression of Bcl-2 decreased. A total of 54 miRNAs (20 upregulated and 34 downregulated) and 1,423 mRNAs (685 upregulated and 738 downregulated) were differentially expressed in the 400 µmol/L CoCl2 medium group compared to the control basal medium group. Further bioinformatics analysis demonstrated that the differentially expressed mRNAs and miRNAs were mainly enriched in the synthesis of extracellular matrix. In addition, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ConclusionsOur results presented the profiles of mRNAs and miRNAs in hypoxia-induced HSC-T6 cells in rats, the signaling pathways, and co-expression networks. These findings may suggest novel insights for the early diagnosis and treatment of HSC activation and liver fibrosis.

Role of Cytokines Released During Pyroptosis in Non-Small Cell Lung Cancer.

PurposePyroptosis is a recently discovered highly inflammatory form of programmed cell death, during which the N-terminus of the cleaved Gasdermin protein family forms pores in the cell membrane, leading to cell disintegration and the release of certain intracellular factors, including caspase3, gasdermin E (GSDME), and high mobility group proteins (HMGB1), which trigger a series of secondary inflammatory reactions. Specifically, caspase3 can lyse GSDME and induce pyrolysis, while HMGB1 is released passively after cell membrane destruction. In this study, the roles of these proteins in lung cancer tissues as well as their clinical significance were investigated.Patients and MethodsThe expression levels of GSDME, caspase3, and HMGB1 proteins in lung cancer and paracancerous tissues were determined via immunohistochemical staining, and their relationship with the clinical stage, pathological grade, and survival prognosis of the patients was analyzed. Further, CD8+ T cell accumulation in the above-mentioned tissues was also determined, and differences between them with respect to CD8+T cell distribution were also investigated. Furthermore, the relationships between CD8+ T cell abundance and the expression levels of the above-mentioned proteins were determined via statistical analyses.ResultsLung cancer and paracancerous tissues showed significantly different GSDME, caspase3, and HMGB1 protein expression levels. GSDME expression level and the presence or absence of lymph node invasion were identified as prognostic indicators of survival in patients with lung cancer. Surprisingly, however, HMGB1, which showed a certain level of correlation with the presence or absence of lymph node metastasis, could not be used as a prognostic indicator of survival.ConclusionGSDME may be an important prognostic indicator of survival in patients with lung cancer. However, the effects of HMGB1 expression level and CD8+ T cell abundance on the prognosis of patients with lung cancer still need further investigation.

Royal jelly abrogates flouride-induced oxidative damage in rat heart tissue by activating of the nrf-2/NF-κB and bcl-2/bax pathway

Royal jelly is known to strengthen memory, provide antioxidative, antidiabetic, antitumor, anticancer, antibacterial, antiinflammatory, antihypertensive. In this study, 42 rats (n = 42) were used, and these rats were divided into 6 groups of 7 rats each. Groups: (i) Control Group: Group fed with standard diet; (ii) Royal Jelly (RJ) Group: RJ (100 mg/kg bw, gavage); (iii) F50 Group: Fluoride (50 mg/kg bw, drinking water); (iv) F100 Group: F (100 mg/kg bw, drinking water); (v) F50 + RJ Group: F (50 mg/kg bw, drinking water) + RJ (100 mg/kg bw, gavage); (vi) F100 + RJ Group: F (100 mg/kg bw, drinking water) + RJ (100 mg/kg bw, gavage). The rats were decapitated after 8 weeks, and their heart tissues were taken and examined. Lipid peroxidation by MDA (malondialdehyde) analyzes, GSH (glutathione) level and catalase activity were determined by spectrophotometer. Protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, BDNF, Gsk-3, Nrf-2 and NF-κB proteins in heart tissue were determined by western blotting technique and hearth tissue evaluated by histopathologically. As a result, MDA levels, Bcl-2, Gsk-3 and NF-κB protein expression levels were reduced, whereas GSH levels, caspase-3, caspase-9, caspase-6, Bax, BDNF and Nrf-2 protein levels were increased in the F50 + RJ and F100 + RJ groups compared to the F50 and F100 groups. According to the results of this study, it has been concluded that Royal jelly has the potential to be developed in to a drug for treatment of heart diseases in addition to providing protection against heart damage.

Effect of bone marrow mesenchymal stem cell transplantation combined with lugua polypeptide injection on osteoarthritis in rabbit knee joint

ABSTRACT Purpose This study aimed to elucidate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation combined with the administration of Lugua polypeptide injection into the knee joint cavity to treat knee osteoarthritis (KOA) in rabbits. Material and methods Sixty white New Zealand rabbits were randomly divided into the blank, model, Lugua polypeptide, BMSC, and combined (Lugua polypeptide plus BMSC) groups, with 12 rabbits in each group. The mRNA and protein expression levels of cyclin D1, bcl-2, TIMP-1, p21, caspase-3, Bax, MMP-1, MMP-13, TLR-4, and NF-κB p65 in chondrocytes, and levels of IL-1, NO, TNF-α, and IL-6 in the synovial fluid were compared. Results The severity of cartilage damage in the combined group was significantly less (P <0.01). Compared to the MG, the mRNA and protein expression levels of cyclin D1, bcl-2 and TIMP-1 in chondrocytes of the three other groups were significantly increased, while those of p21, caspase-3, Bax, MMP-1, MMP-13, TLR-4, and NF-κB p65 in the chondrocytes and levels of IL-1, NO, TNF-α, and IL-6 in the synovial fluid of the three other groups were significantly reduced (P <0.05). The aforementioned indicators in the combined group were significantly better than those of the Lugua polypeptide and BMSCs groups (P <0.05). Conclusions BMSC transplantation combined with Lugua polypeptide injection may improve KOA-related cartilage tissue damage in rabbits.

Icariin inhibits oral squamous cell carcinoma cell proliferation and induces apoptosis via inhibiting the NF-κB and PI3K/AKT pathways.

Oral squamous cell carcinoma (OSCC), one of the most common types of human cancer, has a high mortality rate and a poor prognosis due to its high rates of recurrence and metastasis. In recent years, icariin (ICA) has been reported to play an important role in a variety of malignancies, such as gastric, colorectal, pancreatic and ovarian cancer. However, its role and mechanism in OSCC remains to be elucidated. The present study aimed to investigate the effect of ICA in OSCC cells and to reveal its underlying mechanisms. The OSCC cell lines SCC9 and Cal 27 were used to explore the effect of different concentrations of ICA on the biological behavior of OSCC cells. The effect of ICA on OSCC cell proliferation and apoptosis was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometric assays, respectively. Subsequently, the protein expression levels of caspase-3 and cleaved-caspase-3 were detected using western blot analysis. Additionally, the protein and mRNA expression levels of nuclear factor-κB (NF-κB) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway-related factors were determined using western blot analysis and reverse transcription-quantitative PCR, respectively. The results demonstrated that ICA inhibited OSCC cell proliferation and significantly increased the apoptosis rate in a dose-dependent manner. In addition, treatment of OSCC cells with ICA upregulated the protein expression of cleaved-caspase-3 and increased the cleaved-caspase-3/caspase-3 ratio. The protein expression levels of phosphorylated (p)-p65, p-PI3K and p-AKT were decreased in OSCC cells treated with ICA. The aforementioned findings revealed that ICA could attenuate the proliferation of OSCC cells and induce apoptosis via inhibiting the NF-κB and PI3K/AKT signaling pathways. Therefore, the current study provided a new insight into the clinical treatment of OSCC.

Effects of miR-335-5p targeting G6PD on proliferation and apoptosis of colon cancer cells

Objective: To investigate the effects of miR-335-5p targeting glucose-6-phosphate dehydrogenase (G6PD) on the proliferation and apoptosis of colon cancer cells. Methods: Normal colon cell group, blank control group, NC group and miRNA-335-5p mimic group were set up. Colonic epithelial cells (IEC) and human colon cancer cells SW480 were cultured in vitro, and the cells in the NC group and miRNA-335-5p mimic group cells were transfected. RT-qPCR was used to detect the expression levels of miR-335-5p and G6PD mRNA in each group of cells. The targeting effect of miR-335-5p on G6PD was verified by Double Luciferase Report experiment. MTT assay was used to detect cell proliferation. Flow cytometry was used to detect the apoptosis rate. The expressions of G6PD, Bax, Bcl-2 and caspase-3 were detected by Western blot. Results: Compared with normal colon cells, the relative expression levels of miR-335-5p in SW480 cells of colon cancer in the blank control group and NC group were decreased, and the relative expression level of G6PD mRNA was increased (P＜0.05); compared with the blank control group and NC group, the expression level of miR-335-5p in miR-335-5p mimic group was increased significantly, and the expression of G6PD mRNA was decreased significantly (P＜0.05). Compared with the blank control group and NC group, the proliferative activity of colon cancer SW480 cells in miR-335-5p mimic group was decreased significantly, and the apoptosis rate was increased significantly (P＜0.05). The relative activity of luciferase in miR-335-5p mimic + WT-G6PD 3 '- UTR group was lower than that in miR-335-5p NC + WT-G6PD 3' - UTR group (P＜0.05). Compared with the blank control group, the relative expression levels of G6PD and bcl-2 protein in miR-335-5p mimic group were decreased significantly, and the expression levels of Bax and caspase-3 protein were increased significantly (P＜0.05). Conclusion: MiR-335-5p may inhibit the proliferation and promote apoptosis of colon cancer cells by targeting G6PD.

Palbociclib induces cell senescence and apoptosis of gastric cancer cells by inhibiting the Notch pathway.

Palbociclib (PD0332991), a selective cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has been reported to exert anticancer activity in some cancers, including gastric cancer (GC). However, the role of palbociclib in GC remains largely unknown. The present study aimed to investigate the effects of palbociclib on the progression of GC and the potential mechanisms underlying its effects. The colony formation, proliferation, senescence, as well as apoptosis and cell cycle progression of AGS and HGC-27 cells following treatment with palbociclib were analyzed using colony formation assays, MTT assays, senescence-associated β-galactosidase (SA-β-gal) staining and flow cytometry, respectively. The protein expression levels of Bax, Caspase-3, Bcl-2, p16, p21, p53, Notch1, Notch2 and hairy and enhancer of split 1 (Hes1) were measured in AGS and HGC-27 cells using western blotting. Moreover, the mRNA expression levels of Notch1, Notch2 and Hes1 in AGS and HGC-27 cells were determined by reverse transcription-quantitative PCR. In the present study, palbociclib significantly inhibited cell proliferation and induced cell senescence, cell cycle arrest and apoptosis in both cell lines in a dose-dependent manner. Additionally, palbociclib significantly increased the expression levels of Bax, Caspase-3, p16, p21 and p53, whilst decreasing the expression of Bcl-2, Notch1, Notch2 and Hes1 in AGS and HGC-27 cells. Furthermore, the Notch pathway activator Jagged-1/FC reversed the effects of palbociclib on cell proliferation, apoptosis, senescence and cell cycle progression. These findings demonstrated that palbociclib could inhibit proliferation and induce senescence, cell cycle arrest and apoptosis in GC cells by inhibiting the Notch pathway.

The compound AST-003 could effectively promote apoptosis of renal cell carcinoma cells in vitro.

BackgroundThe toxicity of Sunitinib limits its clinical application. A new compound AST-003 was designed and synthesized based on the structure of Sunitinib, and previous study has confirmed that AST-003 has the same efficacy and less toxicity as Sunitinib. We conducted this study to further verify the effect of AST-003 on renal cell carcinoma (RCC) cells in vitro and its mechanism.MethodsFive RCC cell lines, A498, 786-0, SW-13, Caki-1 and ACHN, were used. Cells were treated with different concentrations of AST-003, and the effect of AST-003 on cell viability was detected by MTT assay and IC50 was determined. Blank control group and AST-003 group were set to evaluate the effect of AST-003 on cell apoptosis and cell cycle. The effect of AST-003 on cell protein expression was detected by western blot.ResultsAfter treatment with AST-003, the viability of the above five RCC cells was significantly inhibited. The IC50 of AST-003 on A498, 786-0, SW-13, Caki-1 and ACHN were 7.396, 6.592, 3.803, 12.05 and 3.422 µmol/L, respectively. Compared with the blank control group, the apoptotic rates were 52.37% and 13.34% in A498 cells (P<0.001), 59.23% and 6.66% in 786-0 cells (P<0.001), 45.67% and 4.19% in SW13 cells (P<0.001), 51.67% and 2.33% in Caki-1 cells (P<0.001), 55.40% and 5.50% in ACHN cells (P<0.001). Flow cytometry revealed that the proportion of cells in G0/G1 phase was significantly increased and the proportion of cells in S phase was significantly decreased in AST-003 group compared with blank control group, suggesting that AST-003 could block cells in G0/G1 phase. Western blot indicated that AST-003 could induce the up-regulation of the protein expression levels of apoptotic genes Caspase3, Caspase8, Caspase9, Bax and tumor suppressor p53, and inhibit the phosphorylation of STAT3, mTOR, AMPK and ERK, as well as the expression of Bcl-2, MKK, MKKK, c-jun and Ras proteins.ConclusionsWe found that AST-003 could effectively promote the apoptosis of RCC cells in vitro and block the cells in the intercellular phase, and its mechanism was similar to that of Sunitinib, suggesting that AST-003 has the value for further research.

Hydroxyurea affects in vitro porcine oocyte maturation through increased apoptosis and oxidative stress.

Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus–oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.

The effects of human dermal-derived mesenchymal stem cells on the keratinocyte proliferation and apoptosis in psoriasis.

Psoriasis is a common chronic inflammatory skin disease, characterized by epidermal hyperproliferation. Mesenchymal stem cells (MSCs) regulate inflammation and vascular proliferation in the psoriasis lesions. Whether dermal-derived mesenchymal stem cells (DMSCs), the main MSCs in the dermis, regulate keratinocyte proliferation and apoptosis remains unknown. In the present study, we assessed the proliferation and apoptosis of keratinocytes cocultured with DMSCs isolated from either normal orpsoriatic involved skin. Cell growth and apoptotic rates were determined using Cell Count Kit-8 and annexin V-FITC staining, respectively. In addition, EDU kit was also used to measure the rate of keratinocyte proliferation. Our results showed that psoriatic DMSCs (pDMSCs) were more potent than normal DMSCs (nDMSCs) in stimulating keratinocyte proliferation. In contrast, the apoptotic rate and expression levels of caspase-3 protein were lower in pDMSC-treated than nDMSC-treated keratinocytes (p<0.001). Moreover, significantly higher contents of IL-6, IL-8, TNF-α and IFN-γ were found in the culture medium of pDMSCs than in that of nDMSCs. In conclusion, pDMSCs were more potent than nDMSCs in stimulation of keratinocyte proliferation and secretion of proinflammatory cytokines, but weaker in promoting apoptosis.

Inhibition effect of active fraction from clove on PI3K/Akt/mTOR signaling pathway to induce apoptosis of human colon cancer HCT116 cells

To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 μg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P&lt;0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/β-actin after treatment of AFC(100 μg·mL~(-1)) were significantly different from those in the control group(P&lt;0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 μg·mL~(-1)) were significantly lower than those in the control group(P&lt;0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P&lt;0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P&lt;0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.

The curative effects of dietary yeast beta-1,3/1,6-glucan on oxidative stress and apoptosis in laying quails (Coturnix coturnixjaponica) exposed to lead

The objective of this study was to investigate the effects of supplementation of beta-glucan to the basal diet of laying quails exposed to lead (Pb) toxicity. A total of 112 birds were randomly divided into 4 groups. The first group was fed a corn-soybean based basal diet without any supplementation (control). The second (Pb) and third (beta-glucan) groups were supplemented with 100 mg/kg of Pb (as Pb acetate) and 100 mg/kg of beta-glucan, respectively. The fourth group (Pb + beta-glucan) was fed a basal diet supplemented with 100 mg/kg of Pb and 100 mg/kg of beta-glucan. It was determined that serum total protein, albumin, and creatinine values of the Pb + beta-glucan group partially improved (P < 0.05) with the addition of beta-glucan, but globulin and blood urea nitrogen (BUN) values were similar to the Pb group (P < 0.001). Globulin and albumin/globulin ratio were not affected by the supplements. Aspartate aminotransferase (AST) (P < 0.01) and alanine transaminase (ALT) (P < 0.001) enzyme activities in the beta-glucan and Pb + betaglucan groups were similar to the control group, unlike the Pb group. However, ALP enzyme activity was similar in all supplemented groups, unlike the control group (P < 0.01). Malondialdehyde (P < 0.001) was reduced in the liver, heart, and kidney tissues of betaglucan supplemented groups compared to the Pb group. Glutathione levels of liver and heart tissues in the Pb + beta-glucan group were significantly higher than in the Pb group (P < 0.001). Catalase and glutathione peroxidase activities were also significantly increased in all tissues by supplementation of beta-glucan as compared to the Pb group. The caspase-3 and caspase-9 protein expression levels in the liver tissue of the beta-glucan group decreased (P < 0.001) compared to the Pb group. As a result, we conclude that beta-glucan helps reduce the harmful effects of Pb toxicity in laying quails.

Protective Effects of Shenfuyixin Granule on H2O2-Induced Apoptosis in Neonatal Rat Cardiomyocytes.

Shenfuyixin granule (SFYXG, i.e., Xinshuaikang granule) is a prescription, commonly used in the clinical experience, which plays a significant role in the treatment of heart failure. The purpose of this present research was to investigate the protective effect of SFYXG, and the mechanism about anti-H2O2-induced oxidative stress and apoptosis in the neonatal rat cardiomyocytes. Myocardial cells, as is well known, were divided into 4 groups: normal, model, SFYXG, and coenzyme Q10 group, respectively. Cells viability was determined by MTT assay. Flow cytometry and AO/EB staining were implemented to test the apoptosis rate and intracellular reactive oxygen species (ROS) level. Mitochondrion membrane potential (MMP) was evaluated by JC-1 fluorescence probe method. The myocardial ultrastructure of mitochondrion was measured by electron microscope. The related mRNA expression levels of Bax, Bcl-2, Caspase-3, caspase-8, and caspase-9 were detected by real-time polymerase chain reaction (PCR). Also, the expression levels of Bax and Bcl-2 protein were detected by Western blot, and the expression levels of caspase-3, caspase-8, and caspase-9 protein were tested by caspase-Glo®3 Assay, caspase-Glo®8 Assay, and caspase-Glo®9 Assay, respectively. GAPDH was used as the internal reference gene/protein. The results revealed that SFYXG (0.5 mg/ml) raised the viability of myocardial cell, weakened the apoptosis rate and ROS level, corrected the mitochondrion membrane potential stability, and improved cell morphology and ultrastructure of myocardial mitochondrion. Furthermore, SFYXG upregulated the antiapoptosis gene of Bcl-2, but downregulated the proapoptosis genes of Bax, caspase-3, and caspase-9. In conclusion, SFYXG could appear to attenuate myocardial injury by its antioxidative and antiapoptosis effect.

Hydrogen-Rich Water Ameliorates Murine Chronic Graft-versus-Host Disease through Antioxidation.

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important treatment option for various hematopoietic diseases and certain hereditary diseases. Chronic graft-versus-host disease (cGVHD) has become the main life-threatening complication and cause of death in later stage postallo-HSCT. Current treatment options for cGVHD are limited. Hydrogen gas (H2) has been demonstrated that has antioxidative, anti-inflammatory, and antifibrosis effects. The aim of this study was to confirm whether oral administration hydrogen-rich water exerted therapeutic effects on a scleroderma cGVHD mouse model and tried to explain the mechanism underly it. Methods A mouse cGVHD model was established by haploidentical bone marrow transplantation. To evaluate therapeutic effects of H2 on cGVHD, survival rate, changes in clinical scores, and skin pathologic characteristics of cGVHD mice were observed. To evaluate its therapeutic mechanism, we detected the expression levels of antioxidative enzymes heme oxygenase-1(HO-1) and NAD (P)H: quinone acceptor oxidoreductase 1(NQO1) in skin homogenates. We also detected the expression level of the apoptotic protein caspase-3 in skin homogenates. Results 1-month survival rate of cGVHD mice in the hydrogen group reached 93.3%, significantly higher than 66.7% in the nonhydrogen group (p < 0.05). Clinical score of cGVHD mice was improved by hydrogen-rich water at 96 days posttransplantation (2.2 versus 4.5, p < 0.05). The skin pathological condition of cGVHD mice was significantly improved by hydrogen-rich water. At 96 days posttransplantation, average skin pathological hematoxylin and eosin (HE) staining score in the hydrogen group was 1.05, which was significantly lower than 3.2 in the nonhydrogen group (p < 0.01). Average Masson staining score was 0.6 point in the hydrogen group, lower than 0.9 point in the nonhydrogen group (p < 0.05). Both the relative expression levels of HO-1 and NQO1 proteins in skin specimens of cGVHD mice in the hydrogen group were lower than that in the nonhydrogen group (2.47 versus 6.21 and 1.83 versus 3.59, p < 0.05). The relative expression level of caspase-3 protein in skin specimens of cGVHD mice increased to 7.17 on the 96th day after transplantation, significantly higher than 4.36 in the hydrogen group. Conclusion In this study, we found that oral hydrogen-rich water improved the survival rate and clinical symptoms of cGVHD mice by antioxidant and antiapoptosis. This study would pave the way for further clinical study, which may provide a new treatment option for cGVHD.

Anticancer Activity of &lt;i&gt;Holothuria atra&lt;/i&gt; Extracts on the Triple-Negative Breast Cancer Cell Line MDA-MB-231

Holothuria atra (HA), black sea cucumber, has been widely used as a traditional medicine in several Asian countries. A previous study showed the potent anticancer effects of HA extracts on cell proliferation and apoptosis in various types of cancer cell lines, including breast cancer cells. However, the molecular mechanism by which HA extracts exert anticancer effects on the triple-negative breast cancer cell line MDA-MB-231 remains unclear and poorly understood. In the present study, we assessed the inhibitory effects of body wall (BW) and visceral (VS) extracts of HA on proliferation, adhesion, migration, invasion, and tube formation in an in vitro model of the highly metastatic triple-negative breast cancer cell line MDA-MB-231. BW and VS extracts were prepared by the ethyl acetate extraction process. Anticancer properties, including the inhibition of proliferation, adhesion, migration, invasion, and tube formation, were examined by MTT assays, cell adhesion assays, wound healing migration assays, Matrigel invasion assays, and tube formation assays, respectively. The protein expression levels of Bax, Bcl-2, Caspase-3, Akt, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9) were examined by Western blot analysis. Our findings demonstrated that BW extract treatment exhibited significantly greater inhibitory effects on MDA-MB-231 cell proliferation in a time- and dose-dependent manner than VS extract treatment. Moreover, dose-dependent suppression of MDA-MB-231 cell adhesion, migration, invasion, and tube formation was observed in the BW extract-treated group. The inhibition of MDA-MB-231 cells invasion in the BW extract-treated group was achieved by suppressing the levels of MMP-2 and MMP-9 through the Akt/MMP pathway. MDA-MB-231 cell apoptosis was promoted after treatment with BW extract by downregulating Bcl-2 levels and upregulating Bax and Caspase-3 expression through Akt/Bad signaling. Our study revealed the anticancer effects of BW extracts of HA. This study provides a potential therapeutic strategy using beneficial HA components for treating patients with breast cancer. Funding Information: This research was funded by Mahidol University and the Thailand Research Fund (Grant IRG5780011). Declaration of Interests: The authors declare no conflict of interest.