Decrease of lncRNA MEG3 in Acute Myeloid Leukemia Affected the Expression of p53 and MDM2.

BACKGROUND: The aim was to determine the clinical role of long non coding maternally expressed gene 3 (lncRNA MEG3) in adult acute myeloid leukemia (AML). METHODS: The expression levels of lncRNA MEG3 in bone marrow of AML patients and healthy donors were determined by qPCR. The correlation between expression levels of lncRNA MEG3 and clinical features was also analyzed. MTT assay and flow-cytometric assay were employed to determine the role of lncRNA MEG3 on the cell viability and apoptosis of AML cell lines. Expression levels of caspase-9, Bcl-2, MDM2, and p53 in those cells were determined by western blot. RESULTS: The expression levels of lncRNA MEG3 was significantly down-regulated in AML patients. Expression levels of lncRNA MEG3 were positively correlated with overall survival of patients. Up-regulation of lncRNA MEG3 could not only inhibit the cell growth and promoted apoptosis, but also increased the expression of caspase-9 and p53 and decreased the expression of Bcl-2 and MDM2. CONCLUSIONS: These results suggest that down-regulation of lncRNA MEG3 in AML patients might promote tumor progression and affect the p53-MDM2 pathway.

[Impacts of LncRNA NORAD on the Proliferation, Apoptosis, and Chemosensitivity of Non-small Cell Lung Cancer Cells by Regulating ZNF217 through MiR-199a-3p].

BACKGROUND: The treatment and diagnosis of non-small cell lung cancer (NSCLC) is still a difficult problem in the medical community, and exploring the molecular mechanism of the occurrence and development of NSCLC is a hot topic of the current research. Long non-coding RNA (lncRNA) NORAD is highly expressed in a variety of cancer cells. It may be a molecular target that promotes NSCLC. The aim of this study was to investigate the impacts of lncRNA NORAD on the proliferation, apoptosis, and chemosensitivity of NSCLC by regulating zinc finger protein 217 (ZNF217) through miR-199a-3p. METHODS: Real-time quantitative polymerase chain reaction (qRT-PCR) method was applied to detect the expressions of NORAD, miR-199a-3p and ZNF217 genes in normal lung epithelial cells BEAS-2B, lung cancer H460 cells, and Cisplatin (DDP) resistant cell lines H460/DDP. H460/DDP cells were devided into control group, si-NC group, si-NORAD group, miR-NC group, miR-199a-3p mimic group, si-NORAD+inhibitor NC group and si-NORAD+miR-199a-3p inhibitor group. Cell proliferation, apoptosis, the expression of NORAD, miR-199a-3p and ZNF217 genes of cells in each group were detected and the expression levels of Ki-67, caspase-9 and ZNF217 proteins in different cells were also observed. The relationship between miR-199a-3p, NORAD and ZNF217 was vefified. RESULTS: Compared with BEAS-2B cells, the expressions of NORAD, ZNF217 mRNA were significantly increased in H460 and H460/DDP cells (P<0.05) but the expression of miR-199a-3p was significantly reduced (P<0.05). Compared with H460 cells, the expression of NORAD and ZNF217 mRNA in H460/DDP cells was significantly increased (P<0.05) and the expression of miR-199a-3p was significantly reduced (P<0.05). Compared with the control group and si-NC group, the proliferation rate, NORAD and ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD group were significantly reduced (P<0.05), but the apoptosis rate, miR-199a-3p expression and caspase-9 expression were significantly increased (P<0.05). Compared with the miR-NC group, the proliferation rate, NORAD and ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the miR-199a-3p mimic group were significantly reduced (P<0.05), but the apoptosis rate, miR-199a-3p expression and caspase-9 expression were significantly increased (P<0.05). Compared with the si-NORAD+inhibitor NC group, the proliferation rate, ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD+miR-199a-3p inhibitor group were significantly increased (P<0.05), the apoptosis rate, miR-199a-3p expression and caspase-9 expression were obviously increased reduced (P<0.05). CONCLUSIONS: Down-regulating NORAD expression can enhance miR-199a-3p expression and inhibit ZNF217 expression, thereby inhibiting H460/DDP cell proliferation, promoting apoptosis and enhancing its DDP chemotherapy sensitivity.

L-asparaginase from human breast milk Lactobacillus reuteri induces apoptosis using therapeutic targets Caspase-8 and Caspase-9 in breast cancer cell line.

BACKGROUND: Breast cancer is the most fatal type of cancer in women worldwide. Many chemotherapeutics targeted breast cancer however, they have frightening side effects. One method of controlling cancer cell growth is targeting apoptosis. OBJECTIVE: This study aimed to induce apoptosis in breast cancer cells by purifying L-asparaginase from human breast milk Lactobacillus reuteri isolates via inhibition of Caspases 8 and 9. METHODS: The best L. reuteri isolates producing L-asparagine with the highest enzyme activity were identified from human breast milk and chosen for L-asparaginase purification. The MTT cell viability assay used for measure the toxicity of the enzyme. Breast cancer cell line was used to study the effect of the enzyme on the caspase 8 and caspase 9 gene expression. RESULTS: The MTT cell viability assay showed the inhibition rates ranged between 30% and 80%, of cell death, occurred when 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml of the enzyme used and IC50 was 4.305 µg/ml. The breast cell lines were treated with the enzyme at a concentration of IC50 value. The Cas8 and Cas9 genes expression in L-asparagine treated breast cancer cell line at a concentration of IC50 value were upregulated (the fold of gene expression are 2.071 and 1.197 respectively). CONCLUSIONS: Breast milk L. reuteri L-asparaginase induces apoptosis via Cas8 and Cas9 upregulation in the breast cancer cell line. L. reuteri L-asparaginase treatment may be the hopeful approach for the management of breast cancer. Furthermore, the results may highlight the fact that the presence of L-asparaginase-producing L. reuteri isolates in human breast milk may aid in breast cancer improvement or even prevention.

A New Diterpenoid of Indonesian Scoparia dulcis Linn: Isolation and Cytotoxic Activity against MCF-7 and T47D Cell Lines.

Scoparia dulcis Linn plays an important role in treatment because it contains active compounds that are proven to have a variety of activities, including cytotoxicity on various cancer cells. The objective of this study is to isolate and identify the cytotoxic compounds in the ethyl acetate fraction of Scoparia dulcis, observe cell cycle inhibition and induction of apoptosis in vitro, and carry out molecular studies using in silico studies. A new diterpene compound was isolated from the ethyl acetate fraction of Scoparia dulcis L. of Indonesian origin. Chromatographic methods were used to isolate the compound, spectroscopic methods were used to elucidate its structure, and these data were compared with those reported in the literature. The compound was tested for its cytotoxic activity against two breast cancer cells (MCF-7 and T47D). The results of the isolated compound showed a cytotoxic effect on MCF-7 and T47D breast cancer cells at IC50 70.56 ± 1.54 and <3.125 ± 0.43 µg/mL, respectively. The compound inhibited the growth of MCF-7 and T47D breast cancer cells and the accumulation of cells in the G1 phases, and it induced apoptosis. Based on a spectroscopic analysis, the isolated compound was identified as 2α-hydroxyscopadiol, which is a new diterpenoid. A docking study revealed that the isolate's hydroxyl groups are essential for interacting with crucial residues on the active sites of the ER and PR and caspase-9. The isolate inhibits ER and PR activity with binding energies of -8.2 kcal/mol and -7.3 kcal/mol, respectively. In addition, the isolate was also able to induce apoptosis through the activation of the caspase-9 pathway with an affinity of -9.0 kcal/mol. In conclusion, the isolated compound from S. dulcis demonstrated anticancer activity based on in vitro and in silico studies.

Xuebijing injection protects sepsis induced myocardial injury by mediating TLR4/NF-κB/IKKα and JAK2/STAT3 signaling pathways.

OBJECTIVE: Compelling evidence has demonstrated that Xuebijing (XBJ) exerted protective effects against SIMI. The aims of this study were to investigate whether TLR4/IKKα-mediated NF-κB and JAK2/STAT3 pathways were involved in XBJ's cardio-protection during sepsis and the mechanisms. METHODS: In this study, rats were randomly assigned to three groups: Sham group; CLP group; XBJ group. Rats were treated with XBJ or sanitary saline after CLP. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6 and TNF-α in serum were measured using ELISA kits. Cardiomyocyte apoptosis were tested by TUNEL staining. The protein levels of Bax, Bcl-2, Bcl-xl, Cleaved-Caspase 3, Cleaved-Caspase 9, Cleaved-PARP, TLR4, p-NF-κB, p-IKKα, p-JAK2 and p-STAT3 in the myocardium were assayed by western blotting. And finally, immunofluorescence was used to assess the level of p-JAK2 and p-STAT3 in heart tissue. RESULTS: The results of echocardiography, myocardial enzyme and HE test showed that XBJ could significantly improve SIMI. The IL-1ß, IL-6 and TNF-α levels in the serum were markedly lower in the XBJ group than in the CLP group (p<0.05). TUNEL staining's results showed that XBJ ameliorated CLP-induced cardiomyocyte apoptosis. Meanwhile, XBJ downregulated the protein levels of Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, Cleaved-PARP, TLR4, p-NF-κB, p-IKKα, p-JAK2 and p-STAT3, as well as upregulated the protein levels of Bcl-2, Bcl-xl (p <0.05). CONCLUSIONS: In here, we observed that XBJ's cardioprotective advantages may be attributable to its ability to suppress inflammation and apoptosis via inhibiting the TLR4/ IKKα-mediated NF-κB and JAK2/STAT3 pathways during sepsis.

Anti-Cancer Effect of Chlorophyllin-Assisted Photodynamic Therapy to Induce Apoptosis through Oxidative Stress on Human Cervical Cancer.

Photodynamic therapy is an alternative approach to treating tumors that utilizes photochemical reactions between a photosensitizer and laser irradiation for the generation of reactive oxygen species. Currently, natural photosensitive compounds are being promised to replace synthetic photosensitizers used in photodynamic therapy because of their low toxicity, lesser side effects, and high solubility in water. Therefore, the present study investigated the anti-cancer efficacy of chlorophyllin-assisted photodynamic therapy on human cervical cancer by inducing apoptotic response through oxidative stress. The chlorophyllin-assisted photodynamic therapy significantly induced cytotoxicity, and the optimal conditions were determined based on the results, including laser irradiation time, laser power density, and chlorophyllin concentration. In addition, reactive oxygen species generation and Annexin V expression level were detected on the photodynamic reaction-treated HeLa cells under the optimized conditions to evaluate apoptosis using a fluorescence microscope. In the Western blotting analysis, the photodynamic therapy group showed the increased protein expression level of the cleaved caspase 8, caspase 9, Bax, and cytochrome C, and the suppressed protein expression level of Bcl-2, pro-caspase 8, and pro-caspase 9. Moreover, the proposed photodynamic therapy downregulated the phosphorylation of AKT1 in the HeLa cells. Therefore, our results suggest that the chlorophyllin-assisted photodynamic therapy has potential as an antitumor therapy for cervical cancer.

HSP27 Inhibitory Activity against Caspase-3 Cleavage and Activation by Caspase-9 Is Enhanced by Chaperone O-GlcNAc Modification in Vitro.

One of the O-GlcNAc modifications is the protection of cells against a variety of stressors that result in cell death. Previous experiments have focused on the overall ability of O-GlcNAc to prevent protein aggregation under stress as well as its ability to affect stress-response signaling pathways. Less attention has been paid to the potential role for O-GlcNAc in the direct inhibition of a major cell-death pathway, apoptosis. Apoptosis involves the sequential activation of caspase proteases, including the transfer of cell-stress information from initiator caspase-9 to effector caspase-3. Cells have multiple mechanisms to slow the apoptotic cascade, including heat shock protein HSP27, which can directly inhibit the activation of caspase-3 by caspase-9. We have previously shown that O-GlcNAc modification increases the chaperone activity of HSP27 against amyloid aggregation, raising the question as to whether this modification may play important roles in other facets of HSP27 biology. Here, we use protein chemistry to generate different versions of O-GlcNAc modified HSP27 and demonstrate that the modification enhances this antiapoptotic function of the chaperone, at least in an in vitro context. These results provide additional molecular insight into how O-GlcNAc functions as a mediator of cellular stress with important implications for human diseases like cancer and neurodegeneration.

Water-soluble Yb3+, Er3+ codoped NaYF4 nanoparticles induced SGC-7901 cell apoptosis through mitochondrial dysfunction and ROS-mediated ER stress.

BACKGROUND: Nanoparticles are potential luminescent probes; among them, upconversion nanoparticles (UCNP) are currently being developed as fluorescent probes for biomedical applications. However, the molecular mechanisms of UCNP in human gastric cell lines remain poorly understood. Here, we aimed to examine UCNP cytotoxicity to SGC-7901 cells and explore its underlying mechanisms. METHODS: The effects of 50-400 µg/mL UCNP on human gastric adenocarcinoma (SGC-7901) cells were investigated. Flow cytometry was used to evaluate reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), intracellular Ca2+ levels, and apoptosis. Activated caspase-3 and nine activities were measured; meanwhile, cytochrome C (Cyt C) in the cytosol and B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), protein kinase B (Akt), phosphorylated-Akt (p-Akt), 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), calpain-1, and calpain-2 protein levels were also detected. RESULTS: UCNP inhibited the viability of SGC-7901 cells in a concentration- and time-dependent manner and increased the proportion of cell apoptosis. Exposure to UCNP enhanced the ratio of Bax/Bcl-2, elevated the level of ROS, decreased ΔΨm, increased intracellular Ca2+ and Cyt C protein levels, decreased the levels of phosphorylated Akt, increased the activity of caspase-3 and caspase-9, and upregulated the protein expression of GRP-78, GRP-94, calpain-1 and calpain-2 in SGC-7901 cells. CONCLUSION: UCNP induced SGC-7901 cell apoptosis by promoting mitochondrial dysfunction and ROS-mediated endoplasmic reticulum (ER) stress, initiating the caspase-9/caspase-3 cascade.

Polystyrene microplastics modulated bdnf expression triggering neurotoxicity via apoptotic pathway in zebrafish embryos.

A ubiquitous presence of microplastics and nanoplastics created a new toxicological research area arising concept of "plastic rivers". But, the precise molecular mechanisms by which its exposure affects developmental neurotoxicity are poorly understood. Hence, in the present investigation, healthy zebrafish embryos were exposed to different concentrations of 500 nm polystyrene microplastics (0.1 ppm, 1 ppm and 10 ppm) to assess the neurotoxicity and the underlying biomolecular mechanism. On the last day of exposure, behaviour, accumulation, embryotoxicity, acridine orange staining, antioxidant enzyme assay, acetylcholinesterase assay, nitric oxide (NO) estimation, along with neurotransmitter (serotonin, dopamine) quantification and gene expression using qRT-PCR (bdnf, p53, bcl-2, caspase-3, caspase-9) were performed. As a result, we found that zebrafish embryos ingest and bioaccumulate microplastic without causing any morphological changes and lethality. The survival and hatching rates of the embryos were also unaffected but the swimming behaviour patterns were found to be altered. Further, acridine orange staining exhibited more apoptosis in treated groups with increased p53, caspase-3, caspase-9 and decreased bcl-2 gene expression. Moreover, polystyrene microplastics exposure resulted in reduced acetylcholinesterase activity leading to elevated NO concentration along with altered serotonin and dopamine levels and subsequently leading to down-regulated bdnf gene expression and modulated downstream apoptotic signalling, confirming the neurotoxicity potential of microplastics causing neuronal dysfunction. This study also compared the binding affinities between styrene and human proteins (Bdnf, p53 and Bcl-2) using bioinformatics methods, and docking results showed negative binding energy resulting in high binding affinities of Bcl-2 then p53 and Bdnf with styrene.

[Effect of autophagy in cadmium chloride induced apoptosis of mouse spermatogenic cells].

OBJECTIVE: To study the effect of autophagy in cadmium chloride(CdCl_2)-induced apoptosis of mouse spermatocytes(GC-2 spd) cells and explore the underlying molecular mechanisms. METHODS: The cells were treated with different concentrations of CdCl_2(0, 5 and 10 µmol/L) for 24 h. Hoechst33342 staining and monodansylcadaverine(MDC) were performed to explore the formation of autophagosomes and apoptotic bodies. The apoptosis of cadmium-treated cells was examined by TUNEL staining. Autophagy inhibitor 3-methyladenine(3-MA)(60 µmol/L), apoptotic inhibitorCaspase inhibitor Z-VAD-FMK( zVAD-FMK)(50 nmol/L), autophagy inducer rapamycin(RAPA)(50 nmol/L) and lysosomal inhibitor chloroquine(CQ)(10 µmol/L) were added to cell culture in the presence/absence of CdCl_2(10 µmol/L) to treat GC-2 spd cells for 24 h. The expression levels of autophagy-related proteins LC3, P62, and pro-apoptotic proteins cleaved Caspase-3 and cleaved Caspase-9 were examined by Western blot. RESULTS: Autophagosomes aggregated and the number of apoptotic cells increased after exposure to CdCl_2 for 24 h. Western blot result showed that in the 5 and 10 µmol/L CdCl_2 exposure groups, the protein expression levels of LC3II/LC3I increased to 9.23±0.81 and 12.15±0.80 compared with the control group(5.50±0.56)(P<0.05), LC3II protein expression level increased to 3.35±0.14 and 3.47±0.32 compared with the control group(2.35±0.34)(P<0.05), P62 protein expression level increased to 1.48±0.12 and 1.80±0.22 compared with the control group(0.83±0.09)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of LC3II/LC3I, LC3II, P62, cleaved Caspase-9 and cleaved Caspase-3 after 3-MA treatment decreased to 0.90±0.07(CdCl_2 group: 1.47±0.06), 1.57±0.14(CdCl_2 group: 2.45±0.29), 0.82±0.05(CdCl_2 group: 1.44±0.18), 0.18±0.01(CdCl_2 group: 0.28±0.01) and 0.61±0.84(CdCl_2 group: 1.15±0.04)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of cleaved Caspase-9 and cleaved Caspase-3 after zVAD-FMK treatment decreased to 0.12±0.01(CdCl_2 group: 0.28±0.01) and 0.34±0.01(CdCl_2 group: 1.15±0.04)(P<0.05), while those of LC3II/LC3I, LC3II and P62 had no significant change(P>0.05). Compared with the CdCl_2-treated group, RAPA enhanced cadmium-induced LC3II/LC3I, LC3II and P62 protein expressions to 2.22±0.21(CdCl_2 group: 1.56±0.06), 3.72±0.21(CdCl_2 group: 2.97±0.15) and 2.41±0.19(CdCl_2 group: 1.52±0.35)(P<0.05). Western blot result showed that compared with the CdCl_2 group, the protein expressions of LC3II/LC3I, LC3II, P62 and cleaved Caspase-3 in the CdCl_2 and CQ treatment groups increased to 3.21±0.31(CdCl_2 group: 2.09±0.25), 4.49±0.43(CdCl_2 group: 2.72±0.26), 2.59±0.19(CdCl_2 group: 1.84±0.19) and 2.43±0.23(CdCl_2 group: 1.50±0.27)(P<0.05). CONCLUSION: Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.

Dexmedetomidina, un agonista de alfa 2, incrementa el efecto analgésico de la morfina y reduce el desarrollo de tolerancia a la morfina suprimiendo el estrés oxidativo y la vía de señalización de TNF/IL-1 en ratas / Dexmedetomidine, an alfa 2 agonist, increases the morphine analgesic effect and decreases morphine tolerance development by suppressing oxidative stress and TNF/IL-1 signalling pathway in rats

Antecedentes: El objetivo del presente estudio es examinar el posible efecto de dexmedetomidina en el desarrollo de tolerancia a la morfina en ratas, incluyendo nocicepción, analgesia con morfina, apoptosis, estrés oxidativo, y las vías del factor de necrosis tumoral (TNF)/interleucina-1 (IL-1). Materiales y métodos: En este estudio se utilizaron 36 ratas Wistar Albino (225–245 g) dividiéndose a los animales en seis grupos: solución salina (S), 20 mcg/kg de dexmedetomidina (D), 5 mg/kg de morfina (M), M + D, tolerancia a la morfina (MT), y MT + D. El efecto analgésico se midió mediante las pruebas analgésicas de placa caliente (hot-plate) y de retirada de la cola (tail-flick). Tras dichas pruebas, se extirparon los ganglios de la raíz dorsal (GRD), y se midieron en los tejidos de los mismos los parámetros del estrés oxidativo (estado antioxidante total [TAS], estado oxidante total [TOS]), TNF, IL-1 y enzimas de la apoptosis (Caspasa-3, Caspasa-9). Resultados: Dexmedetomidina reflejó un efecto antinociceptivo al administrarse en solitario (p < 0,05 a p < 0,001). Además, dexmedetomidina incrementó el efecto analgésico de la morfina (p < 0,001), y también redujo la tolerancia a la morfina a un nivel significativo (p < 0,01 a p < 0,001), reduciendo también los niveles de estrés oxidativo (p < 0,001) y TNF/IL-1 al administrarse como fármaco adicional al grupo de dosis única de morfina y tolerancia a la morfina (p < 0,001). Además, dexmedetomidina redujo los niveles de Caspasa-3 y Caspasa-9 tras el desarrollo de tolerancia (p < 0,001). Conclusión: Dexmedetomidina tiene propiedades antinociceptivas, e incrementa el efecto analgésico de la morfina, previniendo también el desarrollo de tolerancia. Estos efectos se producen probablemente debido a la modulación del estrés oxidativo, la inflamación y la apoptosis.(AU)

Background: The aim of the present study is to examine the possible effect de dexmedetomidine on the development of morphine tolerance in rats including nociception, morphine analgesia, apoptosis, oxidative stress, and tumour necrosis factor (TNF)/ interleukin-1 (IL-1) pathways. Materials and methods: In this study, 36 Wistar Albino (225–245 g) rats were used. Animals were divided into 6 groups: saline (S), 20 mcg/kg dexmedetomidine (D), 5 mg/kg morphine (M), M + D, morphine tolerance (MT), and MT + D. The analgesic effect was measured with hot plate and tail-flick analgesia tests. After the analgesia tests, the dorsal root ganglia (DRG) tissues were excised. Oxidative stress parameters [total antioxidant status (TAS), total oxidant status (TOS)], TNF, IL-1 and apoptosis enzymes (Caspase-3, Caspase-9), were measured in DRG tissues. Results: Dexmedetomidine showed an antinociceptive effect when given alone (p < 0.05 to p < 0.001). In addition, dexmedetomidine increased the analgesic effect of morphine (p < 0.001), and also decreased the tolerance to morphine at a significant level (p < 0.01 to p < 0.001). Moreover, it decreased oxidative stress (p < 0.001) and TNF/IL-1 levels when given as an additional drug of single-dose morphine and morphine tolerance group (p < 0.001). Furthermore, dexmedetomidine decreased Caspase-3 and Caspase-9 levels after tolerance development (p < 0.001). Conclusión: Dexmedetomidine has antinociceptive properties, and it increases the analgesic effect of morphine and also prevents tolerance development. These effects probably occur by the modulation of oxidative stress, inflammation and apoptosis.(AU)

Comparison of Cardioplegic Solutions in Coronary Bypass Surgery Over Autophagy and Apoptosis Mechanisms. / Comparação de Soluções Cardioplégicas em Cirurgia de Revascularização Miocárdica sobre Mecanismos de Autofagia e Apoptose.

BACKGROUND: Coronary artery disease (CAD) due to myocardial ischemia causes permanent loss of heart tissue. OBJECTIVES: We aimed to demonstrate the possible damage to the myocardium at the molecular level through the mechanisms of autophagy and apoptosis in coronary bypass surgery patients. METHODS: One group was administered a Custodiol cardioplegia solution, and the other group was administered a Blood cardioplegia solution. Two myocardial samples were collected from each patient during the operation, just before cardiac arrest and after the aortic cross-clamp was released. The expressions of autophagy and apoptosis markers were evaluated. The level of statistical significance adopted was 5%. RESULTS: The expression of the BECLIN gene was significant in the myocardial tissues in the BC group (p=0.0078). CASPASE 3, 8, and 9 gene expression levels were significantly lower in the CC group. Postoperative TnT levels were significantly different between the groups (p=0.0072). CASPASE 8 and CASPASE 9 gene expressions were similar before and after aortic cross-clamping (p=0.8552, p=0.8891). In the CC group, CASPASE 3, CASPASE 8, and CASPASE 9 gene expression levels were not found to be significantly different in tissue samples taken after aortic cross-clamping (p=0.7354, p=0.0758, p=0.4128, respectively). CONCLUSIONS: With our findings, we believe that CC and BC solutions do not have a significant difference in terms of myocardial protection during bypass operations.

FUNDAMENTO: A doença arterial coronariana (DAC) devido à isquemia miocárdica causa perda permanente de tecido cardíaco. OBJETIVOS: Nosso objetivo foi demonstrar o possível dano ao miocárdio em nível molecular através dos mecanismos de autofagia e apoptose em pacientes submetidos à cirurgia de revascularização miocárdica. MÉTODOS: Um grupo recebeu uma solução de cardioplegia Custodiol e o outro grupo uma solução de cardioplegia sanguínea. Duas amostras miocárdicas foram coletadas de cada paciente durante a operação, imediatamente antes da parada cardíaca e após a liberação do pinçamento aórtico. Foram avaliadas as expressões de marcadores de autofagia e apoptose. O nível de significância estatística adotado foi de 5%. RESULTADOS: A expressão do gene BECLIN foi significativa nos tecidos miocárdicos do grupo CS (p=0,0078). Os níveis de expressão dos genes CASPASE 3, 8 e 9 foram significativamente menores no grupo CC. Os níveis pós-operatórios de TnT foram significativamente diferentes entre os grupos (p=0,0072). As expressões dos genes CASPASE 8 e CASPASE 9 foram semelhantes antes e depois do pinçamento aórtico (p=0,8552, p=0,8891). No grupo CC, os níveis de expressão gênica de CASPASE 3, CASPASE 8 e CASPASE 9 não foram significativamente diferentes em amostras de tecido coletadas após pinçamento aórtico (p=0,7354, p=0,0758, p=0,4128, respectivamente). CONCLUSÕES: Com nossos achados, acreditamos que as soluções CC e CS não apresentam diferença significativa em termos de proteção miocárdica durante as operações de by-pass.

The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8).

Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers.Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.

INTRINSIC/EXTRINSIC APOPTOSIS AND PYROPTOSIS CONTRIBUTE TO THE SELECTIVE DEPLETION OF B CELL SUBSETS IN SEPTIC SHOCK PATIENTS.

ABSTRACT: The depletion of peripheral blood B cells is associated with immunosuppression and poor prognosis during sepsis, and selective depletion occurs when B cell subsets are specifically targeted. In this study, we examined the mechanisms underlying the selective depletion of B cell subsets in the immunosuppressive phase of septic shock patients. Thirty-two septic shock patients were recruited as a septic shock group and 10 healthy volunteers as a control group. The expression of Bcl-2, CD95, cleaved caspase-9/8, and activated caspase-3/1 in the B cell subsets were measured by flow cytometry. Another 23 septic shock patients were recruited to test the remission of caspase-3 (Z-DEVD-FMK) and caspase-1 (VX-765) inhibitors on B cell subset depletion in vitro . In septic shock patients, the Bcl-2 levels in immature/transitional (IM) B cells decreased and the levels of cleaved caspase-9 in IM B cells increased; the levels of CD95 in IM, naive, resting memory (RM), and activated memory (AM) B cells and the levels of cleaved caspase-8 in IM, RM, and AM B cells increased; the levels of activated caspase-3 and caspase-1 in IM, RM, and AM B cells increased. Activated caspase-1 levels in IM B cells were higher compared with activated caspase-3 in septic shock patients, whereas the levels of activated caspase-1 in AM B cells were lower compared with activated caspase-3. Moreover, in vitro experiments showed that Z-DEVD-FMK and VX-765 could alleviate the depletion of IM, AM, and RM B cells. The selective reduction of circulating B cell subsets in septic shock patients could be attributed to intrinsic and extrinsic apoptosis as well as pyroptosis.

[Formononetin improves cognitive behavior in aging rats with chronic unpredictable mild in hippocampal tissue stress by blocking the NF-κB pathway and inhibiting the release of inflammatory factors].

Objective To investigate the effects of formononetin (FMN) on cognitive behavior and inflammation in aging rats with chronic unpredictable mild stress (CUMS). Methods SD rats aged about 70 weeks were divided into healthy control group, CUMS model group, CUMS combined with 10 mg/kg FMN group, CUMS combined with 20 mg/kg FMN group and CUMS combined with 1.8 mg/kg fluoxetine hydrochloride (Flu) group. Except for healthy control group, other groups were stimulated with CUMS and administered drugs for 28 days. Sugar water preference, forced swimming experiment and open field experiment were used to observe the emotional behavior of rats in each group. HE staining was used to observe the pathological injury degree of brain equine area. The contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were detected by the kit. The apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in the brain tissue. The levels of tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6) in peripheral blood were measured by ELISA. Western blot analysis was used to detect Bcl2, Bcl2 associated X protein (BAX), cleaved caspase-9, cleaved caspase-3, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and phosphorylated nuclear factor κB p65 (p-NF-κB p65) in brain tissues. Results Compared with CUMS model group, sugar water consumption, open field activity time, open field travel distance and swimming activity time significantly increased in the CUMS combined with 20 mg/kg FMN group and the CUMS combined with 1.8 mg/kg Flu group. The number of new outarm entry increased significantly, while the number of initial arm entry and other arm entry decreased significantly. The pathological damage of brain equine area was alleviated, and the contents of 5-HT and 5-HIAA were significantly increased. The ratio of BAX/Bcl2 and the expression of cleaved caspase-9 and cleaved caspase-3 protein as well as the number of apoptotic cells were significantly decreased. The contents of TNF-α, iNOS and IL-6 were significantly decreased. The protein levels of TLR4, MyD88 and p-NF-κB p65 were significantly decreased. Conclusion FMN can inhibit the release of inflammatory factors by blocking NF-κB pathway and improve cognitive and behavioral ability of CUMS aged rats.

A Review on Caspases: Key Regulators of Biological Activities and Apoptosis.

Caspases are proteolytic enzymes that belong to the cysteine protease family and play a crucial role in homeostasis and programmed cell death. Caspases have been broadly classified by their known roles in apoptosis (caspase-3, caspase-6, caspase-7, caspase-8, and caspase-9 in mammals) and in inflammation (caspase-1, caspase-4, caspase-5, and caspase-12 in humans, and caspase-1, caspase-11, and caspase-12 in mice). Caspases involved in apoptosis have been subclassified by their mechanism of action as either initiator caspases (caspase-8 and caspase-9) or executioner caspases (caspase-3, caspase-6, and caspase-7). Caspases that participate in apoptosis are inhibited by proteins known as inhibitors of apoptosis (IAPs). In addition to apoptosis, caspases play a role in necroptosis, pyroptosis, and autophagy, which are non-apoptotic cell death processes. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits. This review covers the different types of caspases, their functions, and their physiological and biological activities and roles in different organisms.

Burn-Induced Apoptosis in the Livers of Aged Mice Is Associated With Caspase Cleavage of Bcl-xL.

INTRODUCTION: Older adult burn victims have poorer outcomes than younger burn victims. The liver is critical for the recovery of patients with burns. Postburn hepatic apoptosis in young individuals compromises liver integrity; however, this pathway has not yet been studied in older individuals. Because aged animals with burns suffer significant liver damage, we hypothesized that apoptosis is altered in these animals and may affect liver function. Understanding postburn hepatic apoptosis and its effects on liver function in aged animals may help improve outcomes in older patients. METHODS: We compared the protein and gene expression levels in young and aged mice after a 15% total-body-surface-area burn. Liver and serum samples were collected at different time points after injury. RESULTS: Caspase-9 expression in liver tissue was downregulated by 47% in young animals and upregulated by 62% in aged animals 9 h postburn (P < 0.05). The livers of aged mice showed a Bcl-extra-large (Bcl-xL) transcription increase only after 6 h; however, the livers of young mice exhibited 4.3-fold, 14.4-fold, and 7.8-fold Bcl-xL transcription increases at 3, 6, and 9 h postburn, respectively (P < 0.05). The livers of young mice showed no changes in Caspase-9, Caspase-3, or Bcl-xL protein levels during the early postburn period. In contrast, the livers of aged mice contained cleaved caspase-9, reduced full-length caspase-3, and an accumulation of ΔN-Bcl-x at 6 and 9 h postburn (P < 0.05). p21 expression decreased in aged mice; however, it was significantly increased in the liver tissue of young mice postburn (P < 0.05). Serum amyloid A1 and serum amyloid A2 serum protein levels were 5.2- and 3.1-fold higher in young mice than in aged mice, respectively, at 6 and 9 h postburn (P < 0.05). CONCLUSIONS: Livers of aged mice exhibited different apoptotic processes compared to those of young mice early after burn injury. Collectively, burn-induced liver apoptosis in aged mice compromises hepatic serum protein production.

[Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7].

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.

The effects of non-functionalized polystyrene nanoparticles of different diameters on the induction of apoptosis and mTOR level in human peripheral blood mononuclear cells.

Particles of various types of plastics, including polystyrene nanoparticles (PS-NPs), have been determined in human blood, placenta, and lungs. These findings suggest a potential detrimental effect of PS-NPs on bloodstream cells. The purpose of this study was to assess the mechanism underlying PS-NPs-induced apoptosis in human peripheral blood mononuclear cells (PBMCs). Non-functionalized PS-NPs of three diameters: 29 nm, 44 nm, and 72 nm were studied used in this research. PBMCs were isolated from human leukocyte-platelet buffy coat and treated with PS-NPs at concentrations ranging from 0.001 to 200 µg/mL for 24 h. Apoptotic mechanism of action was evaluated by determining the level of cytosolic calcium ions, as well as mitochondrial transmembrane potential, and ATP levels. Furthermore, detection of caspase-8, -9, and -3 activation, as well as mTOR level was conducted. The presence of apoptotic PBMCs was confirmed by the method of double staining of the cells with propidium iodide and FITC-conjugated Annexin V. We found that all tested NPs increased calcium ion and depleted mitochondrial transmembrane potential levels. The tested NPs also activated caspase-9 and caspase-3, and the smallest NPs of 29 nm of diameter also activated caspase-8. The results clearly showed that apoptotic changes and an increase of mTOR level depended on the size of the tested NPs, while the smallest particles caused the greatest alterations. PS-NPs of 26 nm of diameter activated the extrinsic pathway (increased caspase-8 activity), as well as intrinsic (mitochondrial) pathway (increased caspase-9 activity, raised calcium ion level, and decreased transmembrane mitochondrial potential) of apoptosis. All PS-NPs increased mTOR level at the concentrations smaller than those that induced apoptosis and its level returned to control value when the process of apoptosis escalated.

[LncRNA DRAIC regulates the proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells by targeting let-7i-5p].

Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.