Caspase-3 Protein Expression Research Articles

Flammulina velutipes stem regulates oxidative damage and synthesis of yolk precursors in aging laying hens by regulating the liver–blood–ovary axis

Egg production levels in late laying hens are negatively correlated with increasing age. Decreased liver and ovarian function in aging laying hens is accompanied by decreased antioxidant capacity, reproductive hormone levels, and follicular development, resulting in decreased synthesis of yolk precursors. The golden needle mushroom (Flammulina velutipes) has been reported to exhibit anti-inflammatory, antioxidant, and hypolipidemic properties. We aimed to reveal the therapeutic effects of F. velutipes stem (FVS) on liver-blood-ovary axis and investigate the underlying mechanisms. A total of 360 sixty-seven-wk-old laying hens were randomized into 4 treatment groups: 1) basal maize-soybean meal diet (CON); 2) basal maize+20 g/kg FVS (2% FVS); 3) basal maize+40 g/kg FVS (4% FVS); and 4) basal maize+60 g/kg FVS (6% FVS). FVS groups demonstrated significantly increased egg production and ovarian development compared with the CON group. The addition of FVS increased the levels of antioxidant enzymes (GSH-Px, T-SOD, and T-AOC) in the liver, serum, and ovaries and decreased malondialdehyde levels by regulating the expression of proteins related to the Keap1-Nrf2/ARE signaling pathway. Additionally, FVS significantly decreased ovarian apoptosis by regulating Bax, Bcl-2, and caspase3 mRNA and protein expression levels. FVS significantly increased the expression levels of estradiol, progesterone, luteinizing hormone, and follicle stimulating hormone and their respective receptors. With increased levels of estradiol transported to the liver through the bloodstream, targeted binding to estrogen receptor (ER)-α and ER-β led to significant increases in ApoVLDL II, ApoB, and VTG II mRNA expression associated with yolk precursor synthesis. FVS decreased the levels of triglyceride and total cholesterol and significantly increased the expression of lipid metabolism, and transport-related mRNAs (FAS, PPAR-a/γ, and MTTP) in the liver. Therefore, the dietary supplementation of FVS can maintain the productive performance of aging laying hens by alleviating the degree of oxidative stress and regulating the transport of functional substances along the liver-blood-ovary axis, thereby improving the synthesis of yolk precursors.

Ameliorative Effect of Curcumin Nanoparticles against Monosodium Iodoacetate-Induced Knee Osteoarthritis in Rats.

This study is aimed at evaluating the use of curcumin-loaded polylactic-co-glycolic acid nanoparticles (CUR-loaded PLGA NPs) as a treatment against monosodium iodoacetate- (MIA-) induced knee OA. Eighteen rats were assigned to three groups (n = 6), namely, normal control group that received intra-articular injections (IAIs) of saline, an OA control group that received an IAIs of MIA (2 mg/50 μL), and a treatment group (MIA+CUR-loaded PLGA NPs) that received IAIs of CUR-loaded PLGA NPs (200 mg/kg b.wt). The CUR NP treatment against knee OA alleviated radiographic alternations and histopathological changes and inhibited the upregulation in the serum levels of interleukin-1β, tumor necrosis factor-α, interleukin-6, and transforming growth factor-beta and the downregulation in interleukin-10. CUR NP-treated joints also decreased the mRNA expression of nuclear factor-kappa B and inducible nitric oxide synthase and the protein expression of matrix metalloproteinase-13 and caspase-3. Finally, CUR-loaded PLGA NP treatment mitigated the loss of type II collagen, which resulted in a significant reduction in malondialdehyde level and increased the glutathione content and superoxide dismutase activity compared with that of the OA group. This study demonstrated that the administration of CUR NPs could provide effective protection against MIA-induced OA and knee joint histological deteriorated changes due to its anti-inflammatory, antioxidant, and antiapoptotic properties.

An optimal medicinal and edible Chinese herbal formula attenuates particulate matter-induced lung injury through its anti-oxidative, anti-inflammatory and anti-apoptosis activities

ObjectiveIdentifying novel strategies to prevent particulate matter (PM)-induced lung injury is crucial for the reduction of the morbidity of chronic respiratory diseases. The combined intervention represented by herbal formulae for simultaneously targeting multiple pathological processes can provide a more beneficial effect than the single intervention. The aim of this paper is therefore to design a safe and effective medicinal and edible Chinese herbs (MECHs) formula against PM-induced lung injury. MethodsPM-induced oxidative stress, inflammatory response and apoptosis A549 cell model were used to screen anti-oxidant, anti-inflammatory and anti-apoptotic MECHs, respectively. A network pharmacology method was utilized to rationally design a novel herbal formula. Ultra performance liquid chromatography-mass spectrometer was utilized to assess the quality control of MECHs formula. The excretion of magnetic iron oxide nanospheres of the MECHs formula was estimated in zebrafish. The MECH formula against PM-induced lung injury was investigated with mice experiments. ResultsFive selected herbs were rationally designed to form a new MECH formula, including Citri Exocarpium Rubrum (Juhong), Lablab Semen Album (Baibiandou), Atractylodis Macrocephalae Rhizoma (Baizhu), Mori Folium (Sangye) and Polygonati Odorati Rhizoma (Yuzhu). The formula effectively promoted the magnetic iron oxide nanospheres excretion in zebrafish. The mid/high dose formula significantly prevented PM-induced lung damage in mice by enhancing the activity of SOD and GSH-Px, reducing the MDA and ROS level and attenuating the upregulation of pro-inflammatory cytokine (IL-6, IL-8, IL-1β and TNF-α), down regulating the protein expression of NF-κB, STAT3 and Caspase-3. ConclusionOur findings suggest that the effective MECHs formula will become a novel strategy for preventing PM-induced lung injury and provide a paradigm for the development of functional foods using MECHs.

To Analyze the Mechanism of SalB Regulating SIRT1 to Inhibit NLRP3 and Its Ameliorative Effect on Tubulogastric Junction Tumor Lesions Complicated with Myocardial Injury.

The objective of this research is to investigate the mediating impact of salvianolic acid B (SalB) on SIRT1 signaling pathway and the mechanism by which it inhibits Nod-like receptor protein 3 (NLRP3), as well as to examine how SalB affects myocardial injury brought on by tumor lesions at the junction of the tube and the stomach. Through the establishment of the integration of a stomach tube tumor lesion rats combined with the experimental rat model, this study establishes the normal group, model group, and different SalB dose groups. For each group of cells, cell activity and cell apoptosis were determined and compared using colorimetry and enzyme-linked immunosorbent method about lactate dehydrogenase (LDH). Interleukin-1 beta levels are measured. DCFH-DA fluorescent probe was applied to identify intracellular “reactive oxygen species” (ROS). “Western blot” was used to determine NLRP3, caspase-1, and apoptosis-related spotted protein (ASC) in each group of cells. And SIRT1 signaling pathway related to SIRT1, phosphorylated AMP protein-activated kinase α (P-AMPK α), AMP protein-activated kinase α (AMPKα), and “peroxisome-proliferator-activated receptor γ coactivator 1α (PGC-1α) protein expression” are used. According to the final findings, SalB mediated the SIRT1 signaling pathway and had a beneficial impact on the upregulation of SIRT1, P-AMPK/AMPK, and PGC-1 protein expressions. SalB positively affects the downregulation of NLRP3 inflammasome-related proteins. Caspase-1 and ASC protein expression suggesting that SalB may inhibit the activation of NLRP3 inflammasome induced by oxidative stress by activating SIRT1/AMPK/PGC-1α signaling pathway. This plays an antimyocardial injury effect.

Baicalin inhibits oxidative injures of mouse uterine tissue induced by acute heat stress through activating the Keap1/Nrf2 signaling pathway

Heat stress effect the physiological functions of body, and reproductive system is one of the most sensitive. It's imperative to find out suitable measures to alleviate harmful effects of heat stress. Baicalin is well-known with its antioxidative property. To examine whether Baicalin could reduce oxidative injures of uterine tissue in heat-stressed mice. The mice were divided into four groups: control (Con), Baicalin (Bai), heat stress (H) and heat stress plus Baicalin (H + Bai). The oxidative damage of uterine tissue was detected by ELISA, H&E staining, tunnel assay and immunohistochemical staining. The protein/mRNA expressions of Keap1/Nrf2 related factors were detected by Western blot or QPCR. The results showed that mice heat-stressed at 41 °C for 2 h induced macroscopic changes, significantly increased MDA content and reduced activities of antioxidant enzymes including SOD, CAT and GSH-Px of the uterine tissue. Compared with Con group, heat stress up-regulated caspase-3 and caspase-9, enhanced the apoptosis of endometrial epithelial and glandular epithelial cells, improved the HO-1 mRNA/protein and NQO1 protein expressions, while down-regulated the mRNA/protein of Keap1. Compared with H group, antioxidant enzyme activities, Nrf2 protein and Nrf2, NQO1 and GCLC mRNA expressions were significantly increased in the H + Bai group. While the uterine epithelial cells apoptosis, MDA contents, caspase-3, caspase-9 and Keap1 protein and HO-1 mRNA expressions were decreased in the H + Bai group of mice compared with that in H group. Briefly, acute heat stress causes oxidative injures and apoptosis of mouse uterine tissue and Baicalin protects uterine tissue from the damages possibly through Keap1/Nrf2 signaling pathway.

Neurotoxicity of sanguinarine via inhibiting mitophagy and activating apoptosis in zebrafish and PC12 cells

Sanguinarine, a plant-derived phytoalexin, displays various biological activities, such as insecticidal, antimicrobial, anti-inflammatory, anti-angiogenesis and antitumor effects. But its potential neurotoxicity and the underlying mechanisms has rarely been investigated. Therefore, we aimed to assess the neurotoxicity of sanguinarine using zebrafish model and PC12 cells in this study. The results showed that sanguinarine induced the reduction of the length of dopamine neurons and inhibited the blood vessel in the head area of the zebrafish. Further studies demonstrated that the behavioral phenotype of the larval zebrafish was changed by sanguinarine. In addition, there were more apoptotic cells in the larval zebrafish head area. The mRNA expression levels of β-syn, th, pink1 and parkin, closely related to the nervous function, were changed after sanguinarine treatment. The in vitro studies show that notably increases of ROS and apoptosis levels in PC12 cells were observed after sanguinarine treatment. Moreover, the protein expression of Caspase3, Parp, Bax, Bcl2, α-Syn, Th, PINK1 and Parkin were also altered by sanguinarine. Our data indicated that the inhibition of mitophagy, ROS elevation and apoptosis were involved in the neurotoxicity of sanguinarine. These findings will be useful to understand the toxicity induced by sanguinarine.

Exercise Preconditioning Ameliorates Cognitive Impairment in Mice with Ischemic Stroke by Alleviating Inflammation and Modulating Gut Microbiota.

Several studies have demonstrated that exercise preconditioning is an effective means of alleviating poststroke cognitive impairment (PSCI). Mechanisms of regulating cognitive function have not been fully elucidated. Herein, the present study is aimed at exploring the effect of the microbiota-gut-inflammasome-brain axis in the process of exercise preconditioning moderating cognitive impairment after ischemic stroke. We observed that exercise preconditioning decreased infarct size, reduced the degree of neuronal damage, and alleviated cognitive impairment in mice with ischemic stroke. In addition, exercise preconditioning also reduced the expression of inflammatory cytokines, as well as NLRP3, Caspase-1, IL-18, and IL-1β protein expressions. Ischemic stroke could downregulate the abundance of Roseburia while increasing the abundance of the Helicobacter at the level of genus. As a comparison, exercise preconditioning increased the abundance of the Lactobacillus, which was beneficial for mice at the genus level. In conclusion, exercise preconditioning can improve cognitive dysfunction after ischemic stroke through alleviating inflammation and regulating the composition and diversity of the gut microbiota, which might provide a new strategy for the prevention of PSCI.

Proteomics reveals that Di Dang decoction can regulate the Jak2/Stat5 signaling pathway and inhibit apoptosis by reducing the oxidative stress response in rats with acute intracerebral hemorrhagic stroke

Ethnopharmacological relevanceDi Dang decoction (DDD) is a prescription used for the treatment of cerebral hemorrhage. Its use is derived from the theory of typhoid fever, it has an obvious clinical effect and it has been used in the clinic for a long time. The results of early quantitative proteomics and targeted proteomics studies showed that the administration of high-dose DDD 7 days may regulate the expression of the proteins S100A8, S100A9, Col1a1 and Col1a2. The first 3 days after bleeding begins is the critical period for intervention, what occurs within approximately 3 days after AICH is unclear. Aim of the studyTo explore the effects of Di Dang decoction (DDD) on the Jak2/Stat5 signaling pathway and apoptosis-related gene expression in rats with acute hemorrhagic stroke via the oxidative stress response by proteomics to reveal its neuroprotective mechanism. Materials and methodsNinety healthy Sprague–Dawley (SD) rats were randomly divided into the control, model, and low-, medium-, and high-dose DDD groups, with 18 rats in each group. An acute intracerebral hemorrhage (AICH) model was established by injecting autologous blood into the caudate nucleus. The low-, medium- and high-dose groups were intragastrically administered 0.15625 g/mL, 0.3125 g/mL and 0.625 g/mL DDD, respectively, for 1 or 3 days. The control and model groups were given the same amount of normal saline. Neurological deficits were evaluated by the modified neurological severity score (mNSS) test, brain water content was measured to assess brain tissue damage, and pathological changes in the lesion site were observed by hematoxylin and eosin (HE) staining. The cerebral cortex was selected for quantitative proteomics, and >1.2/1 and <1/1.2 were used as the thresholds for upregulated and downregulated proteins, respectively. KEGG pathway and Gene Ontology (GO) enrichment analyses of the differentially expressed proteins were conducted. The levels of the oxidative stress markers malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to assess p-Jak2, Jak2, p-Stat5, Stat5, Bax, Bcl-2, and Caspase-3 protein expression. ResultsCompared with the model group, the group treated with high-dose DDD for 3 days exhibited significant improvements in neurological defects, brain histopathology, and brain edema; reduced the level of MDA and significantly increased the levels of CAT and SOD; significantly decreased p-Jak2 and p-Stat5 protein expression and expression of the pro-apoptotic genes Bax and c-Caspase-3; and significantly increased expression of the anti-apoptotic gene Bcl-2 (all p＜0.05). ConclusionsHigh-dose DDD administration for 3 days reduces the oxidative stress response, regulates the Jak2/Stat5 signaling pathway and inhibits apoptosis to exert a neuroprotective effect in rats with acute hemorrhagic stroke.

Follicle-Stimulating Hormone Glycosylation Variants Distinctly Modulate Pre-antral Follicle Growth and Survival.

Follicle-stimulating hormone (FSH) is a key endocrine regulator of ovarian function. FSH is secreted as 2 macroglycosylation variants: partially glycosylated FSH (FSH21/18) and fully glycosylated FSH (FSH24). FSH21/18 is more potent than FSH24 at binding to and activating the FSH receptor (R). The ratio of FSH21/18:FSH24 has been shown to change with age, with FSH21/18 predominant at reproductive prime, and FSH24 predominant during perimenopause/menopause. How these FSH glycosylation variants modulate ovarian follicle functions remains largely unknown. The aim of this study was to investigate the effect of FSH glycosylation variants of pre-antral follicle function. Pre-antral follicles were isolated from 3- to 5-week-old C57BL/6 mice and treated ±10 ng/mL FSH21/18, FSH24, a ratio of 80:20 FSH21/18:FSH24 (to mimic reproductive prime), 50:50 FSH21/18:FSH24 (perimenopause), or 20:80 FSH21/18:FSH24 (menopause) for up to 96 hours. FSH21/18 and 80:20 FSH21/18:FSH24 increased follicle growth, in comparison with control, contrasting with FSH24 and 20:80 FSH21/18:FSH24. Survival rates were decreased in follicles treated with FSH24 or 20:80 FSH21/18:FSH24, with follicles undergoing basement membrane rupture and oocyte extrusion, increased Caspase3 gene and protein expression, and decreased markers of cell proliferation in FSH24 or 20:80 FSH21/18:FSH24-treated follicles. Moreover, this correlated with differential regulation of key genes modulating follicular functions. Pharmacological inhibitors of key FSH signal pathways suggests FSH21/18 and FSH24 initiate different FSHR signal pathway activation, which may determine their differential effects on follicle growth and survival. These data suggest that the nature of FSH glycosylation modulates the follicular cellular environment to regulate follicle growth and survival and may underpin the increasing ovarian resistance to FSH observed during aging.

Establishment of the first cell line from the small yellow croaker (Larimichthys polyactis) and its application in unraveling the mechanism of ROS-induced apoptosis under hypoxia

Cell lines play an important role in studying biological processes in vitro. In this study, we aimed to establish and characterize the first cell line isolated from the small yellow croaker (Larimichthys polyactis), an economically important marine fish in China. The small yellow croaker fry (SYCF) cells proliferated well in Leibovitz's 15 medium containing 2% antibiotics, 0.1 M NaCl, and 10% fetal bovine serum at 27 °C. Sequence analyses of mitochondrial genes confirmed the origin of SYCF cells. Furthermore, we unraveled the mechanism of hypoxia-induced apoptosis, a major threat to aquatic animals using the SYCF cell line. The HIF-PHD pathway of the SYCF cells can be activated under hypoxic stress, suggesting that this cell line is impressible for hypoxia and can serve as a superb model in vitro to study the cell physiology in L. polyactis in a hypoxic setting. We found that hypoxia results in morphological abnormalities, excess reactive oxygen species (ROS) production, and dysfunctional redox homeostasis, indicating the occurrence of oxidative stress in the SYCF cells. In addition, decreased cell viability and severe apoptosis were detected under hypoxic conditions. Through monitoring the indicators of the intrinsic and extrinsic apoptosis pathways, including the mitochondrial membrane potential, the expression of bax, bcl2, bnip3, and fas, Bcl2, and Caspase-3 protein, and activity of Caspases-3/8/9, we found that both pathways are involved. Considering that ROS-induced oxidative stress may be the main reason of apoptosis, we investigated whether the addition of an antioxidant, N-Acetyl-L-cysteine (NAC), will ameliorate the apoptosis. Interestingly, we found that both intrinsic and extrinsic apoptosis indicators were markedly recovered by adding NAC in culture media, significant remissions were also observed in the cell viability and apoptosis rate. These results suggested that hypoxia-induced apoptosis of SYCF cells may be mediated by the overproduction of ROS, and the antioxidant may be a useful material to relieve cell damage under hypoxia in fish. In general, this study established a marine fish cell line of SYCF cells, which can be used as an in vitro research model of L. polyactis and for revealing the mechanisms of ROS-induced apoptosis under hypoxic conditions in this species. This provides important insights into the cellular physiology of marine fish under hypoxia.

NLRP3 neuroinflammatory factors may be involved in atopic dermatitis mental disorders: an animal study.

Background: Numerous clinical studies have shown that atopic dermatitis (AD) is often associated with mental disorders. This could contribute to the overall burden of atopic dermatitis. However, the underlying mechanism of mental health symptoms in AD has not been fully elucidated. Methods: An AD mouse was induced by 2,4-dinitrofluorobenzene (DNFB), which was repeatedly applied to the back skin of the BALB/C mice to establish an atopic dermatitis mental disorder model. The role of neuroinflammation in the pathogenesis of atopic dermatitis mental disorders was then explored. Results: After the stimulation of DNFB for 35 days, the skin lesions, the HE staining of skin lesions, and the behavioral experiments (including elevated plus maze assay and tail suspension test) suggested that the AD mental disorder mouse model was successfully replicated. The expression of neuroinflammatory factors in the hippocampus was then investigated through Western blotting. The results showed a significant increase in the protein expression of NLRP3, caspase-1, and IL-1β. Conclusion: Mental disorders in AD might be related to the neuroinflammatory response in the hippocampus. An alternative yet essential approach to promoting AD recovery could be through reducing neuroinflammation and improving mental disorders.

Rosiglitazone Alleviates Contrast-Induced Acute Kidney Injury in Rats via the PPARγ/NLRP3 Signaling Pathway.

Background This study investigated the effect and mechanism of rosiglitazone on a rat model with contrast-induced acute kidney injury (CI-AKI). Materials and Methods The CI-AKI rat model was established from Sprague Dawley rats by furosemide injection (10 ml/kg) to the caudal vein followed by iohexol (11.7 ml/kg). The experimental grouping was randomly allocated into control, model, rosiglitazone, and T0070907 groups. Blood samples were collected from the abdominal aorta. Serum creatinine, urea nitrogen, MDA, and SOD contents were detected by biochemical analysis. TNF-α and IL-10 expression was detected by ELISA. Urine creatinine and urine protein were measured following 24-h urine biochemistry testing. Cell pathology and apoptosis were detected by H&E and TUNEL staining, respectively. PPARγ, NLRP3, eNOS, and caspase-3 mRNA expression were detected by qPCR. Caspase-3 and NLRP3 expression were detected by immunohistochemistry. Results The CI-AKI rat model was successfully established because the results showed that compared with control, serum creatinine, urea nitrogen, MDA, SOD, TNF-α, and IL-10, urine creatinine and urine protein levels were significantly increased in the model group, indicating AKI, but was significantly decreased with rosiglitazone treatment, indicating recovery from injury, while opposite results were obtained with SOD. Apoptosis rate was significantly increased in the model group and significantly decreased with rosiglitazone treatment. NLRP3 and eNOS increased significantly in the model group and decreased significantly with rosiglitazone treatment, while opposite results were obtained with PPARγ. NLRP3 and caspase-3 protein expression was significantly increased in the model group and significantly decreased with rosiglitazone treatment. Conclusion Rosiglitazone could alleviate acute renal injury in the CI-AKI rat model by regulating the PPARγ/NLRP3 signaling pathway and should be further investigated as a potential treatment in clinical studies.

Pathogenesis and Diagnostic Significance of EBV-miR-BARTs in Nasopharyngeal Carcinoma.

Objective Examining the role of EBV-miR-BARTs in nasopharyngeal cancer etiology and diagnosis. Method As the subjects of this study, nasopharyngeal cancer cell lines were chosen and then randomly assigned to one of four groups: the control group, EBV-miR-BART5-3p NC, EBV-miR-BART5-3p mimics, and EBV-miR-BART5-3p inhibitor groups. Utilizing reverse transcription polymerase chain reaction, we determined the levels of gene expression in nasopharyngeal cancer cells that had been treated with EBV-miR-BART5-3p (RT-PCR). The MTT, Transwell, and scratch tests were used to determine the degree to which cells underwent apoptosis, invasion, and migration. The Western blotting method was used in order to examine the protein expression. Result Compared with normal nasopharyngeal cells, P 0.05 showed that nasopharyngeal cancer cells had greater EBV-miR-BART5-3p expressions and proliferation rates in the control, EBV-miR-BART5-3p NC, and EBV-miR-BART5-3p No statistically significant differences were seen between the mimic groups (P > 0.05); compared with the control group, the proliferation rate of the EBV-miR-BART5-3p inhibitor group was lower with P < 0.05. At a significance threshold of P 0.05, there was no difference in the rates of apoptosis between the control group and the EBV-miR- BART5-3p NC group. Comparing the control group to the EBV-miR-BART5-3p mimics group and the EBV-miR-BART5-3p inhibitors group revealed that the rate of apoptosis was dramatically enhanced in the EBV-miR-BART5-3p inhibitors group but significantly decreased in the control group (P 0.05). When comparing the control group to the EBV-miR-BART5-3p NC group, there was no statistically significant change in the total number of invasive cells (P > 0.05). When comparing the EBV-miR-BART5-3p mimics group to the control group, we found a statistically significant increase in the former and a decrease in the latter (P 0.05). The migration rates of the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimics group did not vary from one another in a way that was statistically significant (P > 0.05). When compared to the control group, the migration rate was considerably (P 0.05) lower in the EBV-miR-BART5-3p inhibitor group. There were no discernible changes identified (P > 0.05) in the levels of Bcl-2 protein expression in the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimic group in a research that compared these three groups. Protein levels of BCL-2 were significantly decreased (P 0.05) in the EBV-miR-BART5-3p inhibitor group, in comparison to the control group. When comparing the control and EBV- miR-BART5-3p NC groups, we found no statistically significant differences in Bax and Caspase-3 protein expression levels (P > 0.05). The protein expressions of Bax and Caspase-3 were statistically significantly greater in the EBV-miR-BART5-3p contrast between the inhibitor and control groups. When comparing the protein expressions of MMP-2 and MMP-9 between the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimics group, there was no statistically significant change (P > 0.05). Protein levels of MMP-2 and MMP-9 were inhibited by EBV-miR-BART5-3p to a greater extent (P 0.05) in the experimental group compared to the control group. Conclusion The understanding that inhibiting expression of EBV-miR-BART5-3p might reduce the risk of developing nasopharyngeal cancer may help direct clinical treatment for the condition.

Apoptosis of NG108-15 cells induced by fluorine combined with aluminum exposure and the effect of curcumin intervention

The mechanism by which the combination of fluorine and aluminum (FA) cause learning and memory impairment is still unclear. Curcumin is a yellow polyphenol extracted from the rhizome of the plant curcuma longa linn, which has anti-apoptotic and antioxidant functions; studies have shown that the PKC-NMDAR pathway plays an important role in the process of cell apoptosis. Based on the above, we designed an in vitro study on the role of curcumin in cell apoptosis caused by coexistence of FA. In this study, the cell viability of NG108-15 cell was detected by CCK-8; cell apoptosis was detected by AO/EB dual fluorescence staining kit; mRNA expressions of PKC and NMDAR were evaluated by qRT-PCR and protein expressions of caspase3, Bax, Bcl-2, PKC and NMDAR were evaluated by WB. Our results showed that curcumin alleviated FA-induced apoptosis of NG108-15 cells, possibly by inhibiting the increase of caspase3 protein and Bax protein, and the decrease of Bcl-2 protein, thereby upregulating the expression of PKC and NMDAR to exert neuroprotective effect.

Effect of Curcumin on the Proliferation, Apoptosis, and Cell Cycle of Human Acute Myeloid Leukemia Cell Line K562

To investigate the effects of curcumin on the proliferation, apoptosis, and cell cycle of human acute myeloid leukemia cell line K562. MTT method was used to detect the proliferation inhibition of logarithmic growth phase human acute myeloid leukemia K562 cells, flow cytometry was used to detect the cell cycle, Annexin V-FITC was used to detect the apoptosis rate, and real-time fluorescent quantitative PCR and Western blot were used to detect the expression of Bax, BCL-2 and caspase-3 mRNA and protein, respectively. The inhibition rate of cell proliferation in curcumin 10, 20, and 40 μmol/L group for 24 h and 48 h were higher than that in the control group (curcumin 0 μmol/L), and the cell proliferation inhibition rate was concentration-time dependent (r=0.879, r=0.914). The proportion of G0/G1 cells and apoptosis rate of K562 cells in the curcumin 10, 20, and 40 μmol/L group were higher than those in the control group, and showed drug concentration dependent (r=0.856, r=0.782). The expression of Bax and Caspase-3 mRNA in the curcumin 10, 20, and 40 μmol/L group was higher, while BCL-2 mRNA was lower than those in the control group, and showed drug concentration dependent (r=0.861, r=0.748, r=-0.817). The gray value of Bax protein expression in the curcumin 10, 20, and 40 μmol/L group was higher than that in the control group, while the gray value of BCL-2 and Caspase-3 protein expression was lower than that in the control group, and showed drug concentration dependent (r=0.764, r=-0.723, r=-0.831). Curcumin can inhibit the proliferation of human acute myeloid leukemia cell line K562 cells, block the cell cycle at G0/G1 phase, promote cell apoptosis, and induce apoptosis by regulating Bax, BCL-2, and Caspase-3.

Protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid on OGD/R-damaged HT22 cells via regulation of ASK1/MKK4/JNK signaling pathway

To investigate the protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid(TQHXD-CSF) on HT22 cells damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) and whether the mechanism is related to the regulation of ASK1/MKK4/JNK signaling pathway. HT22 cells were subjected to OGD/R to simulate cerebral ischemia-reperfusion injury(CIRI). Then the cells were randomly divided into five groups: blank cerebrospinal fluid(control group), OGD/R group, TQHXD-CSF group, Z-VAD-FMK group(20 μmol·L~(-1)) and TQHXD-CSF+Z-VAD-FMK group. Except the control group, cells in the other groups were reoxygenated for 12 h after 6 h of oxygen and glucose deprivation for modeling OGD/R, and group administration was performed. Cell viability and cytotoxicity were detected by CCK8 and LDH assay kit, respectively and the morphology of HT22 cells was observed by inverted microscope. Western blot and qRT-PCR were employed to detect the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3, respectively. Then HT22 cells were assigned into the control group, OGD/R group, si-NC group, si-ASK1 group, TQHXD-CSF group and TQHXD-CSF+si-ASK1 group. Cell viability, proliferation and apoptosis were determined by CCK8, electric cell-substrate impedance sensing(ECIS), and Hoechst staining and flow cytometry, respectively. The protein expression of MKK4, p-MKK4, JNK, p-JNK, c-Jun, p-c-Jun, Cyt C, Bax, Bcl-2 and caspase-3 was tested by Western blot. The results showed that compared with OGD/R group, TQHXD-CSF significantly enhanced cell viability, improved cell morphology and reduced the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3. In addition, when ASK1 was silenced, compared with OGD/R group, TQHXD-CSF remarkably improved cell viability, and decreased apoptosis rate and the protein expression levels of p-MKK4, p-JNK, p-c-Jun, Cyt C, Bax/Bcl-2 and caspase-3, but the effect was not as good as that of TQHXD-CSF+si-ASK1 group. In conclusion, TQHXD-CSF can inhibit apoptosis mediated by ASK1/MKK4/JNK signaling pathway in OGD/R-damaged HT22 cells, and has protective effect on ischemia-reperfusion injury.

Apoptotic susceptibility of pancreatic alpha cells to environmentally relevant dose levels of bisphenol-A versus dibutyl phthalate is mediated by HSP60/caspase-3 expression in male albino rats.

Unfortunately, humanity is exposed to mixed plasticizers such as bisphenol-A (BPA) and dibutyl phthalate (DBP) that are leached from the daily used plastic products. Previous studies have demonstrated their potential in pancreatic beta cell injury and diabetes induction. The study hypothesized that both compounds would affect the pancreatic alpha cells in albino rats when administered at environmentally relevantdoses. Heat shock protein 60 (HSP60) and caspase-3 protein expression was also investigated as potential mechanisms. Thirty-six male Wistar albino rats were separated into four equal groups: control, BPA alone, DBP alone, and BPA + DBP combined groups. BPA and DBP were given in drinking water for 45 days in a dose of 4.5 and 0.8 µg/L, respectively. Fasting blood glucose, serum insulin, pancreatic tissue levels of malondialdehyde, and superoxide dismutasewere measured. Pancreatic sections were subjected to hematoxylin & eosin (H & E)staining, glucagon, HSP60, and caspase-3 immunohistochemistry. Although all three experimental groups showed diffuse islet cell HSP60 immunoreactivity, rats exposed to BPA alone showed α-cell-only apoptosis, indicated by H & E changes and caspase-3 immunoreactivity, associated with reduced glucagon immunoreaction. However, rats exposed to DBP alone showed no changes in either α or β-cells. Both combined-exposed animals displayed α and β apoptotic changes associated with islet atrophy and reduced glucagon expression. In conclusion, the study suggested HSP60/caspase-3 interaction, caspase-3 activation, and initiation of apoptosis in α-cell only for BPA-alone exposure group, meanwhile DBP alone did not progress to apoptosis. Interestingly, both α/β cell effect was observed in the mixed group implying synergetic/additive action of both chemicals when combined.

Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis rats

To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion (combination) groups (12 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension （10 mL/kg）. EA (10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion (3 cones) was applied to "Zhongwan" (CV12), bilateral "Neiguan" (PC6) and bilateral "Sanyinjiao" (SP6) of the related groups, once a day for 3 weeks. The blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The apoptosis index of gastric antrum cells were observed by TUNEL staining. The protein and mRNA expressions of Caspase-3, B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in gastric antrum were detected by Wes-tern blot and real-time quantitative PCR, respectively. Compared with the normal group, the blood glucose, the apoptosis index, the protein and gene expressions of Caspase-3 and Bax were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsive rate, the protein and gene expressions of Bcl-2 were considerably decreased (P<0.01) in the model group. In contrast to the model group, the blood glucose in the EA, moxibustion and combination groups, the apoptosis index in the 4 treatment groups, as well as Caspase-3 protein, Bax protein and mRNA expressions in the medication, EA and combination groups, Caspase-3 protein and mRNA, Bax mRNA expressions in the moxibustion group were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate in the 4 treatment groups, and Bcl-2 protein and mRNA expressions in the medication and combination groups, Bcl-2 mRNA expressions in the EA and moxibustion groups were obviously increased (P<0.01). The effects of EA+moxibustion were significantly superior to those of simple EA, moxibustion or medication in increasing gastric emptying rate and intestinal propulsive rate, and in lowering blood glucose (P<0.05, P<0.01). And the effects of the combination treatment were better than those of EA in lowering Caspase-3 protein and Bax mRNA expressions (P<0.01), and in increasing Bcl-2 protein and mRNA expressions (P<0.05, P<0.01). Also the effects of the combination treatment were better than those of moxibustion in lowering the apoptosis index, Caspase-3 protein, and Bax protein and mRNA expressions (P<0.01, P<0.05), and in increasing Bcl-2 protein expression (P<0.05). EA combined with Zhuang-medicine-thread moxibustion can reduce blood glucose and improve gastrointestinal motility in DGP rats, which may be related to its effect in regulating of Caspase-3, Bax and Bcl-2 expression.

Protein Kinase N2 Reduces Hydrogen Peroxide-inducedDamage and Apoptosis in PC12 Cells by AntiOxidative Stress and Activation of the mTOR Pathway.

Objective To investigate the role and mechanism of protein kinase N2 (PKN2) in hydrogen peroxide (H2O2)-induced injury of PC12 cells. Methods. PC12 cells were transfected with lentivirus to knock down or overexpress PKN2 and then were treated with 300 μM H2O2 to establish a cell model of oxidative stress injury. The cell viability of PC12 cells in each group was determined by the CCK-8 method. Biochemical assays were used to measure reactive oxygen species (ROS), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. Western blot was used to detect the protein expressions of PKN2, caspase-3, cleaved-caspase-3, PARP, cleaved-PARP, p-mTOR, and mTOR in PC12 cells in each group. Results H2O2 treatment could significantly reduce PC12 cell viability and promote cell apoptosis and oxidative stress. PKN2 overexpression inhibited H2O2-induced apoptosis and oxidation damage by increasing PC12 cell viability, SOD activity, and p-mTOR protein expression, reducing intracellular ROS and MDA levels, and cleaved-caspase-3 and cleaved-PARP protein expression. Conclusion PKN2 overexpression can alleviate H2O2-induced oxidative stress injury and apoptosis in PC12 cells by activating the mTOR pathway.

Effects of Moderate-Intensity Continuous Training and High-Intensity Interval Training on Testicular Oxidative Stress, Apoptosis and m6A Methylation in Obese Male Mice.

Exercise is an effective way to improve reproductive function in obese males. Oxidative stress and apoptosis are important pathological factors of obesity-related male infertility. Accumulating studies have demonstrated that N6-methyladenosine (m6A) methylation is associated with obesity and testicular reproductive function. Our study aimed to investigate and compare the effect of 8 weeks of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on testicular oxidative stress, apoptosis and m6A methylation in obese male mice. Male C57BL/6 mice were randomly allocated into the four groups: normal diet (ND) group, high-fat diet (HFD) group, high-fat diet with moderate-intensity continuous training (HFD-MICT) group and high-fat diet with high-intensity interval training (HFD-HIIT) group. Mice in the HFD-MICT and HFD-HIIT groups were subjected to 8 weeks of MICT or HIIT treadmill protocols after 12 weeks of HFD feeding. We found that MICT and HIIT increased the protein expression of Nrf2, HO-1 and NQO-1 in the testes of obese mice, and HIIT increased it more than MICT. The Bax/Bcl-2 ratio, Cleaved Caspase-3 protein expression and TUNEL-positive cells were consistently up-regulated in the testes of obese mice, but MICT and HIIT restrained these HFD-induced effects. In addition, HFDs increased m6A levels and the gene expression of METTL3, YTHDF2 and FTO in the testes, but these effects were reversed by MICT and HIIT. However, HIIT was more effective than MICT in reducing m6A methylation in the testes of obese mice. These results demonstrate that both MICT and HIIT protected against HFD-induced oxidative stress, apoptosis and m6A methylation in testicular tissues; as a result, testicular morphological and functional impairment improved. In particular, HIIT was more beneficial than MICT in increasing the mRNA expression of steroidogenic enzymes and testicular antioxidant capacity and decreasing m6A methylation in the testes of HFD-fed mice.