Caspase-3 mRNA Expression Research Articles

Toxic effects of polystyrene microbeads and benzo[α]pyrene on bioaccumulation, antioxidant response, and cell damage in goldfish Carassius auratus

Microplastics (MP) are harmful, causing stress in aquatic species and acting as carriers of hydrophobicity. In aquatic environments, benzo[α]pyrene (BaP) is an endocrine-disrupting chemical that accumulates in the body and causes toxic reactions in living organisms. We investigated the effects of single and combined microbead (MB) and BaP environments on goldfish antioxidant response and apoptosis. For 120 h, goldfish were exposed to single (MB10, MB100, and BaP5) and combined (MB10+BaP5 and MB100+BaP5) environments of 10 and 100 beads/L of 0.2 µm polystyrene MB and 5 µg/L BaP. We measured MB and BaP bioaccumulation as well as plasma parameters including ALT, AST, and glucose. The level of oxidative stress was determined by evaluating lipid peroxidation (LPO) and total antioxidant capacity (TAC) in plasma, as well as antioxidant-related genes for superoxide dismutase and catalase (SOD and CAT) and caspase-3 (Casp3) mRNA expression in liver tissue. The TUNEL assay was used to examine SOD in situ hybridization and apoptosis in goldfish livers. Except for the control group, plasma LPO levels increased at the end of the exposure period in all experimental groups. TAC increased up to 24 h of exposure and then maintained a similar level until the trial ended. SOD, CAT, and Casp3 mRNA expression increased substantially up to 120 h as the exposure concentration and time increased. The TUNEL assay revealed more signals and apoptotic signals in the combined exposure environments as a consequence of SOD in situ hybridization than in single exposure environments. These results suggest that combined exposure to toxic substances causes oxidative stress in organisms, which leads to apoptosis.

Effects of nanoselenium supplementation on lactation performance, nutrient digestion and mammary gland development in dairy cows

The objective of this experiment was to evaluate the influence of nanoselenium (NANO-Se) addition on milk production, milk fatty acid synthesis, the development and metabolism regulation of mammary gland in dairy cows. Forty-eight Holstein dairy cows averaging 720 ± 16.8 kg of body weight, 66.9 ± 3.84 d in milk (dry matter intake [DIM]) and 35.2 ± 1.66 kg/d of milk production were divided into four treatments blocked by DIM and milk yields. Treatments were control group, low-Se (LSe), medium-Se (MSe) and high-Se (HSe) with 0, 0.1, 0.2 and 0.3 mg Se, respectively, from NANO-Se per kg dietary dry matter (DM). Production of energy- and fat-corrected milk (FCM) and milk fat quadratically increased (p < 0.05), while milk lactose yields linearly increased (p < 0.05) with increasing NANO-Se addition. The proportion of saturated fatty acids (SFAs) linearly decreased (p < 0.05), while proportions of monounsaturated fatty acids (MUFAs) linearly increased and polyunsaturated fatty acids (PUFAs) quadratically increased. The digestibility of dietary DM, organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) quadratically increased (p < 0.05). Ruminal pH quadratically decreased (p < 0.01), while total VFA linearly increased (p < 0.05) with increasing NANO-Se addition. The acetic to propionic ratio decreased (p < 0.05) linearly due to the unaltered acetic molar percentage and a quadratical increase in propionic molar percentage. The activity of CMCase, xylanase, cellobiase and pectinase increased linearly (p < 0.05) following NANO-Se addition. The activity of α-amylase increased linearly (p < 0.01) with an increase in NANO-Se dosage. Blood glucose, total protein, estradiol, prolactin, IGF-1 and Se linearly increased (p < 0.05), while urea nitrogen concentration quadratically decreased (p = 0.04). Moreover, the addition of Se at 0.3 mg/kg from NANO-Se promoted (p < 0.05) mRNA and protein expression of PPARγ, SREBP1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2 and the ratios of p-ACACA/ACACA and BCL2/BAX4, but decreased (p < 0.05) mRNA and protein expressions of Bax, Caspase-3 and Caspase-9. The results suggest that milk production and milk fat synthesis increased by NANO-Se addition by stimulating rumen fermentation, nutrients digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

Effects of hypoxia stress on oxidative stress, apoptosis and microorganisms in the intestine of large yellow croaker (Larimichthys crocea)

In order to understand the physiological response of large yellow croaker under hypoxia stress, large yellow croaker with body weight of (31.67 ± 3.69) g was used as the research object. The 96 h hypoxia (2.0 ± 0.1) mg / L stress group and normoxia (7.8 ± 0.5) mg / L control group were set up. The changes of oxidative stress index, apoptosis rate, apoptosis-related gene expression, tissue morphology and microorganisms of large yellow croaker intestine in hypoxia stress group and normoxia control group were analyzed by biochemical detection, paraffin section, qRT-PCR and 16S rDNA sequencing. The results showed that the oxidative stress indexes such as ROS, SOD, CAT, MDA and GSH in the intestinal tissue of the hypoxic stress group were significantly higher than those in the control group. The expression of bax, caspase-3 and caspase-9 mRNA was significantly higher than that of the control group, and the expression of bcl2 mRNA was significantly lower than that of the control group. After 96 h of hypoxia stress, the apoptosis index of intestinal cells in in the hypoxia stress group was significantly higher than that in the control group, the number of intestinal goblet cells decreased, and the height, width of intestinal villi and muscle thickness were significantly lower than those in the control group. There were significant differences in intestinal microorganisms between the hypoxia stress group and the control group. The dominant bacteria involved in energy metabolism under hypoxia stress were Firmicutes and Bacteroidetes, and the dominant bacteria involved in intestinal inflammation were Proteobacteria. Oxidative stress enzyme activity and apoptosis gene expression were significantly correlated with the abundance changes of conditional pathogenic bacteria such as Mycoplasma, Staphylococcus and Pseudomonas, and beneficial bacteria such as Lactobacillus and Clostridium. The above results showed that hypoxia stress caused oxidative stress and apoptosis in large yellow croaker, and changed the intestinal tissue structure and intestinal microorganisms. Intestinal probiotics help fish to resist oxidative stress and apoptosis under hypoxic stress, while conditional pathogenic bacteria have the opposite effect. This study has accumulated basic biological data for the study of the physiological response mechanism of large yellow croaker to hypoxia stress, and provided new understanding for understanding the mechanism of hypoxia affecting fish intestinal health.

Scolicidal and apoptotic effects of phyto- and chemically synthesized silver/boehmite nanocomposites on Echinococcus granulosus protoscoleces

Cystic hydatid disease (CHD) is a zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus). This study aimed to synthesize silver nanoparticles (Ag NPs), silver boehmite nanocomposite (Ag/Bhm NC), and silver boehmite nanocomposite modified with chitosan (Ag/Bhm/Chit NC) using Rosmarinus officinalis (R. officinalis) extract and chemical method, and to evaluate their scolicidal and apoptotic effects on protoscoleces (PSCs) in vitro. The nanomaterials (NMs) were characterized by XRD, FTIR, FESEM, EDS, DLS, PDI, and zeta potential (ZP). The NMs were tested against PSCs at different concentrations (0.2–1.6 mg/mL) and exposure times (10–60 min). The size of Ag NPs, phytosynthesized Ag/Bhm NC, Ag/Bhm/Chit NC, and chemically synthesized Ag/Bhm NC were 25.55, 43, 72.3, and 60.8 nm, respectively. Ag NPs and phytosynthesized Ag/Bhm NC showed the highest scolicidal effect, with 65.34 % and 51.60 % mortality rate at 1.6 mg/mL and 60 min, respectively. Caspase-3 mRNA expression was higher in PSCs treated with Ag NPs and Ag/Bhm NC than in control groups (P < 0.05). Phytosynthesized Ag/Bhm NC had stronger scolicidal and apoptotic effect than chemically synthesized Ag/Bhm NC. Ag/Bhm/Chit NC had a weaker scolicidal effect but higher gene expression than Ag/Bhm NC. In conclusion, this study demonstrates the potential of phytosynthesized Ag NPs and Ag/Bhm NC as effective scolicidal and apoptotic agents against PSCs of hydatid cysts, which may be useful for the treatment of this disease.

Dose-dependent renoprotective effect of vanillic acid on methotrexate-induced nephrotoxicity via its anti-apoptosis, antioxidant, and anti-inflammatory properties.

Methotrexate-induced nephrotoxicity is a medical emergency which is associated with a variety of side effects. Vanillic acid (VA), as an antioxidant, removes free radical oxygen to protect cell defense. Therefore, this study investigated VA's beneficial effects on nephrotoxicity induced by methotrexate through its anti-apoptosis, antioxidant, and anti-inflammatory properties. Our study included five groups of male Wistar rats (n = 8): sham, MTX (Methotrexate) group: rats receiving methotrexate (20 mg/kg, intraperitoneally) on Day 2. Moreover, the remaining groups consisted of animals that received vanillic acid (25, 50, and 100 mg/kg, orally for seven days) plus MTX on the 2nd day. The rats were deeply anesthetized on the eighth day to obtain blood and renal tissue samples. The results showed that MTX can increase blood urea nitrogen and creatinine. However, VA (50 and 100 mg/kg) improved renal function as approved by histological findings. Compared with MTX-treated rats, VA enhanced the contents of total antioxidant capacity (TAC) and reduced renal malondialdehyde (MDA). Moreover, VA reduced mRNA expressions of caspase-3 and Bcl-2-associated x protein (Bax) and caused mRNA overexpression of the renal B-cell lymphoma-2 (Bcl-2), and Nrf-2 (Nuclear factor erythroid 2-related factor 2) compared to the MTX group. Also, VA administration significantly reduced inflammatory agents. Overall, VA protects the kidneys against methotrexate-induced nephrotoxicity via anti-apoptosis, antioxidant, and anti-inflammatory properties. Our results revealed that the most effective dose of VA was 100 mg/kg.

Genistein Induces Antiproliferative Activity and Apoptosis in Human Osteosarcoma Saos-2 Cells.

Genistein (4', 5, 7-trihydroxyisoflavone) and daidzein (4', 7-dihydroxyisoflavone) are isoflavones derived from soybean and have anti-cancer effects in various cells. However, the effects of genistein and daidzein on the human osteosarcoma cell line Saos-2 has not been investigated before. Human osteosarcoma Saos-2 cells were treated with genistein for 24 and 48 hours. Cytotoxicity and apoptosis were measured. Genistein significantly inhibited proliferation of Saos-2 cells stronger than daidzein in a dose-dependent manner (0 to 80 μM). Genistein also significantly suppressed Saos-2 cell viability in a dose-dependent manner (0 to 100 μM). In contrast, daidzein did not affect Saos-2 cell viability. Real-time PCR revealed that genistein caused G1-arrest by increasing the expression of p21 and p27 mRNAs in Saos-2 cells. In addition, genistein induced apoptosis through the up-regulation of effector caspase-3/7 activity in Saos-2 cells. Genistein also enhanced initiator caspase-9 and TNF-α mRNA expression in cells. Genistein may inhibit proliferation through the up-regulation of p21 and p27 and viability by inducing apoptosis in Saos-2 cells.

Chlorogenic Acid Alleviated AFB1-Induced Hepatotoxicity by Regulating Mitochondrial Function, Activating Nrf2/HO-1, and Inhibiting Noncanonical NF-κB Signaling Pathway.

Aflatoxin B1 (AFB1), a kind of mycotoxin, imposes acute or chronic toxicity on humans and causes great public health concerns. Chlorogenic acid (CGA), a natural phenolic substance, shows a powerful antioxidant and anti-inflammatory effect. This study was conducted to investigate the effect and mechanism of CGA on alleviating cytotoxicity induced by AFB1 in L-02 cells. The results showed that CGA (160 μM) significantly recovered cell viability and cell membrane integrity in AFB1-treated (8 μM) cells. Furthermore, it was found that CGA reduced AFB1-induced oxidative injury by neutralizing reactive oxygen species (ROS) and activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. In addition, CGA showed anti-inflammatory effects as it suppressed the expression of inflammation-related genes (IL-6, IL-8, and TNF-α) and AFB1-induced noncanonical nuclear factor kappa-B (NF-κB) activation. Moreover, CGA mitigated AFB1-induced apoptosis by maintaining the mitochondrial membrane potential (MMP) and inhibiting mRNA expressions of Caspase-3, Caspase-8, Bax, and Bax/Bcl-2. These findings revealed a possible mechanism: CGA prevents AFB1-induced cytotoxicity by maintaining mitochondrial membrane potential, activating Nrf2/HO-1, and inhibiting the noncanonical NF-κB signaling pathway, which may provide a new direction for the application of CGA.

Protective effects of puerarin on liver tissue in Salmonella-infected chicks: a proteomic analysis

Salmonella enterica is a zoonotic bacterium that not only causes serious economic losses to the livestock and poultry industries but also seriously endangers human health. Long-term indiscriminate use of antibiotics has led to drug resistance in Salmonella, and thus the identification of alternatives to antibiotics is crucial. In this study, the effects of puerarin on the S. enterica-infected chickens were investigated. A total of 360 chicks were randomly assigned as the control group (CON), the S. enterica group (S), and puerarin-treatment group (P). Chicks in the P group were fed the basal diet supplemented with 50 (P50), 100 (P100), 200 (P200), and 400 (P400) mg/kg puerarin, respectively. It was found that puerarin treatment markedly altered the serum activities of aspartate aminotransferase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD), together with the malondialdehyde (MDA) and total antioxidant capacity (T-AOC) contents in the serum. The mRNA expression of IL-6, IL-1β, TNF-α, Bcl-2, and caspase-8 in the livers of S. enterica-infected chicks was increased after infection but significantly reduced after treatment with puerarin. Histologic analysis showed that puerarin effectively mitigated morphological damage in the liver caused by S. enterica. Proteomic analysis revealed that S. enterica infection led to metabolic disorders in the liver, resulting in oxidative stress, increased inflammation, and significantly elevated levels of hepatocellular carcinoma biomarkers. The findings of the filtered sequencing were verified by using quantitative PCR (qPCR). Treatment with 100 mg/mL puerarin thus effectively alleviated disordered liver metabolism, reduced inflammation and oxidative damage and significantly reduced the levels of hepatocellular carcinoma biomarkers in the liver. The results suggest that puerarin has the potential to replace antibiotics to control Salmonella infection in poultry and thus improve food safety.

In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Delta-9-tetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity

The potentially adverse effects of cannabis (marijuana), a common leisure compound, on male reproductive performance are a reason for concern. δ-9-tetrahydrocannabinol (THC), the primary active component of marijuana alters testicular cells’ proliferation and function which affects male fertility and causes testicular cells dysfunction and apoptosis. The main objective of this study was to investigate the possible mechanism underlying the toxic effects of THC with a mechanistic insight into Sertoli cell-based reproductive dysfunction. The Mus musculus Sertoli cell line (TM4) was cultured and exposed to different concentrations of THC and, MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was then performed for evaluating cell viability. The expression of caspase-3 gene and genes related to growth factors were analyzed by real-time RT-PCR. Western blotting was performed for evaluating protein expression level. THC concentration-dependently decreased the TM4 viability with a significant effect starting at concentration of 1 μM and reaching about 75% of the control level at the concentration of 50 μM (IC25). Moreover, caspase-3 mRNA expression levels significantly increased while growth factors mRNA levels decreased in THC-exposed cells compared to unexposed cells. There was also a significant reduction in related protein levels in THC group. Administration of the THC promotes cytotoxic and apoptotic effects on TM4 cells partly through down-regulation of growth factors expression. Increased apoptosis, over expression of caspase-3, and down-regulation of growth factors expression in Sertoli cells exposed to THC may be a reflection of THC-induced testicular toxicity, which may be partly involved in infertility associated with marijuana smoking or medical cannabis use.

The alleviating effect of Scutellaria amoena extract on the regulation of gut microbiota and its metabolites in NASH rats by inhibiting the NLRP3/ASC/caspase-1 axis.

Background: Scutellaria amoena (SA) is the root of S. amoena C.H. Wright of Labiatae, also known as Scutellaria southwestern. This is mainly distributed in Sichuan, Yunnan, and Guizhou in China. In southwest China, SA is used as an alternative method to genuine medicine for the treatment of allergy, diarrhea, inflammation, hepatitis, and bronchitis. Thus far, studies on the effects of SA on non-alcoholic steatohepatitis (NASH) are lacking. This paper investigated the effect of SA on the regulation of gut microbiota and its metabolites in NASH rats by inhibiting the NOD-like receptor 3 (NLRP3)/apoptosis-associated speck-like protein (ASC)/caspase-1 axis. Methods: A NASH rat model was induced by a high-fat diet (HFD) for 12weeks, and rats were orally given different doses of SA extracts (150 and 300mg/kg/d) for 6weeks. Changes in histological parameters, body weight, organ indexes, cytokines, and biochemical parameters related to NLRP3 in NASH rats were checked. 16S rRNA gene sequencing and UPLC-MS/MS technology were used to analyze the changes in the gut microbiota composition and its metabolites in NASH rats. Results: SA significantly inhibited the HFD-induced increase in body weight, lipid levels, and inflammatory infiltration. SA notably inhibited the HFD-induced increase in the upper and lower factors of NLRP3, such as transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-18, pro-IL-18, IL-1β, pro-IL-1β, NLRP3, ASC, and caspase-1. Additionally, mRNA expressions of caspase-1, NLRP3, and ASC were significantly downregulated after SA treatment. The results of the intestinal flora showed that SA could increase the diversity of flora and change its structure and composition in NASH rats by reducing Firmicutes/Bacteroidetes (F/B) ratio, Blautia (genus), Lachospiraceae (family), and Christensenellaceae R-7 group (genus), and increasing Muribaculaceae (family) and Bacteroides (genus). The metabolomics revealed that 24 metabolites were possibly the key metabolites for SA to regulate the metabolic balance of NASH rats, including chenodeoxycholic acid, xanthine, and 9-OxoODE. Nine metabolic pathways were identified, including primary bile acid biosynthesis, bile secretion, purine metabolism, and secondary bile acid biosynthesis. Conclusion: SA can regulate the intestinal microbial balance and metabolic disorder by inhibiting the NLRP3/ASC/caspase-1 axis to relieve NASH.

Phenotype and function of smooth muscle cells derived from the human normal great saphenous vein in response to hypoxia.

The contribution of hypoxia to the pathophysiology of vascular smooth muscle cells (VSMCs) has not yet been fully elucidated. This study evaluated the effect of hypoxia on the phenotype and function of SMCs derived from the human normal great saphenous veins (NGSVs). Fifteen NGSV tissue samples were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, senescence, and the structure of cytoskeletal filaments in SMCs were observed. mRNA and protein expression of Bax, Bcl-2, caspase-3, matrix metalloproteinases (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was detected by fluorescent quantitative polymerase chain reaction and immunoblotting in the cobalt chloride (CoCl2) and the control groups. A decrease in the number of cytoskeletal filaments was observed. mRNA and protein expression of Bas and caspase-3 was significantly decreased, while the quantity of proliferation, migration, adhesion, senescence, and mRNA and protein expression of Bcl-2, MMP-2, MMP-9, TIMP-1, and TIMP-2 in SMCs in the CoCl2 group were significantly increased compared with the control group. Under hypoxic conditions, the phenotype and function of SMCs derived from the human NGSVs were dysregulated, suggesting that VSMCs switch from the contractile phenotype to the secretory or synthetic phenotype, and more dedifferentiate, resulting in extracellular matrix deposition and apoptotic decrease through the intrinsic pathway.

Dimethyl Fumarate Attenuates Methotrexate Hepatotoxicity in Mice Via the Nrf2/HO-1/Anti-Apoptotic Signaling Pathway

Background: Methotrexate (MTX), a folate antagonist used to treat cancer and inflammatory diseases, is known to generate reactive oxygen species. Objectives: The research investigates the impact of dimethyl fumarate (DMF), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, on an MTX-induced mouse hepatotoxicity model. Methods: Forty-two mice were divided into 6 groups: Control, MTX, DMF 120, and 3 groups of MTX co-treated with DMF 30, 60, and 120 mg/kg. Dimethyl fumarate was gavaged once daily for 10 days. On the fifth day, the animals received MTX 20 mg/kg intraperitoneally. On the eleventh day, the animals were sacrificed, and serum and liver samples were collected to assess the level of oxidative/anti-oxidative and apoptotic/anti-apoptotic markers. Results: Dimethyl fumarate prevented the increase of liver function enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) induced by MTX (P &lt; 0.001). It prevented the increase in AST and ALT levels, indicating liver recovery (P &lt; 0.001). Furthermore, DMF restored antioxidant markers superoxide dismutase, catalase, glutathione peroxidase, and total thiol while reducing the level of thiobarbituric acid reactive substances (P &lt; 0.001). Dimethyl fumarate also downregulated hepatic mRNA expression of caspase 3 and upregulated Bcl-2, heme oxygenase 1, and Nrf2 genes in MTX co-treated DMF groups. Conclusions: Dimethyl fumarate alleviates oxidative stress and apoptosis, which may be achieved by the Nrf2/HO-1 pathway. Therefore, DMF may be clinically effective in preventing or treating MTX-induced hepatotoxicity.

Phytochemical Profiling and Assessment of Habbe Musaffe Khoon Against Cervical Cancer

Background In India, cancer of the cervix is the second most common cancer in women. Nowadays, various traditional herbal formulations can be used as adjuncts during conventional cancer therapies due to their anticancer activity. Habbe Musaffi Khoon (HMK), a Unani compound formulation, is traditionally used as a blood purifier and thus used to treat boils, scabies, acne, and pimples. It is also indicated in skin diseases, Habis-ud-Dam (styptic), and therapeutically used in Nafz-ud-Dam (hemorrhagic). Objectives The main purpose of this work was to phytochemically characterize HMK aqueous extract (HMKaq) and determine its activity against cervical cancer cells. Materials and Methods HMKaq was subjected to liquid chromatography-mass spectrometry (LC-MS) and evaluated for the presence of phytocompounds. HMKaq was evaluated for its effect on the viability of cervical cancer cells (HeLa, SiHa, and C33A) and was determined by MTT assay. Trypan blue dye exclusion was used to determine the outcome of HMKaq on the growth kinetics of cervical cancer cells. The role of HMK in regulating the cell cycle was analyzed by FACS and its effect on apoptosis was evaluated by checking mitochondrial membrane potential readings of JC-1 aggregates on a microplate reader and on the mRNA expression of caspase-3 and cytochrome C was evaluated by PCR. Results LC-MS analysis revealed 3845 compounds, out of which 58 were the major compounds. These included phenolics, esters, fatty acids, furans, quinones, tannins, flavonoids, and terpenes. HMKaq reduced the viability and growth of the cells. It induced arrest in the cell cycle of HeLa and apoptosis in SiHa and C33A by depolarizing the membrane potential of the mitochondria and upregulation of mRNA expression of Cas3 and Cyt C. Conclusion HMK exhibited anticancer activity against cervical cancer cells, thereby signifying its therapeutic potential.

Decrease of Cellular Communication Network Factor 1 (CCN1) Attenuates PTZ-Kindled Epilepsy in Mice.

To investigate the molecular mechanism of communication network factor 1 (CCN1) regulating pentylenetetrazol (PTZ)-induced epileptogenesis, deepen the understanding of epilepsy seizure pathogenesis, and provide new drug action targets for its clinical prevention and treatment. Differentially expressed genes (DEGs) on microarrays GSE47516 and GSE88992 were analyzed online using GEO2R. Pathway enrichment and protein-protein interaction network (PPI) analysis of DEGs were carried out using Metascape. Brain tissue samples of severe traumatic brain injury patients (named Healthy group) and refractory epilepsy patients (named Epilepsy group) were obtained and analyzed by qRT-PCR and immunohistochemistry (IHC) staining. A PTZ-induced epilepsy mouse model was established and verified. Morphological changes of neurons in mouse brain tissue were detected using hematoxylin and eosin (HE) staining. qRT-PCR was conducted to detect the mRNA expressions of apoptosis-associated proteins Bax, Caspase-3 and bcl2. TUNEL staining was performed to detect brain neuron apoptosis. The levels of myocardial enzymology, GSH, MDA and ROS in blood of mouse were detected by biochemical assay. CCN1 expression was increased in epilepsy brain tissue samples. CCN1 decreasing effectively prolongs seizure incubation period and decreases seizure duration. Silencing of CCN1 also reduces neuronal damage and apoptosis, decreases mRNA and protein expression of proapoptotic proteins Bax and Caspase-3, increases mRNA expression of antiapoptotic protein Bcl2. Moreover, decrease of CCN1 decreases myocardial enzymatic indexes CK and CK-MB levels, reduces myocardial tissue hemorrhage, and relieves oxidative stress response in hippocampal and myocardial tissue. CCN1 expression is increased in epileptic samples. CCN1 decreasing protects brain tissue by attenuating oxidative stress and inhibiting neuronal apoptosis triggered by PTZ injection, which probably by regulating Nrf2/HO-1 pathway.

Mechanism of propofol-induced apoptosis in neonatal rat oligodendrocytes

To explore the molecular mechanism of propofol-induced apoptosis in oligodendrocytes. Fortyfive neonatal (7 days old) SD rats were randomized into 3 groups (n=15) for a single intraperitoneal injection of saline (control), long chain fat emulsion, or propofol (50 mg/kg). Eight hours after the injection, the rats were examined for mRNA and protein expressions of caspase-3, caspase-8, and caspase-9 in the brain tissues using qPCR and Western blotting, and the expression levels of nerve growth factor (NGF) mRNA and P-PI3K/P-PAkt were also detected. Compared with those in the control group, the neonatal rats with propofol injection showed significantly up-regulated mRNA and protein expressions of caspase-3, caspase-8, and caspase-9 in the brain tissue (P<0.05) with significantly down-regulated expressions of NGF mRNA and P-PI3K/P-PAkt (P<0.05). Propofol induces apoptosis in oligodendrocytes by activating the caspase family protein members involved in triggering cell apoptosis and inhibiting the anti-apoptosis mechanism.

Antitumor Activity of Ziziphus Jujube Fruit Extracts in KG-1 and NALM-6 Acute Leukemia Cell Lines

Background: Ziziphus jujube Mill. belongs to the Rhamnaceae family. It has been reported to have a variety of biological activities such as antitumor, antioxidant, and anti-inflammatory effects. This study investigates the antiproliferative effect of Ziziphus jujube on KG-1 and NALM-6 acute leukemia cell lines.&#x0D; Materials and Methods: In this experimental study, the aqueous, ethyl acetate, and hydroalcoholic extracts of the Ziziphus jujube were prepared. Total phenolic and flavonoid components were detected because the presence of these compounds is associated with antioxidant and anticancer effects. Different concentrations of extracts were prepared, and KG-1 and NALM-6 cell lines were treated with them at 12, 24, 36, and 48 hours. Cell viability and IC50 values of the extracts were calculated using MTT assays. BD Cycle TEST PLUS DNA Kit was used for cell cycle progression analysis. Bcl2, Bax, and caspase-3 mRNA expressions were also assessed.&#x0D; Results: Cell viability decreased in a concentration-dependent manner. The best efficacy belonged to the ethyl acetate extract. Investigation of cell cycle progression demonstrated that the number of G0/G1 cells enhanced and the number of G2/M cells decreased when the ethyl acetate extract was applied in its IC50 concentration. A considerable increase in Caspase-3 and Bax and a decrease in Bcl2 gene expression were detected in molecular examination.&#x0D; Conclusion: According to our research, Ziziphus jujube ethyl acetate extract has antitumor properties on KG-1 and NALM-6 cell lines, possibly through induction of apoptosis and cell cycle regulation.

Andrographolide Reverses Doxorubicin Resistance in Human Breast Cancer Stem Cells by Regulating Apoptotic Gene Expressions

BACKGROUND: Breast cancer stem cells (BCSCs) have been identified as playing a crucial role in therapeutic resistance. This resistance can be attributed to the anti-apoptotic protein survivin and the antioxidant MnSOD high expression. To overcome the resistance to doxorubicin (DOX), this study proposed the utilization of andrographolide (ANDRO), the primary bioactive compound in Andrographis paniculata leaves. The objective was to examine the role of andrographolide in regulating survivin, caspase-9, and caspase-3 gene expressions to reverse doxorubicin resistance in human BCSCs.METHODS: BCSCs were exposed to 0.1 µM DOX every two days or 50 µM rotenone (ROT) for 6 hours, subsequently supplemented with 0.3 mM ANDRO. Superoxide and peroxide levels were measured using DHE and DCFH-DA assay. The MnSOD, survivin, caspase-9, and caspase-3 mRNA expression levels were analyzed using qRT-PCR. Protein expressions were evaluated using Western blotting assay. MnSOD activity was determined using xanthine oxidase inhibition assay. The apoptotic cells were determined using Annexin-V/PI staining.RESULTS: This study indicated that the cytotoxic mechanisms of DOX, similar to ROT, in BCSCs were attributed to oxidative stress, as evidenced by an elevation in superoxide rather than peroxide levels, accompanied by a decrease in MnSOD activity. This study also highlighted that ANDRO reversed DOX resistance in BCSCs subjected to repeated DOX treatment by downregulating survivin and upregulating caspase-9 and caspase-3 mRNA expressions, thereby activating the intrinsic apoptotic pathway.CONCLUSION: This study provides insights into the role of ANDRO in modulating the expression of apoptotic genes, such as survivin, caspase-9, and caspase-3, to overcome DOX resistance in BCSCs.KEYWORDS: breast cancer, breast cancer stem cell, andrographolide, doxorubicin, oxidative stress, apoptosis

Antagonistic effect of selenium on programmed necrosis of testicular Leydig cells caused by cadmium through endoplasmic reticulum stress in chicken.

Cadmium (Cd) is a widely distributed environmental contaminant that is highly toxic to animals and humans. However, detailed reports on Cd-induced programmed necrosis have not been seen in chicken testicular Leydig cells. Selenium (Se) is a trace element in the human body that has cytoprotective effects in a variety of pathological damages caused by heavy metals. This study investigated the potential mechanisms of Cd-induced programmed cell necrosis and the antagonistic effect of Se on Cd toxicity. Chicken testis Leydig cells were divided into six groups, namely, control, Se (5µmol/L Na2SeO3), Cd (20µmol/L CdCl2), Se + Cd (5µmol/L Na2SeO3 and 20µmol/L CdCl2), 4-phenylbutyric acid (4-PBA) + Cd (10mmol/L 4-phenylbutyric acid and 20µmol/L CdCl2), and Necrostatin-1 (Nec-1) + Cd (60µmol/L Necrostatin-1 and 20µmol/L CdCl2). The results showed that Cd exposure decreased the activity of CAT, GSH-Px, and SOD and the concentration of GSH, and increased the concentration of MDA and the content of ROS. Relative mRNA and protein expression of GRP78, PERK, ATF6, IRE1, CHOP, and JNK increased in the Cd group, and mRNA and protein expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 significantly increased in the Cd group, while Caspase-8 mRNA and protein expression significantly decreased. The abnormal expression of endoplasmic reticulum stress-related proteins was significantly reduced by 4-PBA pretreatment; the increased expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 caused by Cd toxicity was alleviated; and the expression of caspase-8 was upregulated. Conversely, the increased mRNA and protein expression of endoplasmic reticulum stress marker genes (GRP78, ATF6, PERK, IRE1, CHOP, JNK) caused by Cd was not affected after pretreatment with Nec-1. We also found that these Cd-induced changes were significantly attenuated in the Se + Cd group. We clarified that Cd can cause programmed necrosis of chicken testicular Leydig cells through endoplasmic reticulum stress, and Se can antagonize Cd-induced programmed necrosis of chicken testicular Leydig cells.

Tumor suppressor p73 induces apoptosis of murine peritoneal cell after exposure to hydatid cyst antigens; a possibly survival mechanism of cystic echinococcosis in vivo mice model.

Cystic echinococcosis (CE) is a life-threatening helminthic disease caused by the Echinococcus granulosus sensulato complex. Previous evidence indicates that the host's innate immune responses against CE can combat and regulate the growth rate and mortality of hydatid cyst in the host's internal organs. However, the survival mechanisms of CE are not yet fully elucidated in the human body. In the present study, the apoptotic effects of fertile and infertile hydatid fluid (HF) were tested on murine peritoneal cells in vivo mice model. Mice were divided into five groups including; control group, fertile HF-treated peritoneal cells, infertile HF-treated peritoneal cells, protoscolices (PSCs)-treated peritoneal cells and HF+PSCs-treated peritoneal cells group. Mice groups were intraperitoneally inoculated with PBS, HF, and/or PSCs. Afterwards, peritoneal cells were isolated and mRNA expression of STAT3, caspase-3, p73 and Smac genes were evaluated by quantitative Real-time PCR. After 48 hours of exposure, the protein levels of Smac and STAT3 was determined by western blotting technique. After 6 hours of exposure, Caspase-3 activity was also measured by fluorometric assay. The intracellular reactive oxygen species (ROS) production was examined in all groups. The mRNA expression levels of p73, caspase-3 and also Caspase-3 activity in HF+PSCs-treated peritoneal cells were higher than in the test and control groups (Pv<0.05), while the mRNA expression level of anti-apoptotic STAT3 and Smac genes in HF+PSC-treated peritoneal cells were lower than in the other groups (Pv<0.05). As well, the level of intracellular ROS in the fertile HCF-treated peritoneal cells, infertile HCF-treated peritoneal cells, PSC-treated peritoneal cells and HF+PSC-treated peritoneal cells groups were significantly higher than in the control group (Pv<0.05).Current findings indicates that oxidative stress and p73 can trigger the apoptosis of murine peritoneal cells through modulator of HF-treated PSCs that is likely one of the hydatid cyst survival mechanisms in vivo mice model.

Brain-aging related protein expression and imaging characteristics of mice exposed to chronic hypoxia at high altitude.

To determine changes in protein expression related to brain aging and imaging features in mice after chronic hypoxia exposure at high altitude. A total of 24 healthy 4-week-old mice were randomly divided into high altitude hypoxia (HH) and plain control (PC) groups (n = 8 per group). HH mice were transported from Xi'an (450 m above sea level) to Maduo (4,300 m above sea level) while PC mice were raised in Xi'an. After 6 months, 7.0T magnetic resonance imaging (MRI) was performed. All mice completed T2-weighted imaging (T2WI), diffusion tensor imaging (DTI), resting-state functional MRI (rs-fMRI), arterial spin labeling (ASL), and magnetic resonance angiography (MRA) examinations. Next, brain slices were prepared and Nissl staining was used to observe morphological changes in neurons. Ultrastructural changes in neurons were observed by transmission electron microscopy. Expression changes of Caspase-3, klotho, P16, P21, and P53 at the gene and protein levels were detected by real-time PCR (RT-PCR) and Western blot. The number of neuronal Nissl bodies in the hippocampus and frontal cortex was significantly decreased in the HH group compared to the PC group. Some hippocampal and frontal cortical neurons were apoptotic, the nuclei were wrinkled, chromatin was aggregated, and most mitochondria were mildly swollen (crista lysis, fracture). Compared with the PC group, the HH group showed elevated expression of caspase-3 mRNA, P16 mRNA, P21 mRNA, and P53 mRNA in the hippocampus and frontal cortex. Expression of Klotho mRNA in the frontal cortex was also significantly decreased. Western blot results showed that caspase-3 protein expression in the hippocampus and frontal cortex of the HH group was increased compared with the PC group. Moreover, there was decreased Klotho protein expression and significantly increased P-P53 protein expression. Compared with the PC group, expression of P16 protein in the frontal cortex of the HH group was increased and the gray matter (GM) volume in the left visceral area, left caudate nucleus, and left piriform cortex was decreased. Furthermore, the amplitude of low frequency fluctuation was decreased in the left posterior nongranular insular lobe, right small cell reticular nucleus, left flocculus, left accessory flocculus, and left primary auditory area, but increased in the GM layer of the left superior colliculus. Regional homogeneity was decreased in the left and right olfactory regions, but increased in the left bed nucleus. After exposure to high altitude, functional connectivity (FC) between the bilateral caudate nucleus and thalamus, corpus callosum, cingulate gyrus, anterior limbic cortex, globus pallidus, and hippocampus was weakened. FC between the right caudate nucleus and hypothalamus and entorhinal cortex was also weakened. The fractional anisotropy value of the left hippocampus was decreased in the HH group. Compared with the PC group, the HH group showed significantly increased inner diameters of the bilateral common carotid artery and left internal carotid artery. The cerebral blood flow values of the bilateral cortex and bilateral hippocampus in the HH group did not change significantly. Taken together, our findings show that chronic hypoxia exposure at high altitude may promote neuronal apoptosis and abnormal expression of related proteins, changing the structure and function of brain. These changes may contribute to brain aging.