Bax mRNA Expression Research Articles

Effect of folic acid supplementation on proliferation and apoptosis in bovine mammary epithelial (MAC-T) cells

Folic acid (FA) is known to be an important micronutrient in humans; however, information regarding the effect of FA supplementation on bovine mammary epithelial (BME) cells is insufficient. FA supplementation is reported to increase milk production in dairy cows, but the underlying molecular mechanisms are unknown. This study examined the effects of FA supplementation on the proliferation and apoptosis of a BME cell line (MAC-T). MAC-T cells were treated with various concentrations (deficient in FA (DF) < 0.01 ng/mL; low-level FA (LF) 3.1 ng/mL; normal FA (NF) 15.4 ng/mL; and high-level FA (HF) 30.8 ng/mL) based on serum folate (10–20 ng/mL) in milking cows. HF treatment significantly increased the proliferation of MAC-T cells. Cellular apoptosis was observed mainly in the DF group. The number of apoptotic cells in DF media was significantly higher than that in NF media. The bcl-2/bax mRNA expression ratio was significantly increased in the HF group compared to that in the DF group. FA supplementation significantly increased the ratio of Bcl-2/Bax protein levels in MAC-T cells. FA supplementation increases proliferation and decreases apoptosis in these cells. This study might provide information regarding the molecular mechanism through which FA supplementation is associated with increased milk yield.

Effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury in newborn rats

To explore the effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury (HIBI) in newborn rats. Seventy-two healthy SPF grade SD newborn rats were randomly and equally divided into Normal group (healthy rats) and Sham group (rats underwent sham operation), Model group (HIBI model rats), p38 MAPK Inhibitor group (HIBI model rats treated with p38 MAPK inhibitor) and p38 MAPK Activator group (HIBI model rats treated with p38 MAPK activator). On postnatal day 28, Morris water maze, tail suspension test and inclined plane test were conducted on rats in each group. Twenty-four hours after modeling, the expression of p-p38 MAPK protein and apoptosis related genes in rat hippocampal tissues was detected by TUNEL staining, qRT-PCR and Western blot. Compared with Normal group, escape latency and inclined plane test time were prolonged, the number of passing through the platform and tail suspension time were reduced (all P < 0.05); Bax and Caspase-3 mRNA and protein expression levels and p-p38 MAPK protein level were increased, Bcl-2 mRNA level was decreased, and neuronal apoptosis proportion was increased in Model group (all P < 0.05). Compared with Model group, the above indicators showed reversed and enhanced trends in p38 MAPK Inhibitor and p38 MAPK Activator groups, respectively (all P < 0.05). Inhibition of p38 MAPK signaling pathway can effectively improve the learning and memory ability and motor function of newborn rats with HIBI, and reduce neuronal apoptosis in the hippocampal tissues, thereby promoting neuronal recovery.

Effect of Rehabilitation with Extremely Low Frequency Electromagnetic Field on Molecular Mechanism of Apoptosis in Post-Stroke Patients.

Apoptosis in acute stroke is associated with a negative prognosis and is correlated with the severity of the neurological deficit. However, there is no evidence that indicates that, in the subacute phase of the stroke, the apoptosis process might activate neuroplasticity. Therefore, in this study, we investigated the effect of an extremely low frequency electromagnetic field (ELF-EMF) on the molecular mechanism of apoptosis, as used in the rehabilitation of post-stroke patients. Patients with moderate stroke severity (n = 48), 3–4 weeks after incident, were enrolled in the analysis and divided into ELF-EMF and non-ELF-EMF group. The rehabilitation program in both groups involves the following: kinesiotherapy—30 min; psychological therapy—15 min; and neurophysiological routines—60 min. Additionally, the ELF-EMF group was exposed to an ELF-EMF (40 Hz, 5 mT). In order to assess the apoptosis gene expression level, we measured the mRNA expression of BAX, BCL-2, CASP8, TNFα, and TP53. We found that ELF-EMF significantly increased the expression of BAX, CASP8, TNFα, and TP53, whereas the BCL-2 mRNA expression after ELF-EMF exposition remained at a comparable level in both groups. Thus, we suggest that increasing the expression of pro-apoptotic genes in post-stroke patients promotes the activation of signaling pathways involved in brain plasticity processes. However, further research is needed to clarify this process.

Bioactive compounds from Aspergillus niger extract enhance the antioxidant activity and prevent the genotoxicity in aflatoxin B1-treated rats

This study aimed to identify the bioactive compounds of the ethyl acetate extract of Aspergillus niger SH2-EGY using GC-MS and to evaluate their protective role against aflatoxin B1 (AFB1)-induced oxidative stress, genotoxicity and cytotoxicity in rats. Six groups of male Sprague-Dawley rats were treated orally for 4 weeks included the control group, AFB1-treated group (80 μg/kg b.w); fungal extract (FE)-treated groups at low (140) or high dose (280) mg/kg b.w and the groups treated with AFB1 plus FE at the two tested doses. The GC-MS analysis identified 26 compounds. The major compounds found were 1,2,3,4,6-Penta-trimethylsilyl Glucopyranose, Fmoc-L-3-(2-Naphthyl)-alanine, D-(-)-Fructopyranose, pentakis (trimethylsilyl) ether, bis (2-ethylhexyl) phthalate, trimethylsilyl ether-glucitol, and octadecanamide, N-(2- methylpropyl)-N-nitroso. The in vivo results showed that AFB1 significantly increased serum ALT, AST, creatinine, uric acid, urea, cholesterol, triglycerides, LDL, carcinoembryonic antigen, alpha-fetoprotein, interleukin-6, Malondialdehyde, nitric oxide, Bax, caspase-3 and P53 mRNA expression, chromosomal aberrations and DNA fragmentation. It decreased serum TP, albumin, HDL, Bcl-2 mRNA expression, hepatic and renal TAC, SOD and GPx content and induced histological changes in the liver and kidney. FE prevented these disturbances in a dosage-dependent manner. It could be concluded that A. niger SH2-EGY extract is safe a promising agent for pharmaceutical and food industries.

MiR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4.

Background: microRNAs (miRNAs) are a small, endogenous non-coding RNAs that are involved in post-transcriptional gene regulation of many biological processes, including embryo implantation and placental development. In our previous study, miR-146a-5p was found expressed higher in the serum exosomes of pregnant sows than non-pregnant. The research on miR-146a-5p has been mainly related to human diseases, but there are few studies on its effects on the reproduction of sows in early pregnancy.Objective: In this article, our motivation is to study the role of miR-146a-5p in the early pregnancy of sows on the cell proliferetion and apoptosis by targeting SMAD3 and SMAD4.Methods: Bioinformatics software was used to identify the target genes of miR-146a-5p. The wild-type and mutant-type recombinant plasmids of dual-luciferase reporter with 3'-UTR of Smad3 or 3'-UTR of Smad4 were constructed, and co-transfected in porcine kidney cell (PK-15 cell) with miR-146a-5p mimic, mimic-NC(M-NC), inhibitor and inhibitor-NC(IN-NC), then dual-luciferase activity analysis, qRT-PCR and Western blot were performed to verify the target genes. After the transfection of BeWo choriocarcinoma cell (BeWo cell) with miR-146a-5p mimic, M-NC, inhibitor and IN-NC, the mRNA expression of Caspase-3, BAX and Bcl-2 was measured using qRT-PCR, and the cell proliferation was measured using CCK-8 kit.Results: The luciferase, mRNA and protein expression of Smad3 in PK-15 cells treated by Smad3-3'-UTR-W co-transfected with miR-146a-5p mimic were significantly lower than that with miR-146a-5p M-NC, and the results of Smad4 were similar to Smad3, but the protein expression had a trend to lower in mimic group. The expression level of Bcl-2 in the miR-146a-5p mimic group was significantly lower than that in the miR-146a-5p M-NC group, but the expression pattern of Caspase-3 was just opposite. The mimic of miR-146a-5p reduced the proliferation of BeWo cells, however the inhibitor increased.Conclusion: Smad3 and Smad4 are the direct target genes of miR-146a-5p. The expression of Smad3 and Smad4 were affected by the mimic and inhibitor of miR-146a-5p. miR-146a-5p affects cell apoptosis and proliferation by regulating their target genes. This study provided new data to understand the regulation mechanism of early pregnancy in sows.

BMAL1 knockdown promoted apoptosis and reduced testosterone secretion in TM3 Leydig cell line

Brain and muscle Arnt-like protein-1 (BMAL1) is a clock gene that plays an important role in hormone secretion and apoptosis, but its effect on Leydig cells is unidentified. Here the role of BMAL1 in apoptosis and testosterone secretion in TM3 Leydig cell line were investigated by inhibiting its expression using small interfering RNA (siRNA). Results showed that BMAL1 knockdown promoted the apoptosis of Leydig cells and expression of (BCL2 associated X) BAX mRNA and protein, and reduced the expression of (B-cell lymphoma-2) BCL-2 mRNA and protein. BMAL1 inhibition resulted in decreased testosterone secretion and reduced expression of key genes during hormone synthesis, specifically steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD). In addition, BMAL1 knockdown reduced the expression of phosphorylated p85 and AKT as confirmed by western blot. In conclusion, BMAL1 may affect testosterone secretion and apoptosis in mouse Leydig cells through regulation of the PI3K/AKT signaling pathway.

Neuroprotective effects of mirtazapine and imipramine and their effect in pro- and anti-apoptotic gene expression in human neuroblastoma cells.

Experimental and clinical studies indicate that neuronal death with the presence of high levels of reactive oxygen species are present in depressed patients and antidepressants might display neuroprotective effects against them. However, the mechanisms underlying antidepressant neuroprotection are not completely understood. In our previous study, we showed that mirtazapine modulated the expression of pro- and anti-apoptotic proteins in mouse brain structures, but there are no data in human cells. Thus, this work was designed to study the possible neuroprotective properties of mirtazapine and imipramine, two commercially available antidepressants with different primary mechanisms of action, in human neuroblastoma SH-SY5Y cells against an oxidative insult. SH-SY5Y cells were preincubated with mirtazapine and imipramine (1-20μM) for 24h, then hydrogen peroxide (H2O2) was added into the medium containing the antidepressants for additional 24h, and MTT assay was carried out subsequently. Also, to elucidate the molecular mechanism underlying the neuroprotective properties of antidepressants, we investigated the effects of mirtazapine and imipramine (2μM) in pro- and anti-apoptotic proteins gene expression in SH-SY5Y cells. Mirtazapine (1 and 2μM) and imipramine (1and 2μM) protected against hydrogen peroxide-induced cellular viability impairment. Most importantly, both compounds reduced p53 mRNA expression, but only imipramine enhanced the Bcl-2/Bax ratio. The obtained data indicate that mirtazapine and imipramine have neuroprotective effects against H2O2-induced cell death. Although both antidepressants reduced Bax and p53 mRNA expression, only the protection mediated by imipramine might be due to its ability to enhance Bcl-2/Bax ratio.

Total flavones of Dracocephalum moldavica L. protect astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway

BackgroundThe active components of Dracocephalum moldavica L. (TFDM) can inhibit myocardial ischemia by inhibiting oxidative stress. However, the effects of TFDM on astrocytes have not been investigated in vitro. The current study aimed to explore whether TFDM protects astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway.MethodsThe human glioma cell line U87 was used to investigate the ability of TFDM to protect astrocytes against H2O2-induced apoptosis. The cell counting kit-8 assay and flow cytometry were used to detect cell viability, apoptosis, MMP, Ca2+ influx and reactive oxygen species (ROS). Lactate dehydrogenase (LDH) and malonic dialdehyde (MDA) levels were measured by ELISA. In addition, protein and mRNA expression changes were detected by Western blotting and qRT-PCR.ResultsTFDM (0.78~200 μg/ml) had limited cytotoxic effects on the viability of U87 cells. Compared with the model group (treated with H2O2 only), cells treated with medium- and high-dose TFDM exhibited reduced MDA concentrations (P < 0.05) and ROS production (P < 0.05) and decreased MMP (P < 0.05) and reduced apoptosis (P < 0.05). The percentage of annexin V-FITC-stained cells was markedly suppressed by TFDM, confirming its anti-apoptotic properties. WB results showed that protein expression of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, Caspase-12, and B-cell leukemia/lymphoma 2 (Bcl2) was reduced in the TFDM group compared with that in the model group (P < 0.05) and that expression of these proteins was normalized by TFDM treatment in a dose-dependent manner. According to RT-qPCR results, TFDM pretreatment resulted in reduced mRNA expression of BAX, Caspase-9, Caspase-12, p38MAPK, and CaMKII and increased mRNA expression of mTOR compared with the model group.ConclusionsThe current study revealed the protective effects of TFDM on U87 cells under oxidative stress conditions through the inhibition of a mitochondria-dependent pathway that is associated with the CaMKII/P38MAPK/ERK1/2 and PI3K/AKT/mTOR pathways.

MiR-133a alleviates renal injury caused by sepsis by targeting BNIP3L.

Sepsis is an important cause of acute kidney injury (AKI), seriously jeopardizing the health of patients. This paper's aim was to investigate whether microRNA-133a had a protective effect on sepsis-induced kidney injury. We established a kidney injury model with lipopolysaccharide (LPS) and divided TCMK-1 cells into 4 groups: control group (con); LPS treatment group; LPS + negative control (NC) treatment group; LPS + miR-133a mimic (mim) group. The expressions of miR-133a, TNF-α mRNA, IL-6 mRNA, Bax mRNA, Bcl-2 mRNA, BNIP3L mRNA, IκKα Mrna and IκB-α mRNA were detected by PCR. Western blot was used to detect the protein expression of TNF-α, IL-6, Bax, Bcl-2, BNIP3L, IκKα and IκB-α. Cell viability was measured by cell counting kit-8 (CCK-8). Flow cytometry was utilized to detect apoptosis rate. IL-1β immunofluorescence and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining were used to observe the inflammation and apoptosis in TCMK-1 cells. The miR-133a expression was decreased in TCMK-1 cells treated with LPS. In the LPS treatment group, the expression of TNF-α, IL-6, Bax, BNIP3L and IκKα increased, and the expression of Bcl-2 and IκB-α decreased. When overexpressing miR-133a, the protein and mRNA expression of TNF-α, IL-6, Bax, BNIP3L and IκKα decreased markedly, while the expression of Bcl-2 and IκB-α increased markedly. Compared with the LPS-treated group, the apoptotic rate, the number of TUNEL-positive cells, and the immunofluorescence intensity of IL-1β in LPS+mim group were greatly decreased. The miR-133a expression was decreased in TCMK-1 cells treated by LPS and miR-133a can inhibit inflammation and apoptosis of TCMK-1 cells induced by LPS by targeting BNIP3L via inhibiting NF-κB pathway.

Meriolin1 induces cell cycle arrest, apoptosis, autophagy and targeting the Akt/MAPKs pathways in human neuroblastoma SH-SY5Y cells.

Meriolins, a kind of chemical hybrid between meridianins and variolins, have lately been determined as kinase inhibitors and reportedly have antitumour activity. However, there is currently no in-depth study for the action mechanism. This study aimed to elucidate the potentially antitumour action mechanism of Meriolin1 on human neuroblastoma (SH-SY5Y) cells. Firstly, cell viability was detected by MTT assay. Secondly, cell cycle, cell apoptosis, cell autophagy, reactive oxygen species and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. Then, cell cycle-associated proteins, Bcl-2 family proteins, Akt/MAPK proteins and autophagy-associated proteins expressions were evaluated by Western blot. Bcl-2 and Bax mRNA expressions were also evaluated by qRT-PCR. Furthermore, cell adhesion assay and Hoechst 33258 fluorescent staining were carried out to detect the effect of Meriolin1 on cell adhesion and morphology. Finally, to gain further insight into mechanism of action of Meriolin1 to CDK protein, the molecular docking study was performed by using the CDOCKER module of DS software. Meriolin1 could exert the antitumour activity on SH-SY5Y cells by inducing cell cycle arrest, cell autophagy, the mitochondrion-dependent cell apoptosis and targeting the Akt/MAPKs signalling pathway. Meriolin1 might be a promising therapeutic candidate for neuroblastoma.

LY294002 Enhaces Chemosensitivity of K562 Cells to Daunorubicin

To investigate the proliferation inhibition and pro-apoptosis effect of LY294002 (PI3K/AKT inhibtor) combined with daunorubicin (DNR) on the chronic myeloid leurenia cell line K562 and its possible mechanisms. The effect of LY294002 and DNR on the proliferation of K562 cells in different treating time and concentration were measured by MTT assay. The cell cycle was determined by flow cytometry, the mRNA and protein expression of SKP2 , P27, BCL-2 and BAX were determined by RT-PCR and Western blot. LY294002 and DNR were able to inhibit the growth of K562 cells and promote apoptosis in time- and concentration-dependent manner (P＜0.05), both the cell proliferation-inhibiting rate and apoptosis rate in combination therapy group were higher than that in DNR-monotherapy group (P＜0.05). After K562 cells treated by LY294002 combined DNR for 36 h, the cells were statistically significantly reduced in G2/M phase (P＜0.05), as compared with control group and DNR group. Compared with DNR group, the cell level of G0/G1 phase rased (P＜0.05) and cell level of S phase decreased (P＞0.05). Compared with DNR group, the expresson of SKP2 and BCL-2 mRNA decreased, and the expression of P27 mRNA increased in the combination therapy group (P＜0.05). The expression of BAX mRNA was not significantly different between different groups. The same result was found in the protein expression. LY294002 has the sensibilizative effect on DNR chemotherapy, which may be relative with blocking the cell cycle and inducing cell apoptosis.

LY294002 Enhaces Chemosensitivity of K562 Cells to Daunorubicin

To investigate the proliferation inhibition and pro-apoptosis effect of LY294002 (PI3K/AKT inhibtor) combined with daunorubicin (DNR) on the chronic myeloid leurenia cell line K562 and its possible mechanisms. The effect of LY294002 and DNR on the proliferation of K562 cells in different treating time and concentration were measured by MTT assay. The cell cycle was determined by flow cytometry, the mRNA and protein expression of SKP2 , P27, BCL-2 and BAX were determined by RT-PCR and Western blot. LY294002 and DNR were able to inhibit the growth of K562 cells and promote apoptosis in time- and concentration-dependent manner (P＜0.05), both the cell proliferation-inhibiting rate and apoptosis rate in combination therapy group were higher than that in DNR-monotherapy group (P＜0.05). After K562 cells treated by LY294002 combined DNR for 36 h, the cells were statistically significantly reduced in G2/M phase (P＜0.05), as compared with control group and DNR group. Compared with DNR group, the cell level of G0/G1 phase rased (P＜0.05) and cell level of S phase decreased (P＞0.05). Compared with DNR group, the expresson of SKP2 and BCL-2 mRNA decreased, and the expression of P27 mRNA increased in the combination therapy group (P＜0.05). The expression of BAX mRNA was not significantly different between different groups. The same result was found in the protein expression. LY294002 has the sensibilizative effect on DNR chemotherapy, which may be relative with blocking the cell cycle and inducing cell apoptosis.

Luteolin induces mitochondrial apoptosis in HT29 cells by inhibiting the Nrf2/ARE signaling pathway.

The aim of the current study was to investigate luteolin-induced apoptosis and the molecular mechanisms underlying it in HT29 cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the cytotoxicity of luteolin on HT29 cells, and a dichloro-dihydro-fluorescein diacetate assay was used to measure cellular levels of reactive oxygen species (ROS). The effects of luteolin on the mitochondrial membrane potential were also evaluated. Bax and Bcl-2 mRNA expression were determined using reverse transcription-quantitative PCR. Additionally, western blot analysis was performed to assess changes in cytochrome c and caspase-3 protein expression. Localization of nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus was also assessed using immunofluorescence. Luteolin exhibited cytotoxicity on HT29 cells in a time- and concentration-dependent manner. Additionally, ROS production was indicated to be increased and ROS scavenging was decreased, which resulted in a significant increase in the levels of ROS in the cells. The mitochondrial membrane potential was indicated to decrease following luteolin treatment. At the molecular level, luteolin significantly increased the mRNA expression of Bax and the protein expression of cytochrome c, caspase-3, p47phox and p22phox. The results revealed that luteolin decreased Bcl-2 protein expression and inhibited the nuclear localization of Nrf2. In conclusion, the current study indicated that luteolin inhibited HT29 cell proliferation and induced apoptosis via the mitochondrial pathway.

Erythropoietin promotes functional recovery via anti-apoptotic mechanisms in mouseunilateral ureteral obstruction.

The purpose of the work wasto investigate mechanisms of erythropoietin-induced protection and accelerated recovery of kidneys and ureters from obstructive injury. Unilateral ureteral obstruction was established for 24, 48, and 72h in C57BL/6 mice using a non-traumatic micro-clip followed by the microscopic quantification of ureteral peristalsis pre- and post-obstruction. Expression of erythropoietin, erythropoietin receptor, β-common receptor, and downstream apoptosis-related markers was assessed by RT-PCR and immunohistochemistry in ureters and kidneys and compared to the respective organs on the contralateral side within each animal. Expression of genes in kidneys and ureters from mice treated with 20IU of erythropoietin daily for 72h prior to obstruction was compared to that of untreated mice following obstruction. Apoptosis in ureteral tissues after 72-h obstruction was assessed via TUNEL assay. Ureteral obstruction increased apoptosis in affected ureters, with peristaltic function halted following all periods of obstruction. Erythropoietin treatment suppressed apoptosis in obstructed tissues and increased the percentage of mice retaining ureteral function immediately following obstruction reversal. Erythropoietin, erythropoietin receptor, Bcl-2, and Bcl-xl mRNA expression were down-regulated, while phospho-Nf-ĸb p65 was up-regulated in ureteral epithelia following obstruction. Erythropoietin treatment induced anti-apoptotic signaling via down-regulated Bax mRNA expression and abrogated phospho-Nf-ĸb p65. Erythropoietin-induced protection of ureteral function and accelerated recovery post-obstruction removal is mediated via anti-apoptotic mechanisms. Ureteral function is disrupted even following obstruction removal, negatively affecting renal function due to delayed recovery. Thus, our results represent a potential target for the development of safe therapeutic agents aimed at improving functional recovery from obstructive injury.

Resveratrol Reduces Kidney Injury in a Rat Model of Uremia and is Associated with Increased Expression of Heat Shock Protein 70 (Hsp70).

BackgroundThis study aimed to investigate the effects of resveratrol on kidney function in a rat model of uremia and the expression of heat shock proteins.Material/MethodsThe rat model of uremia was developed by 5/6 nephrectomy of Sprague–Dawley rats. The Hsp70 inhibitor MKT-077, a rhodacyanine dye, was used. The study groups included rats with sham surgery (the sham group), the rat model of uremia (the model group), the solvent-treated control group (the control group), the rat model treated with resveratrol group (the resveratrol group), the rat model treated with MKT-077 (the MKT-077 group), and the resveratrol+MKT-077 group. Kidney tissues were studied histologically. Renal cell apoptosis was detected by the TUNEL method. Expression of p53, Bax, and Bcl-2 mRNA and protein were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry, respectively.ResultsCompared with the sham group, the expression levels of heat shock proteins Hsp70, Hsp90, Hsp27, Hsp25, Hsp40, and Hsp60 in the kidney of the rat model group increased to different degrees. Compared with the model group, the Hsp70 levels in the resveratrol group were significantly increased (p<0.05). Compared with the model group, treatment with MKT-077 reduced the survival rate of rats, which was increased following resveratrol treatment. Compared with the resveratrol group, renal function in the resveratrol+MKT-077 group was significantly reduced (p<0.05).ConclusionsIn a rat model of uremia, resveratrol reduced renal injury and improved both renal function and survival, which were associated with increased expression of Hsp70.

High-Concentrate Feeding to Dairy Cows Induces Apoptosis via the NOD1/Caspase-8 Pathway in Mammary Epithelial Cells

(1) Background: The effects of a high-concentrate (HC) diet in inducing mammary epithelial cell apoptosis in dairy cows via the NOD1/Caspase-8 pathway have never been investigated before the current study. (2) Methods: Twelve Holstein Frisian cows at mid-lactation were selected to conduct this research. The animals were randomly allocated to two groups (n = 6), and both groups received one of two diets: a low-concentrate (LC) (forage: concentrate 6:4) or a high-concentrate (HC) (forage: concentrate 4:6) diet. Furthermore, an enzyme activity assay, tunnel cell assay, RT-qPCR, western blotting, and an immunofluorescence antibody (IFA) assay were performed to elucidate the effect of an HC diet in the mammary gland of dairy cows. (3) Results: The tunnel cell assay revealed a significant number of apoptotic cells in HC group, and the concentration of Caspase-3, and Caspase-8 was higher in the HC group than in the LC group. NOD1, Rip-2, Caspase-3, Caspase-8, Caspase-9, and Bax mRNA expressions, and NOD1, Caspase-3, Caspase-8, and Bax protein expressions, in the HC group were markedly higher than those in the LC group. Furthermore, Bcl-2 mRNA and protein expressions were markedly decreased in the HC compared to those in the LC group. (4) Conclusions: A HC diet fed to dairy cows incites subacute ruminal acidosis (SARA), which increases the iE-DAP concentration and induces apoptosis in the mammary gland via the NOD1/Caspase-8 pathway.

Bio-functionalized CuO nanoparticles induced apoptotic activities in human breast carcinoma cells and toxicity against Aspergillus flavus: An in vitro approach

This study demonstrates a comprehensive and cost-effective one-step rapid bio-fabrication of Eucalyptus globulus leaf extract (ELE) bio-actives functionalized copper oxide nanoparticles (CuONPs); and analyze its apoptotic and toxic effects in human breast cancer (MCF-7) cells and Aspergillus flavus. The state-of-art characterization data confirmed the thermal-stability of (i) ELE-CuONPs and (ii) surface associated ELE bio-active compounds bearing characteristic functional groups (aromaticOH, polymericOH, CO, and CO) over a range of conventional temperatures (30 °C–80 °C), and microwave (MW) heat (140 °C). The MW accelerated ELE-CuONPs (16.9 nm) treated (25−100 μg/ml) MCF-7 cells showed a significant (p < 0.05) reduction in viability due to the dysfunction of mitochondrial membrane potential, intracellular ROS accumulation, and disruption in normal cell cycle progression. Moreover, transcriptional up-regulation of mRNA expression of p53 (8.8 fold), bax (1.42 fold), caspase-3 (2.42 fold), and caspase-9 (3.34 fold) genes in the MCF-7 cells confirmed a mechanistic anti-cancer landscape of synthesized ELE-CuONPs. Furthermore, ELE-CuONPs inhibited sclerotia germination and growth of A. flavus. Conclusively, this study is novel in better demonstrating the formation of ELE-CuONPs that meets the principles of green chemistry. These findings can be used to develop strategies for the management of cancer and fungal infections affecting human health.

Synergistic effect of ascorbic acid and taurine in the treatment of a spinal cord injury-induced model in rats.

Spinal cord injury (SCI) results in severe damage, which causes functional alterations together with loss of autonomic functions, sensations, and muscle functioning. This injury leads to apoptosis of neurons and oligodendrocytes, which further leads to dysfunction of the spinal cord due to axonal degeneration and demyelination. Taurine is non-proteogenic and an essential amino acid, which plays a major role in the growth and development of brain cells. Ascorbic acid, also known as vitamin C, is found in various foods and is known to prevent scurvy. In this study, we have investigated the therapeutic effect of ascorbic acid and taurine against SCI-induced rats. The rats were divided into the following groups: sham, control, 100mg/kg of taurine, 100mg/kg of ascorbic acid, and 100mg/kg of taurine + 100mg/kg of ascorbic acid. Treatment was continued daily for 45 consecutive days. The combined treatment of taurine and ascorbic acid decreased caspase-3, bax, pro-NGF, and p53 mRNA expression by more than 30% compared to individual treatments. The combined treatment of taurine and ascorbic acid reduced caspase-3 and p53 expression by 33.7% and 44%, respectively, compared to individual treatments. The combined treatment of taurine and ascorbic acid decreased mRNA expression of interleukin-6 (IL-6), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) compared to the individual treatments of taurine and ascorbic acid. The combined treatment of taurine and ascorbic acid also significantly recovered altered antioxidant markers, and induced lipid peroxidation to near normal levels. In summary, apoptotic, inflammatory and oxidative stress markers were significantly decreased in SCI-induced rats treated with taurine and ascorbic acid.

Moderate intensity intermittent exercise upregulates neurotrophic and neuroprotective genes expression and inhibits Purkinje cell loss in the cerebellum of ovariectomized rats

Decreases in estrogen levels due to menopause or ovariectomy may disrupt cerebellar motor functions. This study aimed at investigating the effects of Moderate Intensity Intermittent Exercise (MIEx) on the cerebellum of ovariectomized rats by analyzing neurotrophic and neuroprotective markers, as well as cerebellar motor functions. Thirty-two female Sprague Dawley rats were divided into four groups, i.e. Sham and ovariectomy (Ovx) of non-MIEx (NMIEx) groups, and Sham and Ovx with MIEx groups. MIEx was performed 5 days a week on treadmill for 6 weeks. Motor functions were assessed using rotarod, footprint, open field, and wire hanging tests. Real-time polymerase chain reaction was performed to determine messenger RNA (mRNA) expressions of Pgc-1α, BDNF, synaptophysin, Bcl-2, and Bax. Unbiased stereology was used to estimate the total number of cerebellar Purkinje cells. The Ovx MIEx group had higher Pgc-1α and Bcl-2 mRNA expressions, and number of Purkinje cells, but lower Bax mRNA expression than the Ovx NMIEx group. All motor functions of MIEx groups were better than the Sham and Ovx groups without MIEx. Motor functions on rotarod task, OFT, and FPT correlated significantly with the mRNAs expression of Bcl-2, Bax, BDNF, synaptophysin, Pgc-1α, and the number of cerebellar Purkinje cells in ovariectomized rats. MIEx improves cerebellar neurotrophic and neuroprotective markers, as well as motor functions of ovariectomized rats.

The α-Tomatine Exhibits Antiproliferative Activity, Rupture of Cell Membranes and Induces the Expression of APC Gene in the Human Colorectal Adenocarcinoma Cell Line (Ht-29)

The α-tomatine is a steroidal glycoalkaloid found in immature tomatoes (Lycopersicon esculentum) that has important biological functions including the inhibition of cancer cell growth and preventing metastasis. This study aimed to evaluate the effects of α-tomatine on cytotoxicity, cellular proliferation, apoptosis, and mRNA expression of APC, CCNA2, β-catenin, CASP9, BAK, BAX and BCL-XL in colorectal adenocarcinoma cell line HT-29. HT29 cells were treated with three concentrations of α-tomatine (0.1, 1 and 10 µg/mL), although only the 1 µg/mL concentration of α-tomatine was used to evaluate genetic expression patterns by real time-PCR. Results showed that α-tomatine was cytotoxic only at the 10 µg/mL concentration. Cell proliferation was significantly inhibited after the first 24 hours of treatment only with concentrations of 10 µg/mL. In contrast, there were no significant differences in apoptosis for any treatment. In the gene expression studies, only APC expression was significantly altered by α-tomatine treatment. In conclusion, α-tomatine has antiproliferative activity in the first 24h of treatment, does not induce apoptosis in this cell line and causes disruption of cell membranes, thereby increasing the expression of APC gene related to cell cycle.