mRNA Levels Of Caspase-3 Research Articles

Determination of the Cytotoxicity of Capsaicin and its Effects on Caspase-3 in the Clones Selected from H2452 Mesothelioma Cell Line

Mezotelyoma; kalp, akciğer ve karın gibi iç organların yüzeyini kaplayan mezotelyum dokusundan gelişen bir kanser türüdür ve tümörlerinin poliKlon al kökene sahip olduğu düşünülmektedir. Kapsaisinin kanser hücreleri üzerindeki apoptotik etkileri bilinmektedir. Bu çalışmada; H2452 mezoteliyomahücre hattından seçilmiş dört (I, II, III ve IV numaralı) Klon üzerinde kapsaisinin sitotoksisitesi ve kaspaz-3 ifadesine ve aktivasyonuna etkileri tespit edilmeye çalışılmıştır. Bu amaçla; öncelikle, Klon ların IC50 değerleri MTS [3-(4,5)-dimetiltiazol-2-il)-5-(3-karboksimetoksifenil)-2-(4-sülfofenil)-2H-tetrazolyum] tuzunun formazana indirgenmesi metoduyla belirlenmiş ve kapsaisin sitotoksisitesinin bazı Klon lar arasında istatistiksel olarak anlamlı bir fark gösterdiği (p<0.01) ortaya konulmuştur. Daha sonra, seçilen Klon lara ortalama IC50 derişiminde (251.6 µM) kapsaisin uygulanmış ve hücre büyüme eğrileri çizilmiştir. Buna göre; 72 saat sonunda, Klon I’in diğer Klon lardan daha yüksek sayıda (p<0.001) canlı hücre içerdiği gözlenmiştir. Bunu takiben gerçekleştirilen qRT-PCR ve Western Blotlama ile; kapsaisin uygulanmış (KAP), etanol uygulanmış (EtOH) ve herhangi bir uygulama görmemiş, kontrol grubu hücrelerinde kaspaz-3 molekülünün sırasıyla mRNA ve protein seviyesindeki değişimler tespit edilmiştir. Sonuç olarak; kapsaisin sitotoksistesinin seçilen Klon ile anlamlı bir değişiklik gösterdiği tespit edilmiş, ve kaspaz-3 proteinin inaktif ve aktif biçimlerinin miktarı ile bağıl kaspaz-3 mRNA seviyesi her bir Klon için diğerlerinden farklı sonuçlar ortaya koymuştur. Tüm bulgular birlikte değerlendirildiğinde, mezotelyoma tümörlerini oluşturan hücre popülasyonlarının poliKlon al bir kökene sahip olabileceği fikri destekleniyor görünmektedir.

Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis attenuates cadmium-induced apoptosis in duck renal tubular epithelial cells.

Cadmium (Cd) is an occupational and environmental pollutant, which mainly causes nephrotoxicity by damaging renal proximal tubular cells. To evaluate the effects of Cd on pyroptosis and the relationship between pyroptosis and apoptosis in duck renal tubular epithelial cells, the cells were cultured with 3CdSO4·8H2O (0, 2.5, 5.0, or 10.0μM Cd), N-acetyl-L-cysteine (NAC) (100.0μM), Z-YVAD-FMK (10.0μM) or the combination of Cd and NAC or Z-YVAD-FMK for 12h, and then cytotoxicity was assessed. The results evidenced that Cd significantly increased the releases of interleukin-18 (IL-18) and interleukin-1β (IL-1β), lactate dehydrogenase (LDH) and nitric oxide (NO), relative conductivity and cellular reactive oxygen species (ROS) level. Simultaneously, Cd also markedly upregulated NLRP3, Caspase-1, ASC, NEK7, IL-1β and IL-18 mRNA levels and NLRP3, Caspase-1 p20, GSDMD and ASC protein levels. Additionally, NAC notably improved the changes of above indicators induced by Cd. Combined treatment with Cd and Z-YVAD-FMK remarkably elevated Bcl-2 mRNA and protein levels, inhibited p53, Bax, Bak-1, Cyt C, Caspase-9 and Caspase-3 mRNA levels and p53, Bax, Bak-1, Caspase-9/cleaved Caspase-9 and Caspase-3/cleaved Caspase-3 protein levels, increased mitochondrial membrane potential (MMP), decreased apoptosis ratio and cell damage compared to treatment with Cd alone. Taken together, Cd exposure induces duck renal tubular epithelial cell pyroptosis through ROS/NLRP3/Caspase-1 signaling pathway, and inhibiting Caspase-1 dependent pyroptosis attenuates Cd-induced apoptosis.

Restoration of miRNA-143 Expression Inhibits Growth and Migration of MKN-45 Gastric Cancer Cell Line.

Purpose: Gastric cancer (GC) is one of the main causes of death from diseases, especially in developing countries. MicroRNAs (miRNAs) are important modulators of the messenger RNAs expression. Among these miRNAs, MiR-143 is a tumor suppressor miRNA and its irregular expression has been revealed in a diversity of malignancies such as GC. Methods: In this study, we have attempted to restore the miR-143 expression in MKN-45 cells by introducing pCMV-miR-143 plasmid vectors. The consequences of exogenous expression of miR-143 on cell proliferation and migration were assessed by MTT and scratch tests, respectively. In addition, the DAPI staining assay was applied for apoptosisquantification. Following miR-143 transfection, the changes in K-Ras, C-Myc, MMP9, Bax, Caspase-3, and Caspase-9 mRNA levels were assessed. Results: The results indicated that the enhanced expression of miR-143 had negative effects on MKN-45 cells proliferation and invasion. Moreover, decreased expressions of K-Ras, MMP9, and C-Myc and up-regulation of Bax, Caspase-3, and Caspase-9 as downstream targets of miR-143 were recognized. Conclusion: These experimental results indicate that reversing the miR-143 expression, by novel techniques, including miRNA replacement could be considered as an efficient approach to reduce cell survival and metastasis.

Dietary choline deficiency aggravated the intestinal apoptosis in association with the MAPK signalling pathways of juvenile grass carp (Ctenopharyngodon idella)

To investigate the effects and potential mechanisms of apoptosis in the intestines of juvenile grass carp (Ctenopharyngodon idella) for dietary choline, fish were fed different choline-containing diets (142.2, 407.4, 821.6, 1215.8, 1589.3, and 1996.6 mg kg−1) for 70 days. Results revealed that dietary choline deficiency (1) decreased the contents of choline, phosphatidylcholine, betaine, and acetylcholine (not in distal intestine [DI]) in grass crap intestines; down-regulated the mRNA abundances of choline transportations, such as high-affinity choline transporter CHT1 (not in proximal intestine[PI] and DI), choline transporter-like proteins such as CTL-1, CTL-2, CTL-4 (not in DI), and CTL-5 in grass crap intestines; (2) enhanced the degree of DNA fragments. Moreover, it up-regulated the activities of caspase-3, −8, and-9; reduced mRNA levels of caspase-3, −7, −8 (not in DI), and − 9, Fas ligand (FASL) (not in PI), and apoptotic protease activating factor-1(Apaf1), as well as the protein level of cytochrome c (Cty c). Dietary choline deficiency also down-regulated myeloid cell leukemia-1 (Mcl-1) (not in PI), B-cell lymphoma-2 (Bcl-2), and the inhibitor of apoptosis protein (IAP) mRNA levels in grass carp intestines, suggesting that choline inhibited cell apoptosis in fish intestines; (3) increased p38MAPK and JNK(not in mid intestine [MI]) mRNA level, as well as p-p38MAPK and T-p38MAPK protein levels in the PI and the ratio of p-p38MAPK/T-p38MAPK in the MI and DI, indicating that choline deficiency aggravated cell apoptosis in association with the MAPK signalling pathway in fish intestines; In conclusion, choline insufficiency promotion of intestinal apoptosis might be related to MAPK signalling pathways in fish. Moreover, the requirements of choline for resist intestinal apoptosis indices (caspase-8 and capase-9 activities) of grass carp (9.28–108.97 g) were calculated to be (1162.13–1780.93) mg kg−1 diet, respectively.

Emodin Reverses Gemcitabine Resistance of Pancreatic Cancer Cell Lines Through Inhibition of IKKβ/NF-κB Signaling Pathway.

BackgroundPancreatic cancer is one of the most malignant tumors, and gemcitabine has been considered as the standard treatment and been widely utilized as a first-line drug for advanced pancreatic cancer, but gemcitabine-resistance always occurs after a short period of treatment.MethodsTwo pancreatic cancer cell lines Panc-1 and MIA-PaCa-2 were used as the study subject and their gemcitabine-resistant cells were established. Both drug-resistant cells were divided into four groups: blank, emodin, gemcitabine, and emodin+gemcitabine. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis rate and P-gp function. Quantitative real-time polymerase chain reaction and Western blotting were used to detect Survivin, XIAP, Caspase-9/3, NF-κB p65, IKKβ and IκB-α mRNA/protein expressions, respectively. Electrophoretic mobility shift assay (EMSA) was performed to detect NF-κB binding activity. Rhodamine 123 efflux assay was used to detect P-gp function.ResultsEmodin could inhibit cell activity in all cell lines. Both emodin and gemcitabine can significantly increase the apoptosis rate, and the combination of the two drugs can further significantly increase the apoptosis rate in normal pancreatic cancer cell lines. In both drug-resistant pancreatic cancer cell lines, it can be observed that although gemcitabine can increase the apoptosis rate, the effect of promoting apoptosis is significantly lower than that of emodin; the drug combination can still significantly increase the apoptosis rate on the basis of emodin alone. Emodin can significantly reduce the mRNA and protein expression levels of Survivin, XIAP, NF-κB, and IKKβ, and significantly increase the mRNA and protein expression levels of Caspase-3/9 and IκB-α. Emodin significantly reduced NF-κB activity and emodin significantly promoted P-gp fluorescence intensity from Rhodamine 123 efflux assay.ConclusionEmodin inhibits the expression of IKKβ, thereby inhibiting the expression and activity of downstream NF-κB, and inhibits P-gp function at the same time, ultimately achieving the purpose of reversing the drug-resistance of pancreatic cancer cell lines.

Lowered Cyclin E levels increase the efficiency and the specificity of capsaicin against cancerous cells of mesothelium

Capsaicin is one of the most extensively studied phytochemicals and its cytotoxicity on various types of cancer has been demonstrated both in vitro and in vivo. The evaluation of its effect on mesothelioma, however, has remained quite limited. In this study, we investigated the anti-mesothelioma potential of capsaicin by observing its cytotoxicity on healthy, immortalized and cancerous cells of mesothelium in vitro and how this potential be affected by lowered Cyclin E levels, a key regulator of G1/S transition of cell cycle. For this purpose, we determined and compared the IC50 values of capsaicin in both FBS (Fetal Bovine Serum) containing and FBS-deprived medium of each cell population studied. Additionally, we examined the changes in both protein and mRNA levels of caspase-3 upon capsaicin exposure as well as conducted a series of experiments through which the relatively long term effect of capsaicin on the growth rate of the cells was assessed. As a result, the reduced Cyclin E obtained through the absence of FBS in growth medium was found not only to decrease IC50 values for all cell types dramatically (p<0.05) but also to cause a considerable difference between the values determined for cancerous and non-cancerous populations (p<0.05), which had not been observed in regular medium. Moreover, along with the fact that capsaicin exposure did not have an impact on the cell growth in long term in most cases, caspase-3 levels also remained the same when exposed to capsaicin, suggesting a mechanism of cell death independent of caspases.

Zinc attenuates ecstasy-induced apoptosis through downregulation of caspase-3 in cultured TM3 cells: An experimental study.

Background3, 4-Methylenedioxymethamphetamine (MDMA) is commonly known as the most famous amphetamine derivative.ObjectiveTo evaluate the influence of zinc on MDMA-induced apoptosis and caspase- 3 gene expression in Leydig cell line (TM3).Materials and MethodsLeydig cells were studied in differenet treatment groups regarding MDMA (0, 0.5, 1, 3, 5 mM) and zinc (0, 4, 8, 16, 32 μM). By the way, the effective concentration was determined to be 5 mM for MDMA and 8 μM for zinc. Then, TM3 cells were cultured in free medium as control (group I), medium containing MDMA (5 mM) (group II), zinc (8 µM) (group III), and zinc (8 µM) prior to MDMA (5 mM) (group IV) as well as in an untreated group (control). Cell viability was assessed at different times after cell culture by MTT assay. The mRNA expression level of caspase-3 was analyzed using real-time quantitative polymerase chain reaction.Results The cellular viability was significantly reduced in TM3 cells after 24 hr and 48 hr exposure time regarding different concentrations of MDMA as well as high concentration of zinc (16 and 32 μM). Cell viability was increased in the group that received zinc (8 µM) before addition of MDMA (5 mM) compared to the control and MDMA groups. The mean SE of fold was 22.40 7.5, 0.06 0.02, and 0.009 0.003 in MDMA, zinc, and zinc + MDMA groups, respectively. The mean of caspase-3 mRNA level was significantly increased in the MDMA-treated group (5 mM), while the relative expression of caspase-3 gene was significantly decreased in the zinc (8 µM) + MDMA (5 mM) group compared with the MDMA (5 mM) group (p = 0.001).ConclusionDietary intake of zinc has a protective effect against MDMA consumption in mouse.

Niclosamide-loaded polymeric micelles ameliorate hepatocellular carcinoma in vivo through targeting Wnt and Notch pathways

AimNiclosamide (NIC) is an anthelmintic agent repurposed as a potent anticancer agent. However, its use is hindered by its poor solubility. We investigated the underlying mechanisms of NIC anticancer activity employing a novel oral NIC pluronic-based nanoformulation and tested its effect in thioacetamide-induced hepatocellular carcinoma (HCC) in rats. We evaluated its antitumor effect through regulating Wnt/β-catenin and Notch signaling pathways and apoptosis. Main methodsNiclosamide-loaded pluronic nanoparticles (NIC-NPs) were optimally developed and characterized with sustained release properties up to 7 days. Sixteen weeks after HCC induction, NIC (70 mg/kg) and an equivalent dose of NIC-NPs were administered orally for 3 consecutive weeks. Hepatocyte integrity was assessed by measuring serum levels of aminotransferases, ALP, GGT, bilirubin, albumin and total protein. HCC development was detected by measuring AFP expression. Necroinflammation and fibrosis were scored by histopathological examination. Wnt/β-catenin and Notch signaling were evaluated by measuring hepatic mRNA levels of Wnt3A, Lrp5 and Lrp6 Co-receptors, Dvl-2, Notch1 and Hes1 and β-catenin protein levels. Apoptosis was assessed by measuring mRNA and protein levels of cyclin D1 and caspase-3. Key findingThe novel NIC-NPs restored liver integrity, reduced AFP levels and showed improved anticancer and proapoptotic activities compared to drug alone. The inhibitory effect of NIC on Wnt/β-catenin and Notch signaling pathways was potentiated by the NIC-NPs formulation. SignificanceWe conclude that NIC acts by inhibiting Wnt/β-catenin and Notch signaling and inducing apoptosis in HCC. Developing pluronic-based nanoformulations may be a promising approach to improve NIC solubility and offer the possibility of controlled release.

Florfenicol causes excessive lipid peroxidation and apoptosis induced renal injury in broilers

In order to study the effects and mechanism of florfenicol (FFC) on the kidney function of broilers, 180 1-day-old broilers were randomly divided into 6 groups, 30 in each group. Except for the control group, different doses of FFC were added to drinking water in the other 5 groups (0.15 g/L, 0.3 g/L, 0.6 g/L, 1.2 g/L and 1.8 g/L). After continuous administration for 5 days, renal histopathological changes, serum renal function indicators, renal peroxidation products and antioxidant factors, and apoptotic factors were detected in broilers aged 21 and 42 days. The results showed that compared with the control group, the kidney tissue structure was disordered, the glomerulus was atrophic, the cystic cavity was enlarged, and the epithelial cells of renal tubules were seriously vacuolated in broilers of treatment groups. And with the growth of broilers, the kidney injury of broilers in the low-dose FFC group was relieved. FFC significantly increased the contents of uric acid (UA), blood urea nitrogen (BUN), creatinine (CRE) in serum and malondialdehyde (MDA) in kidney of broilers, but significantly reduced the levels of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in kidney. FFC significantly inhibited the mRNA relative transcriptional levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase-1 (NQO-1), and increased the mRNA and protein expression levels of p53, Caspase-3 and Caspase-6 in kidney tissue of broilers. It is concluded that FFC has certain nephrotoxicity to broilers, and its effect on kidney is dose-dependent and reversible. FFC causes intense lipid peroxidation in broiler kidney by inhibiting the expression of related factors in the downstream signal pathway of Nrf2. FFC can also up-regulate the expression of pro-apoptotic factors and accelerate the abnormal apoptosis of renal cells, thus seriously affecting the renal function of broilers.

Inhaled H2 or CO2 Do Not Augment the Neuroprotective Effect of Therapeutic Hypothermia in a Severe Neonatal Hypoxic-Ischemic Encephalopathy Piglet Model.

Hypoxic-ischemic encephalopathy (HIE) is still a major cause of neonatal death and disability as therapeutic hypothermia (TH) alone cannot afford sufficient neuroprotection. The present study investigated whether ventilation with molecular hydrogen (2.1% H2) or graded restoration of normocapnia with CO2 for 4 h after asphyxia would augment the neuroprotective effect of TH in a subacute (48 h) HIE piglet model. Piglets were randomized to untreated naïve, control-normothermia, asphyxia-normothermia (20-min 4%O2–20%CO2 ventilation; Tcore = 38.5 °C), asphyxia-hypothermia (A-HT, Tcore = 33.5 °C, 2–36 h post-asphyxia), A-HT + H2, or A-HT + CO2 treatment groups. Asphyxia elicited severe hypoxia (pO2 = 19 ± 5 mmHg) and mixed acidosis (pH = 6.79 ± 0.10). HIE development was confirmed by altered cerebral electrical activity and neuropathology. TH was significantly neuroprotective in the caudate nucleus but demonstrated virtually no such effect in the hippocampus. The mRNA levels of apoptosis-inducing factor and caspase-3 showed a ~10-fold increase in the A-HT group compared to naïve animals in the hippocampus but not in the caudate nucleus coinciding with the region-specific neuroprotective effect of TH. H2 or CO2 did not augment TH-induced neuroprotection in any brain areas; rather, CO2 even abolished the neuroprotective effect of TH in the caudate nucleus. In conclusion, the present findings do not support the use of these medical gases to supplement TH in HIE management.

Effect of weimaining on apoptosis and Caspase-3 expression in a breast cancer mouse model

Ethnopharmacological relevanceWeimaining (WMN) is a condensed Tannin compound extracted from Fagopyrum cymosum (Trevir.) Meisn., which comes from the roots of buckwheat, a type of Chinese herbal medicine, was first recorded in “Bencao Shiyi”. WMN has inhibitory effects on multiple cancer types and is widely used in clinical practice; however, the mechanism underlying the anti-tumor effect of WMN is still unclear. Aim of the studyTo investigate the effect of WMN on the cellular activity and apoptosis of mouse breast cancer 4T1-luc2 cells, and caspase-3 and cleaved-caspase-3 expression. Materials and methodsLuciferase-labeled mouse breast cancer 4T1-luc2 cells were inoculated into the mouse breast pad to establish a luciferase-labeled mouse breast cancer cell model. BALB/C-nu mice were randomly divided into model, WMN, and low-molecular-weight heparin (LMWH) groups (n = 10). Another 10 mice served as the normal control group (no cancer cell injection). The WMN group was administered WMN 250 mg/kg per day for 14 days, the LMWH group was given LMWH (1500 U/kg) daily for 14 days by intraperitoneal injection, and the model and normal control groups were given an equal dose of 0.9% NaCl. The number and distribution of transplanted tumors in 4T1-luc2 breast cancer cells were observed in nude mice by an in vivo imaging system at the time of inoculation after successful modeling, and on days 7 and 14 after drug administration. Tumor cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method; caspase-3 mRNA expression was detected by RT-PCR and Western blotting was applied to detect the levels of caspase-3 and cleaved-caspase-3 protein expression. ResultsThe apoptosis index (AI) of the WMN group was detected by the TUNEL method, and the AI increased with the increase of treatment time. Compared with the model group, the mRNA expression of caspase-3 and the protein levels of caspase-3 and cleaved-caspase-3 were notably elevated in the WMN group. After in vivo bioluminescent imaging, the total photon number of the WMN group was found to be lower than that of the LWMH group on day 14 after administration. Additionally, the AI and expression levels of caspase-3 mRNA, caspase-3, and cleaved-caspase-3 protein of the WMN group were higher than those of the LWMH group. ConclusionWMN can effectively suppress the growth of 4T1-luc2 breast cancer xenografts in mice, and promote the apoptosis of breast cancer cells by upregulating the expression of caspase-3.

Oxidative stress, cell cycle arrest, DNA damage and apoptosis in the mud crab (Scylla paramamosain) induced by cadmium exposure

Cadmium is one of the most common heavy metal pollutants in the aquatic environment. Mud crab (Scylla paramamosain) is considered a model organism to monitor the impact of heavy metals. However, knowledge about toxicological mechanism of cadmium in crustaceans still remains limited. In this study, mud crabs were exposed to different concentrations of cadmium (0, 1.25, 2.5, 5 and 10 mg/L) for 72 h. Cadmium exposure significantly decreased superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidative capacity (T-AOC), and significantly increased malondialdehyde (MDA) and H2O2 levels. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity significantly increased after cadmium exposure. Moreover, integrated biological responses version 2 (IBRv2) analysis suggested that cadmium exposure exerted stronger toxicity on mud crab. Furthermore, oxidative stress induced by cadmium exposure could decrease total hemocyte count (THC), interrupt Ca2+ homeostasis, and lead to cytological damage. Cadmium exposure induced DNA damage, which activated DNA damage response signaling ATR-CHK1-p53 pathway. Our results also showed that cadmium exposure significantly increased the apoptosis and caspase-3 mRNA levels, which implied that cadmium induced apoptosis through a caspase-3 pathway.

Hydro-alcoholic Extract of Achillea Wilhelmsii C. Koch Reduces the Expression of Cell Death-Associated Genes while Inducing DNA Damage in HeLa Cervical Cancer Cells.

Background: Achillea wilhelmsii C. Koch hydroalcoholic extract (AWHE) is proven to induce cell death. Previous studies suggested that AWHE is an effective inhibitor against the proliferation of prostate cancer cells. The present study aimed to evaluate possible alterations of cell death-associated genes and determine the growth inhibitory activity of AWHE on HeLa cervical cancer cells.Methods: The antiproliferative activity of AWHE was tested using the tetrazolium dye-based colorimetric assay (MTT assay). The mRNA levels of Vascular endothelial growth factor (VEGF), caspase-3, and Breast Cancer Susceptibility gene 1 (BRCA1) were measured using the real-time Polymerase Chain Reaction method. The in-cell levels of phosphorylated H2AX were determined using the in-cell ELISA method. The data were analyzed using the non-parametric ANOVA and Friedman tests. P<0.05 was considered statistically significant.Results: Based on the MTT assay, The half-maximal inhibitory concentration and 81.99 µg/mL, respectively. The mRNA levels of BRCA1 increased after 12 and 24 hours of treatment (P<0.001), while the mRNA levels of VEGF significantly decreased after 12 hours (P=0.003) and 24 hours (P=0.001). Caspase-3 expression was increased in the HeLa cells after 6 and 12 hours (P<0.001) whereas γ-H2AX levels significantly increased after 24 and 48 hours of treatment (P<0.001).Conclusion: AWHE possesses growth inhibitory activity by altering the expression of cell death-associated genes. Using extracts from herbal plants may provide alternative strategies to be deployed in the fight against cancer.

Florfenicol induces renal toxicity in chicks by promoting oxidative stress and apoptosis.

To explore the mechanism of renal toxicity induced by florfenicol (FFC), 120 chicks were randomly divided into 6 groups, 20 in each group. Except for the control group, different doses of FFC (0.15 g/L, 0.3 g/L, 0.6 g/L, 1.2 g/L, and 1.8 g/L) were added to drinking water in the other 5 groups. Five days later, blood was collected from the vein under the wing, and the complete kidneys were obtained as soon as possible, then tested the experimental indicators. The results showed that compared with control group, all doses of FFC significantly reduced the average weight gain of chicks (P < 0.05 or P < 0.01). Except for the 0.15 g/L FFC group, kidney index of chicks in the other doses of FFC groups were significantly increased (P < 0.05 or P < 0.01). The kidney tissues in all FFC groups showed obvious damage, deformities, cell atrophy, and cell gap enlargement. In addition, all doses of FFC significantly increased the contents of uric acid (UA), blood urea nitrogen (BUN), creatinine (CRE) in serum, and malondialdehyde (MDA) in renal tissue (P < 0.05 or P < 0.01), but significantly reduced the levels of glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in renal tissue (P < 0.05 or P < 0.01). FFC significantly inhibited the mRNA and protein expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase-1 (NQO-1), and increased the mRNA and protein expression levels of p53, Caspase-3, and Caspase-6 (P < 0.05 or P < 0.01). The apoptotic rate of renal cells in all doses of FFC groups increased significantly (P < 0.05 or P < 0.01). It was concluded that FFC had a certain degree of nephrotoxicity, and with the increase of FFC concentration, the kidney injury of chicks became more and more serious. FFC promoted oxidative stress response in kidney of chicks by inhibiting the expression of related factors in Nrf2-ARE pathway. Moreover, the expression of pro-apoptotic factors was upregulated to improve the apoptosis rate of renal cells, which resulted in excessive apoptosis of renal cells and seriously affected the kidney function of chicks.

Inhibition of autophagy enhances cadmium-induced apoptosis in duck renal tubular epithelial cells

Increasing evidence indicates autophagy and apoptosis are involved in the toxicity mechanism of heavy metals. Our previous studies showed that cadmium (Cd) could induce autophagy and apoptosis in duck kidneys in vivo, nevertheless, the interaction between them has yet to be elucidated. Herein, the cells were either treated with 3CdSO4·8H2O (0, 1.25, 2.5, 5.0 μM Cd) or/and 3-methyladenine (3-MA) (2.5 μM) for 12 h and the indictors related autophagy and apoptosis were detected to assess the correlation between autophagy and apoptosis induced by Cd in duck renal tubular epithelial cells. The results demonstrated that Cd exposure notably elevated intracellular and extracellular Cd contents, the number of autophagosomes and LC3 puncta, up-regulated LC3A, LC3B, Beclin-1, Atg5 mRNA levels, and Beclin-1 and LC3II/LC3I protein levels, down-regulated mTOR, p62 and Dynein mRNA levels and p62 protein level. Additionally, autophagy inhibitor 3-MA decreased Beclin-1, LC3II/LC3I protein levels and increased p62 protein level. Moreover, co-treatment with Cd and 3-MA could notably elevate Caspase-3, Cyt C, Bax, and Bak-1 mRNA levels, Caspase-3 and cleaved Caspase-3 protein levels, and cell apoptotic rate as well as cell damage, decreased mitochondrial membrane potential (MMP), Bcl-2 mRNA level and the ratio of Bcl-2 to Bax compared to treatment with Cd alone. Overall, these results indicate Cd exposure can induce autophagy in duck renal tubular epithelial cells, and inhibition of autophagy might aggravate Cd-induced apoptosis through mitochondria-mediated pathway.

Hypoxia exacerbates cardiomyocyte injury via upregulation of Wnt3a and inhibition of Sirt3

Ischemic injury is a major cause of several cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy, and ventricular remodeling. Using an in vitro hypoxia model to mimic ischemia, we found that hypoxia stimulated Wnt3a expression. A mechanistic study showed that hypoxia-inducible factor 1α (HIF-1α) was directly recruited to the Wnt3a promoter. Wnt3a overexpression significantly decreased cell viability, promoted the generation of apoptotic cells, and enhanced hypoxia-induced injury in neonatal rat cardiomyocytes. This was partially through the upregulation of Caspase-3 mRNA levels and cleaved PARP-1 protein levels. In addition, we observed that Wnt3a exacerbated hypoxia-induced mitochondrial dysfunction and cytosolic release of cytochrome C. Furthermore, we found that Sirt3, a mitochondrial NAD+-dependent deacetylase that modulates mitochondrial metabolism and homeostasis, was negatively regulated by Wnt3a. Conversely, Sirt3 overexpression repressed Wnt3a expression and ameliorated the hypoxia-induced mitochondrial dysfunction. Overall, our findings suggest that the hypoxia–Wnt3a–Sirt3 regulatory axis might be a potential target for cell protection in cardiac ischemia and hypoxia.

Cardioprotective effects of garcinol following myocardial infarction in rats with isoproterenol-induced heart failure

Myocardial infarction is a clinical form of necrosis in the myocardium caused by an imbalance between the coronary blood supply and myocardial demand. Garcinol is a polyisoprenylated benzophenone found in the fruit of Garcinia indica, which is abundant in tropical regions. This fruit contains high levels of garcinol, isoxanthochymol, isogarcinol, hydroxycitric acid and xanthochymol. Garcinol and hydroxycitric acid have been shown to have antioxidant effects. In this study, rats were assigned to sham, control, low-dose, high-dose and positive control groups. Hemodynamic and apoptotic markers were evaluated, and histopathological analysis was conducted. The mRNA and protein levels of caspase-3, Bax, Bcl-2 and cleaved caspase-3 were quantified. Garcinol treatment increased the heart rate and improved the maximum rate of increase in left-ventricle (LV) pressure (+dp/dtmax), maximum rate of decrease in LV pressure (–dp/dtmax), LV ejection fraction and LV systolic pressure in rats with induced heart failure. Garcinol treatment reversed body, liver and heart weight changes, resulting in returns to near-normal levels. In the garcinol treatment group, the number of broken fibers, extent of inflammatory cell infiltration and rate of apoptosis remained within normal ranges. Garcinol reduced the cross-sectional areas of cardiomyocytes, and reduced interstitial fibrosis to a normal level. The mRNA and protein levels of cleaved caspase-3, caspase-3 and Bax were reduced, whereas those of Bcl-2 were increased, following high-dose (100 mg/kg) garcinol treatment. These findings suggest that garcinol effectively prevents apoptosis in rats with isoproterenol-induced heart failure and in cardiac H9C2 cells.

Effects of circRNA_103993 on the proliferation and apoptosis of NSCLC cells through miR-1271/ERG signaling pathway.

Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer. CircRNA is a new type of non-coding RNA. CircRNA was found to be deeply involved in the regulation of NSCLC cells. However, the principle of CircRNA regulating NSCLC cells needs to be further explored. The relative mRNA expression levels of CircRNA_103993, miR-1271 and ERG were detected by quantificational real-time polymerase chain reaction (qRT-PCR) in NSCLC cells and human bronchial epithelial cell line HBE. Cell proliferation was detected by the Cell Counting Kit (CCK8) when NSCLC cells were transfected with si-CircRNA_103993, si-NC, miR-1271 mimics, miR-NC, LV-ERG, respectively. The apoptotic rate of NSCLC cells was measured by apoptotic assay and flow Cytometry. The relative mRNA and protein expression levels of PCNA and caspase-3 were detected by Western blot and qRT-PCR. CircRNA_103993 and ERG were significantly up-regulated while miR-1271 was significantly down-regulated in NSCLC cells. Knockdown of CircRNA_103993 and high expression of miR-1271 significantly inhibited NSCLC cell proliferation and promoted apoptosis. The double luciferin report showed that CircRNA_103993 served as a sponge of miR-1271 and miR-1271 could directly target ERG in NSCLC cells. More importantly, low expression of CircRNA_103993 increased the expression level of miR-1271, and high expression of miR-1271 decreased the expression level of ERG. Further experiments showed that miR-1271 inhibitors reversed the effect of si-CircRNA_103993 on proliferation and apoptosis of NSCLC cells. However, miR-1271 mimics significantly promoted the effects of si-CircRNA_103993 on the proliferation and apoptosis. Moreover, LV-ERG reversed the effect of miR-1271 mimics on proliferation and apoptosis of NSCLC cells. However, si-ERG significantly promoted the effects of miR-1271 mimics on the proliferation and apoptosis. CircRNA_103993 was highly expressed in NSCLC cells. CircRNA_103993 regulated proliferation and apoptosis of NSCLC cells by acting as a sponge of miR-1271. The CircRNA_103993 /miR-1271/ ERG axis had an important effect on the proliferation and apoptosis of NSCLC cells. Therefore, CircRNA_103993 may be a target for treating lung cancer.

Resveratrol protects against apoptosis induced by interleukin-1β in nucleus pulposus cells via activating mTOR/caspase-3 and GSK-3β/caspase-3 pathways.

Objective: The purpose of the present study was to investigate the specific downstream signaling pathway mediated by PI3K/Akt in resveratrol (RES) anti-apoptosis of nucleus pulposus cells (NPCs).Materials and methods: Human NPCs were cultured and divided into six groups. Interleukin (IL)-1β was used to induce apoptosis and RES to inhibit apoptosis. Fluorescence-activated cell sorting (FACS) analysis was used to test apoptotic incidence of NPCs, cell counting kit-8 (CCK-8) assay was performed to detect cell viability, The expression level of caspase-3 mRNA was detected by RT-qPCR, and protein levels were determined by Western blot.Results: Flow cytometry analysis showed that IL-1β increased the apoptosis rate of NPCs in each group, and RES significantly decreased the apoptosis rate, while rapamycin (RAPA) and SB216763 inhibited the effect of RES and increased the apoptosis rate again. Similarly, CCK-8 showed that IL-1β decreased activity of NPCs in each group, while RES increased cell activity, RAPA and SB216763 inhibited the effect of RES and decreased cell activity. RT-qPCR results showed IL-1β significantly increased the level of caspase-3 expression, but it was significantly decreased by using RES, RAPA and SB216763 respectively attenuated effects of RES. Western blot results showed that activated caspase-3 was inhibited by RES effect, and was up-regulated again after the addition of RAPA and SB216763. In addition, p-mTOR and p-GSK-3β were up-regulated by RES and down-regulated by RAPA and SB216763.Conclusion: RES can inhibit apoptosis induced by IL-1β in human NPCs. PI3K/Akt/mTOR/caspase-3 and PI3K/Akt/GSK-3β/caspase-3 pathways are potential mechanisms underlying this process.

Cloning and characterization of Litopenaeus vannamei cystainB-like in WSSV infection

Cystatins B is an endogenous cysteine cathepsin inhibitor. In shrimp, cystatins B-like (CSTB-L) has not been characterized and its role in WSSV infection is largely unknown. In this study, a full-length 699 bp CSTB-L sequence with 291 bp open reading frame encoding a 96 amino acid from L.vannamei (Lv) was first cloned. The tissue distribution assay indicated that LvCSTB-L presented ubiquitous expression in most examined tissues, with the most predominant expression in the hepatopancreas and the weakest expression in the muscles. LvCSTB-L transcripts could be induced in the intestine and hepatopancreas by WSSV challenge. The relative expression level of IE1 and VP28 in the LvCSTB-L knockdown shrimp were increased significantly. In addition, the shrimp cumulative mortality was remarkably (p < 0.01) increased after LvCSTB-L knockdown. Moreover, following the LvCSTB-L silencing, significant decreases in the mRNA levels of p53, p38, caspase3, STAT and ERK were also observed. The results suggested that LvCSTB-L could play positively roles in antiviral immune response by JAK-STAT, MAPK and apoptotic pathway. These findings would further our understanding of shrimp antiviral response, and therefore help for virus control and prevention.