Isoorientin plays an important role in alleviating Cadmium-induced DNA damage and G0/G1 cell cycle arrest.

Cadmium is a heavy metal pollutant that has been reported to cause oxidative stress, apoptosis, and autophagy in cells, while the flavone isoorientin is a traditional Chinese medicine extract that has proven antioxidant and anti-inflammatory properties. Accordingly, in this study we used the rat proximal tubular cell line NRK-52E and primary rat proximal tubular (rPT) cells as models to investigate the effects of isoorientin against Cadmium-induced cell injury and the mechanism of these effects. Comet assay, Western blot, flow cytometry, immunofluorescence, and transmission electron microscopy were used to evaluate cell damage and cell-cycle-related protein expression. Furthermore, real-time cell analysis, cell-counting kit-8, and ELISA were used to investigate the role of isoorientin in Cadmium-induced cell injury. The results revealed that treatment of rat renal tubular epithelial cells with 2.5 µM Cd for 12 h resulted in DNA damage and G0/G1 cell cycle arrest, while isoorientin attenuated this Cd-induced damage.

Effect of oleic acid on induction of steatosis and cytotoxicity in BRL 3A cells.

Recent studies have shown that monounsaturated oleic acid induces steatosis in cultured hepatocyte steatosis in the form of nonalcoholic fatty liver disease models in vitro. However, the underlying mechanism of steatosis development is not completely understood. Therefore, we investigated the molecular mechanism of steatosis and the role of mitogen-activated protein kinase (MAPK)/toll-like receptor 4-related protein (TLR4) expression in this study. Rat hepatocyte cells were subjected to oleic acid in different concentrations (1.2-2.4 mM) for 24 hours. The cell morphological injury index and the changes in the MAPK/TLR4 signaling pathway-related proteins were evaluated. We found that the microstructure of the cells in the oleic acid treatment group was damaged, and higher phosphorylation levels of the MAPK pathway-related proteins were detected than those in the control group. In addition, the protein expression of TLR4, sterol regulatory element-binding protein-1, and fatty acid synthase were increased in the oleic acid treatment group. Our findings demonstrate that oleic acid causes toxic damage to rat hepatocyte cells, and the MAPK/TLR4 signaling pathway plays a significant role in lipid storage.

N-acetylcysteine delayed cadmium-induced chronic kidney injury by activating the sirtuin 1-P53 signaling pathway.

With the development of modern industrial civilization, cadmium (Cd), a known nephrotoxic metal, has become a growing public safety issue due to its ability to induce various types of kidney disease. Maladaptive proximal tubule repair is a significant cause of Cd-induced chronic kidney disease (CKD), which is characterized by premature senescence and pro-fibrosis. Previously, we demonstrated that cadmium causes DNA damage and cycle arrest in renal tubular epithelial cells, which may be relevant to premature senescence regulated by sirtuin 1 (SIRT1). In this study, in vivo and in vitro studies were conducted to elucidate the role of SIRT1-mediated premature renal senescence in Cd-induced CKD. As oxidative stress is a significant cause of aging, we evaluated whether N-acetylcysteine (NAC) would inhibit Cd-induced premature aging and dysfunction in rat renal tubular epithelial cells. Cadmium induced premature renal senescence and fibrosis, and NAC inhibited premature renal senescence and fibrosis through the SIRT1-P53 pathway and delayed CKD progression. Overall, the results suggested that the SIRT1-P53 pathway mediates oxidative stress, premature renal senescence, and renal fibrosis during cadmium exposure, which may be a potential therapeutic target for Cd-induced CKD.

Protective Effect of Isoorientin on Oleic Acid-Induced Oxidative Damage and Steatosis in Rat Liver Cells.

The harm of nonalcoholic fatty liver disease to human health is increasing, which calls for urgent prevention and treatment of the disease. Isoorientin is an effective ingredient of Chinese herbal medicine with anti-inflammatory and antioxidant effects. However, the effect of isoorientin in nonalcoholic fatty liver disease is still unclear. In this study, combined in vivo and in vitro experiments, through pathological observation, flow cytometry, immunofluorescence and western blot analysis to explore the role of isoorientin in steatosis and reveal its molecular mechanism. The results demonstrated that oleic acid treatment significantly increased the content of ROS and lipid droplets in rat hepatocytes, and promoted the expression of γH2AX, HO-1, PPARγ, SREBP-1c, FAS. The ROS content in the cells of co-treated with isoorientin and oleic acid was significantly reduced compared to the oleic acid group, and the expression of γH2AX, HO-1, PPARγ, SREBP-1c, FAS, and the nuclear translocation of NF-κB p65 were also significantly inhibited. Our data showed that oleic acid induce oxidative damage and steatosis in hepatocytes both in vitro and in vivo, and activate the PPARγ/NF-κB p65 signal pathway. Moreover, isoorientin can significantly reduce oleic acid -induced oxidative damage and steatosis by regulating the PPARγ/NF-kB p65 signal pathway.

Chitosan-Coated Selenium Nanoparticles Attenuate PRRSV Replication and ROS/JNK-Mediated Apoptosis in vitro.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly prevalent and endemic swine pathogen that causes significant economic losses to the global swine industry. Selenium nanoparticles (SeNPs) have attracted increasing attention in the biomedical field, given their antiviral effects. This study aimed to investigate the inhibitory effect of chitosan-coated SeNPs (CS-SeNPs) on PRRSV replication. Methods: In this study, CS-SeNPs were synthesized by chemical reduction and characterized by assessing the morphology, size distribution, zeta potential, and element composition. Marc-145 cells were infected with r-PRRSV-EGFP (0.1 MOI) and inoculated with CS-SeNPs (10 µM). Subsequently, the concentrations of hydrogen peroxide (H2O2) and glutathione (GSH), and glutathione peroxidase (GSH-Px) activity were measured using specific commercial assay kits. ORF5 RNA expression, viral titer, and nucleocapsid (N) protein expression were assessed using qRT-PCR, TCID50, and Western blot. ROS generation, apoptosis rates, and JNK /caspase-3/PARP protein expression were evaluated using dihydroethidium staining, flow cytometry, and Western blot. Results: The results showed that CS-SeNPs treatment significantly suppressed oxidative stress induced by r-PRRSV-EGFP infection by increasing GSH-Px activity, promoting GSH production, and inhibiting H2O2 synthesis. CS-SeNPs treatment significantly inhibited ORF5 gene expression, viral titers, and N protein of r-PRRSV-EGFP at 24 and 48 hours post-infection (hpi) in Marc-145 cells. The increase in apoptosis rates induced by r-PRRSV-EGFP infection was significantly decreased by CS-SeNPs inoculation through inhibiting ROS generation, JNK phosphorylation levels, and cleavage of caspase-3 and PARP mainly at 48 hpi. Conclusion: These results demonstrated that CS-SeNPs suppress PRRSV-induced apoptosis in Marc-145 cells via the ROS/JNK signaling pathway, thereby inhibiting PRRSV replication, which suggested the potential antiviral activity of CS-SeNPs that deserves further investigation for clinical applications.

Role of poly (ADP-ribose) polymerase-1 in cadmium-induced cellular DNA damage and cell cycle arrest in rat renal tubular epithelial cell line NRK-52E.

With the development of modern industry, the problem of cadmium (Cd) pollution cannot be ignored and its toxicity has caused great personal injury to humans. Poly (ADP-ribose) polymerase 1 (PARP-1) protein is a research hotspot in recent years, the research we have published shows that 5 µM of Cd-treated NRK-52E cells activated PARP-1, but the specific effects of PARP-1 on DNA damage and cell cycle is unclear. Therefore, the purpose of this study is to reveal the effect of Cd on DNA damage and cell cycle arrest in NRK-52E cells, in addition, to investigate the role of PARP-1 in mediating this effect. Western blotting, comet assay, QRT-PCR, immunofluorescence, and co-immunoprecipitation were used to detect DNA damage and cell cycle-associated protein expression. Flow cytometry was used to assess cell cycle distribution and the apoptosis rates. Results showed that after the increase in treatment time and Cd concentration, the degree of DNA damage was significantly increased, and a transition from G0/G1 to S phase arrest was observed. In addition, inhibition of PARP-1 expression exacerbated cell damage and cell cycle arrest when DNA damage was low, but attenuated cell damage and even cell cycle arrest when DNA damage was severe. These findings in this study indicate that Cd causes DNA damage in NRK-52E cells, leading to cell cycle arrest at different phases depending on the degree of DNA damage. Moreover, PARP-1 plays an important role in mediating this effect, when DNA damage is low, it functions in DNA repair, however, when DNA damage is severe, it aggravates cell damage and induces cell death.

Cadmium induces apoptosis via generating reactive oxygen species to activate mitochondrial p53 pathway in primary rat osteoblasts.

Cadmium (Cd), a heavy metal produced by various industries, contaminates the environment and seriously damages the skeletal system of humans and animals. Recent studies have reported that Cd can affect the viability of cells, including osteoblasts, both in vivo and in vitro. However, the mechanism of Cd-induced apoptosis remains unclear. In the present study, primary rat osteoblasts were used to investigate the Cd-induced apoptotic mechanism. We found that treatment with 2 and 5 µM Cd for 12 h decreased osteoblast viability and increased apoptosis. Furthermore, Cd increased the generation of reactive oxygen species (ROS), and, thus, DNA damage measured via p-H2AX. The level of the nuclear transcription factor p53 was significantly increased, which upregulated the expression of PUMA, Noxa, Bax, and mitochondrial cytochrome c, downregulated the expression of Bcl-2, and increased the level of cleaved caspase-3. However, pretreatment with the ROS scavenger N-acetyl-l-cysteine (NAC) or the p53 transcription specific inhibitor PFT-α suppressed Cd-induced apoptosis. Our results indicate that Cd can induce apoptosis in osteoblasts by increasing the generation of ROS and activating the mitochondrial p53 signaling pathway, and this mechanism requires the transcriptional activation of p53.

TGF-ß-activated kinase 1 (TAK1) mediates cadmium-induced autophagy in osteoblasts via the AMPK / mTORC1 / ULK1 pathway.

Cadmium (Cd) is one of worldwide environmental pollutants that causes bone homeostasis, which depends on the resorption of bones by osteoclasts and formation of bones by the osteoblasts (OB). However, the Cd toxicity on OB and its mechanism are unclear. Autophagy is an evolutionarily conserved degradation process in which domestic intracellular components are selectively digested for the recycling of nutrients and energy. This process is indispensable for cell homeostasis maintenance and stress responses. Dysregulation at the level of autophagic activity consequently disturbs the balance between bone formation and bone resorption and mediates the onset and progression of multiple bone diseases, including osteoporosis. TAK1 has been recently emerged as an activator of AMPK and hence an autophagy inducer. AMPK is a key molecule that induces autophagy and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by mTORC1. In this study, we found that Cd treatment caused the formation of autophagosomes, LC3-II lipidation and p62 downregulation, and the increased autophagic flux, indicating that Cd treatment induced autophagy in OBs. Cd treatment induced TAK1 activation mediated AMPK phosphorylation, which promoted autophagy via phosphorylation of ULK1 at S317. Meanwhile, Cd treatment dramatically decreased mTORC1, S6K1, 4E-BP1, S6, ULK1S555 and ULK1S757 phosphorylation, suggesting that mTORC1 activity was inhibited and inactive mTORC1 prevents ULK1 activation by phosphorylating ULK1 at SerS555 and Ser757. Our data strongly suggest that TAK1 mediates AMPK activation, which activates ULK1 by phosphorylating ULK1S317 and suppressing mTORC1-mediated ULK1S555 and ULK1S757 phosphorylation. Our study has revealed a signaling mechanism for TAK1 in Cd-induced autophagy in OBs.

Effect of cell cycle synchronization on cadmium-induced apoptosis and necrosis in NRK-52E cells.

Heavy metal pollution is a problem that cannot be ignored. Due to the prevalence of cadmium in the environment and its harmful effects on humans, cadmium pollution has become a research hotspot recently. The mechanism of cadmium-induced toxicity has also drawn much attention and most studies have been conducted using whole cells, but the toxicological mechanism of cadmium remains unclear. In this study, we aimed to obtain NRK-52E cells at different growth stages by various methods and analyze the differences in cadmium toxicity. The results show that the cadmium sensitivity of cells in each phase was different and the late apoptotic rate was increased significantly after 5 µM Cd treatment. In addition, cadmium easily induces apoptosis of G0- and S-phase cells, as well as necrosis of S- and M-phase cells, but has no significant effect on G1-phase cells. Overall, we first explored the differences in the effects of cadmium on NRK-52E cells at various growth phases. Besides, the findings of this study might provide a theoretical basis for further exploration of the toxicological mechanism of cadmium.Abbreviations Cd: cadmium; CDK: cyclin-dependent kinases; DAPI 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; TBST: Tris-buffered saline with Tween-20; PI: propidium iodide; DMEM: Dulbecco's Modified Eagle Medium; BCA: bicinchoninic acid.

ERK1/2 MAPK promotes autophagy to suppress ER stress-mediated apoptosis induced by cadmium in rat proximal tubular cells.

Cadmium (Cd) is a toxic heavy metal and its toxic mechanism is not entirely clear. The goal of the present study was to investigate the toxic mechanism of Cd on rPT cells, and to elucidate the role of ERK1/2 signaling pathway in mediating the relationship between apoptosis and autophagy. We evaluated the cell morphology, cell cycle distribution, apoptosis rates, and the expression of related proteins. We observed that increased Cd concentration disrupted cell morphology, increased apoptosis and induced autophagy. Additionally, activation of JNK1/2 and p38 MAPK promoted apoptosis, while activation of ERK1/2 inhibited apoptosis. Upon inhibition of autophagy, apoptosis rate and the expression of ER proteins related to the apoptosis were increased. Following inhibition of the ERK1/2 signaling pathway, the number of LC3 aggregates, the rate of LC3II/LC3I and the expression of Beclin-1were decreased, but the expression level of ER proteins related to apoptosis were increased. Our results indicated that Cd exposure damages cells also induces apoptosis and autophagy, meanwhile demonstrate that the ERK1/2 signaling pathway plays an important role in this process. Besides, these data suggest that autophagy can inhibit Cd-induced apoptosis and the ERK1/2 signaling pathway can suppress ER stress-mediated apoptosis by activating autophagy.

Beclin-1-mediated Autophagy Protects Against Cadmium-activated Apoptosis via the Fas/FasL Pathway in Primary Rat Proximal Tubular Cell Culture.

The Fas/FasL signaling pathway is one of the primary apoptosis pathways, but the involvement and regulatory mechanism of this pathway by autophagy remain unclear. Here we demonstrated that cadmium (Cd) activated the Fas/FasL apoptosis pathway in rat proximal tubular (rPT) cells; this was accompanied by simultaneous activation of autophagy resulted in reduced apoptosis. In this model, we induced autophagy through RAPA and further demonstrated that autophagy protects against activation of Fas/FasL signaling and apoptosis. The antiapoptotic effect of autophagy was blocked by 3-MA, an autophagy inhibitor. The interactions between Beclin-1 and Fas, FasL, FADD, caspase-8 and BID/tBID were relatively weak, with the exception of cleaved caspase-8, indicated that minimal interactions between these proteins and Beclin-1 are involved in maintaining the balance of autophagy and apoptosis. Beclin-1 precipitated with cleaved caspase-8 in a dose-dependent mannter, and the expression was increased by siRNA against Beclin-1. These data suggested that Beclin-1-mediated autophagy impairs the expression and function of cleaved caspase-8 to protect against Cd-induced activation of apopotosis through Fas/FasL signaling pathway.

PARP-1 overexpression contributes to Cadmium-induced death in rat proximal tubular cells via parthanatos and the MAPK signalling pathway.

Parthanatos is a newly discovered form of PARP-1-dependent programmed cell death. It has been reported to play an important role in several cancer or tumour cells; however, few studies have been performed in normal cells. Cadmium is a highly toxic pollutant and is reported to induce autophagy and apoptosis in multiple cell types. Although cadmium toxicity induces cell death, the underlying mechanism is not fully understood. Therefore, in this study we aimed to investigate the mechanism of Cadmium -induced cell damage using rat proximal tubular cell line NRK-52E and primary rat proximal tubular (rPT) cells. Our results indicated that parthanatos and the MAPK signalling pathway contribute to Cadmium-induced cell death, and that oxidative stress and mitochondrial damage play key roles in this process. In addition, parthanatos with oxidative stress has a synergistic effect on apoptosis, and JNK1/2 and p38 contribute to parthanatos.

Treatment of cadmium-induced renal oxidative damage in rats by administration of alpha-lipoic acid.

Cadmium (Cd) is a toxic heavy metal that is widespread and nephrotoxic, but the mechanism of its toxicity is not well understood. Alpha-lipoic acid (α-LA) has a protective effect on Cd-induced oxidative stress, but the underlying mechanism is also not clear. This study aimed to confirm that Cd causes renal damage and to explore the potential underlying mechanism of α-LA to the kidney. Rats were randomly divided into four groups: control group, Cd group (50 mg/L CdAc2), Cd+α-LA group (50 mg/L CdAc2 + 50 mg/kg body wt/day α-LA), and α-LA group (50 mg/kg body wt/day). The rats were exposed to Cd via drinking water and α-LA in the form of gavage at the same time every day. After 12 weeks, the activity of antioxidant enzymes and the level of Cd in the kidney were analyzed. Renal damage was evaluated based on histopathological and ultrastructure examinations. The apoptosis index was determined based on the results of western blotting and qRT-PCR. Our results indicate that accumulation of Cd causes serious kidney damage and α-LA has a protective effect against Cd-induced oxidative stress and apoptosis. Further, the findings indicate that the antioxidant, Cd chelation, and antiapoptotic activities of α-LA are the key factors that alleviate nephrotoxicity.

Caspase-Dependent and Caspase-Independent Pathways Are Involved in Cadmium-Induced Apoptosis in Primary Rat Proximal Tubular Cell Culture.

We designed this study to investigate whether cadmium induces caspase-independent apoptosis and to investigate the relationship between the caspase-dependent and caspase-independent apoptotic pathways. Cadmium (1.25-2.5 µM) induced oxidative stress in rat proximal tubular (rPT) cells, as seen in the reactive oxygen species levels; N-acetylcysteine prevented this. Cyclosporin A (CsA) prevented mitochondrial permeability transition pore opening and apoptosis; there was mitochondrial ultrastructural disruption, mitochondrial cytochrome c (cyt c) translocation to the cytoplasm, and subsequent caspase-9 and caspase-3 activation. Z-VAD-FMK prevented caspase-3 activation and apoptosis and decreased BNIP-3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) expression levels and apoptosis-inducing factor/endonuclease G (AIF/Endo G) translocation. Simultaneously, cadmium induced prominent BNIP-3 expression in the mitochondria and cytoplasmic AIF/Endo G translocation to the nucleus. BNIP-3 silencing significantly prevented AIF and Endo G translocation and decreased the apoptosis rate, cyt c release, and caspase-9 and caspase-3 activation. These results suggest that BNIP-3 is involved in the caspase-independent apoptotic pathway and is located upstream of AIF/Endo G; both the caspase-dependent and caspase-independent pathways are involved in cadmium-induced rPT cell apoptosis and act synergistically.