Thymol Preserves Spermatogenesis and Androgen Production in Cisplatin-Induced Testicular Toxicity by Modulating Ferritinophagy, Oxidative Stress, and the Keap1/Nrf2/HO-1 Pathway

Nurture vs nature: how can we optimize sperm quality?

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Protective potential of Irosustat, STX140 and the sulfonate derivative 1G in counteracting cisplatin-induced renal and hepatic toxicities: An in vivo comparative study.

Introduction: Sepsis, a common and costly cause of inpatient mortality in both sexes and all age groups (particularly in intensive care units), ensues an inflammatory process involving a variety of pathogen-expressed conserved structures called pathogen associated molecular pattern (PAMP) which are considered the main cause of oxidative stress and increase in inflammatory markers. Methods and Results: In this murine model study, male Wistar rats were assigned into three groups: CLP with gauge 18, CLP with gauge 21, and a sham group (a group without CLP). Subsequently, 24 hours following the surgery, all animals were sacrificed and inflammatory markers such as myeloperoxidase (MPO), reactive oxygen species (ROS) and lipid peroxidation (LPO) were measured in their cardiac tissues. In CLP group with gauge 18, LPO, ROS, and MPO were significantly increased in comparison with the other two groups. Moreover, although lower in comparison with the gauge 18 CLP group, LPO, ROS, and MPO were significantly higher in gauge 21 CLP group compared to the sham group. Conclusions: In murine models of sepsis, the gauge size can be influential in study outcomes and inflammatory changes observed in gauge 18 CLP mice can be considered as the most reliable and clinically-relevant indicators of sepsis-induced inflammation in humans. Key words: Cecal ligation and puncture, Sepsis, Lipid peroxidation, Myeloperoxidase, Reactive oxygen species, Cardiac tissues.

Hepcidin as a Therapeutic Tool to Limit Iron Overload and Improve Anemia In β-Thalassemia

Cancer is still a serious problem in the world. Therefore, it is important to find ways to prevent or cure cancer. Because cancer cells possess abilities to prolong cell growth and invade to other tissues, it is still thorny to control cancer disease. Therefore, how to suppress cell growth in cancer cells and prevent cancer cells invade to other tissues is of great urgency. Phytochemicals are a group of compounds from natural plants. Recent reports show that they are bioactive components in natural plant to execute chemoprevention or other beneficial effects such as prevent diabetes, protect neuron cells and prevent obesity. Rotenone is a phytochemical from the roots of the Derris and Lonchorcarpus species. Rotenone is an inhibitor of electron transfer chain complex Ι (NADH dehydrogenase complex) and it can promote production of reactive oxygen species (ROS). It has been used as a botanical insecticide for at least 150 years to control crop pests and used even longer as a fish poison by native tribes in South America and East Africa. Recent reports show that rotenone can induce apoptosis in human melanoma cells and leukemia cells. Because anti-apoptosis plays an important role in anti-cancer, we want to determine whether rotenone can induce apoptosis in MCF-7 breast cancer cells which threaten women worldwide. In this study, we found that rotenone can induce apoptosis in MCF-7 human breast cancer cells by using MTT assay, trypan blue exclusion assay, alamar blue assay, flow cytometry and Hoechst 33258 staining and they are dose- and time-dependent effects. To further confirm these results, we used western blot analysis to investigate apoptosis-related proteins, PARP, Bcl-2 and Bax. We showed that rotenone can promote PARP cleavage, downregulate anti-apoptotic Bcl-2 and upregulate apoptotic Bax. In addition, rotenone can reduce mitochondria membrane potential. Because rotenone is attributed to irreversible binding and inactivation of mitochondrial electron transport chain complex Ι, we investigated whether rotenone indeed promote production of ROS in MCF-7 cells. Rotenone can result in an increase of the reactive oxygen species (ROS). We treated MCF-7 cells with rotenone and indeed found that ROS was increased. Then, we want to determine the signaling pathways under treatment of EGCG in MCF-7 cells. Using western blotting analysis, we found that rotenone induced JNK and p38 activation, whereas it attenuated ERK1/2 activation. Then, apoptosis was induced. In addition, metastasis is a fundamental property of high malignant cancer cells with poor clinical outcome. Therefore, to investigate how to suppress metastasis is an important urgency. Cleavage of extracellular matrix to allow cells invade into blood and transfer to other tissues is an important step in metastasis and MMPs play important roles in this step. Because lung cancer has the highest rate of cancer mortality worldwide and the highest rate of lung cancer mortality is due to their high potential of metastasis, we used highly invasive CL1-5 human lung cancer cells to investigate possible mechanisms of suppression tumor invasion in cancer cells. Green tea contains anti-cancer effects, so we want to determine whether EGCG, the major bioactive compound in green tea, can attenuate metastasis in highly invasive CL1-5 human lung cancer cells. First, we found that EGCG suppress cell growth at concentration higher than 20 μM by using MTT assay. Further, we want to investigate whether EGCG induce apoptosis or cell cycle arrest at concentrations higher than 20 μM. We used flow cytometry analysis and found that EGCG induced G2/M arrest in CL1-5 cells at concentrations of 30, 40 and 50 μM. Next, we want to investigate whether EGCG can attenuate invasion ability of CL1-5 cells. Using transwell invasion assay, EGCG could suppress invasion of CL1-5 cells. Because MMP-2 plays important roles to degrade extracellular matrix and is closely related to invasion in patients of lung cancer, we want to investigate whether EGCG can affect expression of MMP-2 in CL1-5 cells. Using gelatin zymography, western blot analysis and PCR, we found that EGCG can repress MMP-2 expression at the transcriptional level. Furthermore, by western blot analysis we showed that EGCG could suppress activation of JNK and attenuate translocation of MMP-2 transcription factor, NF-κB and Sp1, from the cytosol into the nucleus. In addition, docetaxel, a clinical drug used in the treatment of lung cancer, performs serious side effect, we want to clarify synergistic effects of EGCG and docetaxel. Combine EGCG and docetaxel can repress MMP-2 expression at low dosages of docetaxel. Taken together, rotenone and EGCG may be potential phytochemicals to chemoprevent breast cancer and lung cancer respectively and they may play synergistic effects with clinical drug.

The curcuminoid, EF-24, reduces cisplatin-mediated reactive oxygen species in zebrafish inner ear auditory and vestibular tissues

Inhibition of NFS1 Induces Ferroptosis and Activates Immune Response in Acute Myeloid Leukemia Cells through STAT1/ISG Pathway

Although recent reports indicate an increasing incidence of patients with reflux esophagitis, its pathomechanism remains unclear. Cytokines and neutrophils, the latter of which produce reactive oxygen species (ROS), have been implicated in the formation of gastrointestinal diseases. This study investigated the roles of neutrophils, ROS, and cytokines in the pathogenesis of experimental reflux esophagitis. Esophagitis was induced in male Wistar rats by ligation at both the limiting ridge of the stomach and lower portion of the duodenum. The esophagus was then removed, and the lesion index, wet weight, thiobarbituric acid-reactive substances (an index of lipid peroxidation), myeloperoxidase activity (an index of neutrophil accumulation), tumor necrosis factor-alpha (TNF-alpha), and cytokine-induced neutrophil chemoattractant (CINC)-1 in the esophageal mucosa were estimated, and a histological study (hematoxylin-and-eosin staining) was performed. The mRNA expression of TNF-alpha and CINC-1 was analyzed. Anti-neutrophil serum (ANS) was injected intraperitoneally prior to the induction of esophagitis, and inflammatory markers were estimated as described above. The values of all markers increased, and the histological study revealed neutrophil infiltration and edema in mucosa and submucosa at both 12 and 18 h after induction. However, the mRNA expression of both cytokines was observed earlier at 3 and 6 h after induction. ANS inhibited the increases in all inflammatory markers. These results indicate that ROS and lipid peroxidation mainly derived from neutrophils, which are stimulated and mobilized by TNF-alpha and CINC-1, are implicated in the pathogenesis of esophageal inflammation induced by the reflux of gastroduodenal contents.

Ceruloplasmin suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma cells

Hyperoxaluria-induced calcium oxalate crystallisation is associated with the generation of reactive oxygen species (ROS) via mitochondria and NADPH oxidase. Endoplasmic reticulum (ER) has emerged as an organelle which could influence mitochondrial functioning and ROS generation. Plugging an upstream pathway of mitochondrial and NADPH oxidase-induced ROS generation may have better prophylaxis. Therefore, we propose to investigate the linkage of hyperoxaluria-induced ROS generation with ER stress by inhibiting the later with 4-Phenylbutyric acid (4-PBA). Male wistar rats were divided into three groups: a normal control group, an ethylene glycol with ammonium chloride-induced hyperoxaluric group (EA) and a third group which has hyperoxaluric animals given 4-PBA at a dose of 300mg/kg. After 9days of treatment, animals were sacrificed and renal tissues were analysed for histopathological examination, ROS, mitochondrial dysfunction, ER stress markers, inflammatory markers and NADPH oxidase subunits expression. Hyperoxaluric rats exhibited a significant increase in the levels of ROS, subsequently up-regulated levels of ER stress markers, inflammatory indicators, NADPH oxidase subunits and compromised mitochondrial functioning. However, ER stress amelioration appreciably curtailed the alterations caused by hyperoxaluric abuse. Therefore, suggesting the major role of ER in hyperoxaluric manifestations thereby providing an opportunity to target ER stress for future therapeutic interventions.

Cisplatin (CDDP) is a chemotherapeutic agent that is widely used in the treatment of lymphomas and solid malignancies. However, its clinical usage is limited by its severe side effects in the kidneys. Glomerular and tubularinjuries in the kidneys commonly progress to interstitial fibrosis and, ultimately, the end stage of renal failure. We previously reported that 3-acetyl-5-methyltetronic acid (AMT) had inhibitory effects on rat renal vitaminK1 2,3-epoxide reductase (VKOR) in vitro and also suppressed mesangial cell proliferation and, consequently, the formation of fibrosis via the vitamin K-dependent activation of the growtharrest-specific 6 (Gas6)/Axl pathway in anti-Thy-1 glomerulonephritis (Thy-1 GN) in rats. In the present study, we demonstrated that AMT alleviated the progression of renal fibrosis in CDDP-treated rats. The repeated intravenousadministration of AMT for 28 days dose-dependently suppressed increases in plasma urea nitrogen and plasma creatinine levels as well as creatinine clearance in CDDP-treated rats. Furthermore, the treatment suppressed theexpression of α-smooth muscle actin (SMA)-positive cells and ameliorated the extracellular matrix accumulation of collagen III, indicating an antifibrotic effect. In conclusion, our toxicological and histopathological resultsdemonstrated quantitatively the pharmacological inhibitory effects of AMT on the progression of renal fibrosis in CDDP-treated rats.

Human immunity involves both innate and adaptive defence mechanisms, with inflammation playing a central role in responding to cellular injury, pathogenic infections, and allergic stimuli. Reactive oxygen species (ROS) are closely associated with the onset and progression of inflammation. While moderate ROS levels function as crucial signalling molecules, excessive ROS can damage cellular components. This study aimed to evaluate the anti-inflammatory and antioxidant potential of plant-derived bioactive compounds including chlorogenic acid, oleuropein, tomatine, and tyrosol using human monocytic cell models (U-937 and THP-1). Differentiation of U-937 and THP-1 cells was induced prior to treatment with the selected bioactive compounds. Cell morphology and integrity were examined utilizing confocal microscopy. Gene expression stability was evaluated using reference genes β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Protein expression levels of key inflammatory markers were determined by Western blot analysis. In addition, molecular docking studies were conducted to assess the binding affinity of the compounds to human target proteins [Interleukin-4 (IL-4), 5-Lipoxygenase (LOX-5), Myeloperoxidase (MPO), and Tumor necrosis factor-alpha ( TNF-α)]. No cytotoxic effects were observed in treated cells, and GAPDH was confirmed as a stable reference gene under all experimental conditions. In U-937 cells, treatment with the bioactive compounds led to increased expression of the anti-inflammatory cytokine IL-4 and decreased expression of MPO. Notably, exposure to chlorogenic acid and tyrosol reduced MPO activity. Oleuropein and tyrosol demonstrated a strong suppressive effect on the expression of LOX-5, an enzyme responsible for leukotriene production. All tested bioactive compounds significantly reduced the phorbol 12-myristate 13-acetate (PMA) induced increase in LOX-5 activity. Molecular docking supported the potential of these compounds to interact with key inflammatory proteins, contributing to reduced oxidative stress. The plant-derived compounds, particularly oleuropein and tyrosol from olives, exhibit promising anti-inflammatory and antioxidant effects by modulating ROS-associated signalling pathways and downregulating inflammatory markers. These findings support the therapeutic potential of agricultural waste-derived bioactive in inflammation management and oxidative stress regulation.

Chemotherapy and radiotherapy are an essential treatment for many cancers, sometimes in combination with cytoreductive surgery. While radiotherapy destroys cancer cells by ionizing radiation that directly damages DNA, most of the chemotherapeutic agents generate Reactive Oxygen Species (ROS) that lead to oxidative damage in various molecules of the cell [1]. However, both processes mainly induce apoptosis to kill cancer cells. Any cancer cells that are not killed by these therapies could proliferate again, resulting in the relapse of cancer. The clinical benefit of using antioxidant supplements along with chemotherapy and radiotherapy is highly debatable, and not conclusive. This is either due to incomplete investigations, lack of enough samples for suitable statistical analysis, or failure to correlate wide ranges of different parameters used in different studies [2]. However, some of the clinical studies suggest that the antioxidant supplemented group had a worst survival rate, than the group who did not use antioxidant supplements [3]. Although in some cases, use of antioxidant has fewer side effects leading to less damage to normal tissues, but with a decrease in the overall survival rate. The ROS generated by anticancer agents, although effective in killing cancer cells, also alters other cellular pathways leading to various side effects [4]. The serious side effects encountered in chemotherapy include nephrotoxicity, ototoxicity or cardiotoxicity [2]. However, it is also believed that antioxidant supplemented reduction of side effects depends, mainly on using specific anticancer drugs for certain cancers.Thus, the intriguing question is whether antioxidant should continue to be used along with chemotherapy, or should be avoided. Also, the question arises whether cancer cells should be killed completely to cure cancers, without considering side effects. Logically, complete cure of cancer by overcoming drug resistance with minimal side effects should be the first priority.A systematic search including chemotherapy and ROS generation yielded more than 13,000 publications in the PubMed database, including mainly clinical studies. Searching anticancer agents mediated ROS generation yielded 307 studies in most recent years, demonstrating an intense focus shift towards understanding the mechanism of drug-mediated cancer cell death, through ROS generation. This shift could be attributed to the use of current genomic and molecular biology tools, to help understand adverse outcomes of chemotherapy and drug resistance that are observed in decades of clinical treatments. Arguably, as most of the recent studies have pointed out, if those anticancer agents generate ROS to kill cancer cells, then why antioxidant would be used along with chemotherapy? Instead, elevation of ROS should be the most effective way to supplement anticancer drugs. In addition, it has been observed in several recent reports that a reduced ROS level is attributed to drug resistance in certain cancer cells for some anticancer drugs [1,5], and indirectly supported by other studies [6,7]. Thus, increase of ROS level along with the drug could help in overcoming drug resistance during chemotherapy.Now what are the means to elevate ROS level in the cancer cell for effective killing with chemotherapy? The simple answers are by manipulating antioxidant genes or ROS maintaining genes/enzymes, or by conjugating nanoparticles that generate ROS, or by introducing external ROS to the tumor sites. Although, several antioxidant genes are known such as SODs (superoxide dismutases), catalase, GSH (glutathione), thioredoxins and peroxiredoxins, the ROS maintaining genes (e.g. ARHGEF6, p53, APEX1 etc.), and their mechanisms are largely unknown. Nanoparticles-mediated ROS generation technologies are also being developed [8]. The most important question is how effective overall would it be to overcome drug resistance by elevating the ROS level? At this point, extensive investigations could be initiated to assess the effect of elevated level of ROS to overcome drug resistance for many anticancer drugs in different types of cancers. After successful characterization in cellular and mouse models, the results could be implemented in systematic clinical trials. Thus, in the coming years, advanced chemotherapeutic treatments could be supplemented by means of ROS elevation, rather than with antioxidants.

Beta-thalassemia is one of the most frequently inherited disorders caused by mutations in the beta globin gene or its promoter, leading to reduced or absent beta globin synthesis. Ineffective erythropoiesis (IE) and consequent extramedullary hematopoiesis, splenomegaly and systemic iron overload are major features of this disease. The disease course can be associated with severe anemia and need for lifelong transfusion therapy (thalassemia major, TM) or relatively less severe anemia (non-transfusion-dependent thalassemia, NTDT, or thalassemia inter-media, TI). Patients affected by beta-thalassemia intermedia do not require chronic blood transfusions for survival. However, transfusion-independence is still associated with a variety of serious clinical morbidities.1–3 In NTDT the master regulator of iron homeostasis, hepcidin (Hamp), is chronically repressed.4–7 Therefore, patients absorb abnormally high levels of iron, requiring iron chelation to prevent the clinical sequelae associated with iron overload. Iron homeostasis needs to be carefully regulated in order to avoid toxicity due to its excess. If untreated, iron overload leads to organ failure and death. For this reason, in beta-thalassemia and other iron-related disorders, the management of iron overload has become the main focus. Chelation therapy, however, does not target the mechanism responsible for abnormal iron absorption, which is low levels of Hamp expression and synthesis. It has been shown that in mice affected by NTDT (Hbbth3/+ or th3/+), second generation antisense oligonucleotides (Tmprss6-ASO) or lipid nanoparticle (LNP)-formulated siRNAs can reduce the expression of transmembrane serine protease Tmprss6, one of the major suppressors of hepcidin expression.8,9 Suppression of Tmprss6 led to an increase in hepcidin synthesis and hemoglobin levels. These observations were also associated with a net reduction in splenomegaly, iron overload, transferrin saturation (TfSat), formation of insoluble membrane-bound globins (hemichromes) and reactive oxygen species (ROS).9 Thus, we hypothesized that the simultaneous use of the iron chelator deferiprone (DFP) with Tmprss6-ASO (Tmprss6-ASO+DFP) could combine the positive effects of Tmprss6-ASO on erythropoiesis and iron absorption with the chelation benefit on organ iron content. In this study, 3- to 4-month-old Hbbth3/+ females were treated with 50 mg/kg of Tmprss6 antisense oligonucleotide (Tmprss6-ASO, twice a week for 6 weeks) or Tmprss6-ASO in combination with the oral iron chelator DFP dissolved in the drinking water at 1.25 mg/ml, using either a commercial diet (normally used in the facility where animals were housed) containing 200 ppm of iron, or a physiological diet containing 35 ppm of iron. The majority of the animals available were treated using the commercial diet and just a few animals per group received the physiological one. With both diets we obtained the same trend in behavior, but considering that the numbers were not comparable, we decided to show only the data obtained from the 200 ppm diet.