Implication of system xc- in neuroinflammation during the onset and maintenance of neuropathic pain.

BACKGROUND: Despite the high prevalence of neuropathic pain, treating this neurological disease remains challenging, given the limited efficacy and numerous side effects associated with current therapies. The complexity in patient management is largely attributed to an incomplete understanding of the underlying pathological mechanisms. Central sensitization, that refers to the adaptation of the central nervous system to persistent inflammation and heightened excitatory transmission within pain pathways, stands as a significant contributor to persistent pain. Considering the role of the cystine/glutamate exchanger (also designated as system xc-) in modulating glutamate transmission and in supporting neuroinflammatory responses, we investigated the contribution of this exchanger in the development of neuropathic pain. METHODS: We examined the implication of system xc- by evaluating changes in the expression/activity of this exchanger in the dorsal spinal cord of mice after unilateral partial sciatic nerve ligation. In this surgical model of neuropathic pain, we also examined the consequence of the genetic suppression of system xc- (using mice lacking the system xc- specific subunit xCT) or its pharmacological manipulation (using the pharmacological inhibitor sulfasalazine) on the pain-associated behavioral responses. Finally, we assessed the glial activation and the inflammatory response in the spinal cord by measuring mRNA and protein levels of GFAP and selected M1 and M2 microglial markers. RESULTS: The sciatic nerve lesion was found to upregulate system xc- at the spinal level. The genetic deletion of xCT attenuated both the amplitude and the duration of the pain sensitization after nerve surgery, as evidenced by reduced responses to mechanical and thermal stimuli, and this was accompanied by reduced glial activation. Consistently, pharmacological inhibition of system xc- had an analgesic effect in lesioned mice. CONCLUSION: Together, these observations provide evidence for a role of system xc- in the biochemical processes underlying central sensitization. We propose that the reduced hypersensitivity observed in the transgenic mice lacking xCT or in sulfasalazine-treated mice is mediated by a reduced gliosis in the lumbar spinal cord and/or a shift in microglial M1/M2 polarization towards an anti-inflammatory phenotype in the absence of system xc-. These findings suggest that drugs targeting system xc- could contribute to prevent or reduce neuropathic pain.

Phenolic acids from Chicory roots ameliorate dextran sulfate sodium-induced colitis in mice by targeting TRP signaling pathways and the gut microbiota.

BACKGROUND: Inflammatory bowel disease (IBD) is a type of immune-mediated condition associated with intestinal homeostasis. Our preliminary studies disclosed that Cichorium intybus L., a traditional medicinal plant, also known as Chicory in Western countries, contained substantial phenolic acids displaying significant anti-inflammatory activities. We recognized the potential of harnessing Chicory for the treatment of IBD, prompting a need for in-depth investigation into the underlying mechanisms. METHODS: On the third day, mice were given 100, 200 mg/kg of total phenolic acids (PA) from Chicory and 200 mg/kg of sulfasalazine (SASP) via gavage, while dextran sodium sulfate (DSS) concentration was 2.5 % for one week. The study measured and evaluated various health markers including body weight, disease activity index (DAI), colon length, spleen index, histological score, serum concentrations of myeloperoxidase (MPO), nitric oxide (NO), superoxide dismutase (SOD), lipid oxidation (MDA), and inflammatory factors. We evaluated the TRP family and the NLRP3 inflammatory signaling pathways by Western blot, while 16S rDNA sequencing was used to track the effects of PA on gut microbes. RESULTS: It was shown that PA ameliorated the weight loss trend, attenuated inflammatory damage, regulated oxidative stress levels, and repaired the intestinal barrier in DSS mice. Analyses of Western blots demonstrated that PA suppressed what was expressed of transient receptor potential family TRPV4, TRPA1, and the expression of NLRP3 inflammatory signaling pathway, NLRP3 and GSDMD. In addition, PA exerted therapeutic effects on IBD by regulating gut microbiota richness and diversity. Meanwhile, the result of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that gut microbiota was mainly related to Membrane Transport, Replication and Repair, Carbohydrate Metabolism and Amino Acid Metabolism. CONCLUSION: PA derived from Chicory may have therapeutic effects on IBD by regulating the TRPV4/NLRP3 signaling pathway and gut microbiome. This study provides new insights into the effects of phenolic acids from Chicory on TRP ion channels and gut microbiota, revealing previously unexplored modes of action.

Effect of sulfasalazine on ferroptosis during intestinal injury in rats after liver transplantation.

Using a rat autologous orthotopic liver transplantation (AOLT) model and liver cold ischemia-reperfusion (I/R)-induced intestinal injury, we clarified whether ferroptosis occurred in rat AOLT cold I/R-induced intestinal injury. Additionally, the role and possible mechanism of the ferroptosis activator sulfasalazine (SAS) in intestinal injury-induced ferroptosis in rats with AOLT liver cold I/R were investigated. Sixty specific pathogen free (SPF)-grade adult male Sprague‒Dawley (SD) rats were randomly divided into 5 groups using the random number table method (n = 12). Six rats were randomly selected at 6 hour (h) and 24 h after I/R. Inferior vena cava blood specimens were collected from the portal vein (PV) opening at 6 h and 24 h. The concentrations of serum malondialdehyde (MDA), serum interleukin 6 (IL-6) were determined by enzyme-linked immunosorbent assay (ELISA). Ileal tissue was obtained from the PV opening in rats in each group at 6 h and 24 h, and ileal tissue sections were observed under light microscopy. The contents of intestinal MDA, superoxide dismutase (SOD), glutathione(GSH), glutathione peroxidase 4 (GPX4), and tissue iron were determined by ELISA, and the expression of GPX4 and the cysteine glutamate reverse transporter light chain protein (xCT) was determined by Western blot. The experimental results show that ferroptosis is involved in the pathophysiological process of intestinal injury induced by cold hepatic ischemia-reperfusion in AOLT rats. In addition, SAS (500 mg/kg) may inhibit the cystine/glutamate antiporters (System Xc¯)/GSH/GPX4 signal axis in intestinal injury induced by cold I/R in rat AOLT liver, or iron overload after reperfusion, causing a massive accumulation of L-ROS and activating cellular ferroptosis, further aggravate the intestinal injury.

Sulfasalazine promotes ferroptosis through AKT-ERK1/2 and P53-SLC7A11 in rheumatoid arthritis.

OBJECTIVE: Ferroptosis has been reported to play a role in rheumatoid arthritis (RA). Sulfasalazine, a common clinical treatment for ankylosing spondylitis, also exerts pathological influence on the progression of rheumatoid arthritis including the induced ferroptosis of fibroblast-like synoviocytes (FLSs), which result in the perturbated downstream signaling and the development of RA. The aim of this study was to investigate the underlying mechanism so as to provide novel insight for the treatment of RA. METHODS: CCK-8 and Western blotting were used to assess the effect of sulfasalazine on FLSs. A collagen-induced arthritis mouse model was constructed by the injection of collagen and Freund's adjuvant, and then, mice were treated with sulfasalazine from day 21 after modeling. The synovium was extracted and ferroptosis was assessed by Western blotting and immunofluorescence staining. RESULTS: The results revealed that sulfasalazine promotes ferroptosis. Compared with the control group, the expression levels of ferroptosis-related proteins such as glutathione peroxidase 4, ferritin heavy chain 1, and solute carrier family 7, member 11 (SLC7A11) were lower in the experimental group. Furthermore, deferoxamine inhibited ferroptosis induced by sulfasalazine. Sulfasalazine-promoted ferroptosis was related to a decrease in ERK1/2 and the increase of P53. CONCLUSIONS: Sulfasalazine promoted ferroptosis of FLSs in rheumatoid arthritis, and the PI3K-AKT-ERK1/2 pathway and P53-SLC7A11 pathway play an important role in this process.

Unravelling pH/pKa influence on pH-responsive drug carriers: Insights from ibuprofen-silica interactions and comparative analysis with carbon nanotubes, sulfasalazine, and alendronate.

This study employs density functional theory to explore the interaction between ibuprofen (IBU) and silica, emphasizing the influence of the trimethylsilyl (TMS) functional group for designing pH-responsive drug carriers. The surface (S) and drug (D) molecules' neutral (0) or deprotonated (-1) states were taken into consideration during the investigation. The likelihood of these states was determined based on the pKa values and the desired pH conditions. To calculate the pH-dependent interaction energy (EintpH), four different situations have been identified: S0D0, S0D-1, S-1D0, and S-1D-1.The electrostatic component of interaction energy aligns favorably with its theoretical value in both the Debye-Hückel and Grahame models. The investigation has gathered first-hand experimental data on the drug loading and release of pH-responsive mesoporous silica nanoparticles. Effective drug loading was observed in the acidic environment of the stomach (pH 2-5), followed by a release in the slightly basic to neutral pH of the small intestine (pH 7.4), These findings align with existing literature. The results revealed horizontal drug adherence on silica surfaces, improving binding capabilities. Comparisons were made with combinations involving carboxylated carbon nanotubes and ibuprofen, silica, and sulfasalazine, and silica and alendronate, exploring drug loading/release dynamics associated with positive/negative interaction energies. The investigation, supported by experimental data, contributes valuable insights into pH-responsive mesoporous silica nanoparticles, offering new design possibilities for drug carriers.

The gut microbiome regulates the clinical efficacy of sulfasalazine therapy for IBD-associated spondyloarthritis.

Sulfasalazine is a prodrug known to be effective for the treatment of inflammatory bowel disease (IBD)-associated peripheral spondyloarthritis (pSpA), but the mechanistic role for the gut microbiome in regulating its clinical efficacy is not well understood. Here, treatment of 22 IBD-pSpA subjects with sulfasalazine identifies clinical responders with a gut microbiome enriched in Faecalibacterium prausnitzii and the capacity for butyrate production. Sulfapyridine promotes butyrate production and transcription of the butyrate synthesis gene but in F. prausnitzii in vitro, which is suppressed by excess folate. Sulfasalazine therapy enhances fecal butyrate production and limits colitis in wild-type and gnotobiotic mice colonized with responder, but not non-responder, microbiomes. F. prausnitzii is sufficient to restore sulfasalazine protection from colitis in gnotobiotic mice colonized with non-responder microbiomes. These findings reveal a mechanistic link between the efficacy of sulfasalazine therapy and the gut microbiome with the potential to guide diagnostic and therapeutic approaches for IBD-pSpA.

Regulatory network of ferroptosis and autophagy by targeting oxidative stress defense using sulfasalazine in triple-negative breast cancer.

AIMS: The cellular defense system against oxidative stress is important for the survival ability and sensitization in chemotherapy; however, the regulatory mechanisms remain unknown in triple-negative breast cancer (TNBC) cells. This study aimed to investigate the relationship between ferroptosis and autophagy by targeting the defense of oxidative stress through the cystine transporter (xCT) using sulfasalazine (SASP), which is a widely employed xCT inhibitor. MAIN METHODS: We analyzed the cell death process of SASP in human TNBC cells, and examined the effects of SASP on tumor progression by using xenograft mouse model. KEY FINDINGS: TNBC cells demonstrated a high defense capacity against reactive oxidative species through xCT. SASP significantly attenuated oxidative stress resistance in MDA-MB-231, which is a generally used model cell as TNBC, through decreased glutathione levels, causing a marked iron-dependent ferroptotic cell death induction. Moreover, autophagy was required to trigger efficient SASP-induced ferroptosis at the early stage of cell death. Tamoxifen, which is currently in clinical use as the gold standard for endocrine therapy of estrogen receptor-positive breast cancer, was a beneficial tool as an autophagy regulator under ferroptotic cell death by SASP. Additionally, SASP suppressed tumor growth and metastasis progression through total glutathione reduction in the primary tumor, indicating high anticancer activity against TNBC without liver injury in vivo. SIGNIFICANCE: We revealed that SASP can efficiently induce ferroptosis associated with autophagy and that an understanding of the mechanism of cell death regulation by SASP is a promising new strategy for TNBC therapy and drug repositioning.

A polymeric iron oxide nanocomplex loaded with sulfasalazine: an approach for inducing ferritinophagy-assisted ferroptosis for anti-cancer therapy.

The approach of using ferroptosis to treat cancer has garnered attention due to its promising potential. However, the effectiveness of this therapy is often limited by the inherent redox system in cancer cells and the presence of ferritin as an iron ion storage molecule. To address this issue, we have designed a polymeric iron oxide nanocomplex loaded with sulfasalazine as a ferritinophagy-assisted ferroptosis inducing agent. The nanocomplex is based on a pH-responsive drug releasing platform that enables improved ferroptosis anti-cancer therapy. The nanocomplex was synthesized using polymerized phenylboronic acid decorated with iron oxide and further loaded with sulfasalazine by interacting with polymerized phenylboronic acid. Upon entering cancer cells, the nanocomplex releases sulfasalazine at the lysosomal acidic pH, which causes the complex to degrade into the labile iron ion (Fe2+). This process inhibits the production of GSH and reproduces the labile iron ion by degrading ferritin. As a result, an excess iron ion pool is formed, and the facilitated Fenton reaction induces an improved ferroptosis anti-cancer effect. Moreover, our research has demonstrated that the nanocomplex effectively regresses tumors, thereby representing significant inhibition of tumor growth using in vivo models. We believe that this ferritinophagy-assisted ferroptosis strategy using the nanocomplex provides a promising solution for iron-based anti-cancer therapy.

The nonessential amino acid cysteine is required to prevent ferroptosis in acute myeloid leukemia.

ABSTRACT: Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing.

Gilaburu (Viburnum opulus L.) fruit extract has potential therapeutic and prophylactic role in a rat model of acetic acid-induced oxidant colonic damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) which has a global impact on the health care system with its recurrent and incompletely curable characteristics, affects the patients' quality of life. Gilaburu (GB; Viburnum opulus L.) is a fruit with rich polyphenol ingredient which is used ethnobotanically in Türkiye for medicinal purposes (for example, to pass kidney stones, to treat stomach, heart, and liver diseases, hemorrhages, hypertension, ulcers, common cold, tuberculosis, rheumatic and menstrual pain, and diabetes). On the other hand, the effects of GB in the experimental UC model have not been studied. AIM OF THE STUDY: This study aimed to explore the potential antioxidant and anti-inflammatory effects of GB fruit extract in improving acetic acid (AA)-induced UC. MATERIALS AND METHODS: Starting immediately after (AA + GB group) or 1 week before (GB + AA + GB group) the colitis induced by intrarectal AA (5%; v/v) administration, the rats orally received GB (100 mg/kg) once per day for 3 days. The control and AA groups were administered orally saline (1 ml), while the AA + SS group were administered sulfasalazine (SS; 100 mg/kg; orally) as a positive control once per day for 3 days. Distal colonic tissue specimens were obtained for the histological and biochemical [myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), chemiluminescence (CL), caspase-3, 8-hydroxy-2'-deoxyguanosine (8-OHdG), matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-ß1, smad-3 and cytokine (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, interferon (IFN)-γ), measurements] evaluations on the 3rd day. RESULTS: Elevated macroscopic and microscopic damage scores, high tissue wet weight values, increased tissue-associated MPO, MDA, CL, caspase-3, 8-OHdG, cytokines (TNF-α, IL-1ß, IL-6, IL-8), MMP-9, TGF-ß1, smad-3 levels, and decreased GSH values of the AA group were all reversed by GB treatments (AA + GB and GB + AA + GB groups) (p < 0.05-0.001). However, sulfasalazine treatment (AA + SS group) did not change the IL-8, 8-OHdG, MMP-9, and TGF-ß1 measurements significantly. CONCLUSIONS: Gilaburu shows both anti-inflammatory and antioxidant effects against AA-induced colonic damage by suppressing neutrophil infiltration, regulating inflammatory mediators, inhibiting reactive species production, lipid peroxidation, and apoptosis, conserving endogenous antioxidant glutathione, and ameliorating oxidative DNA damage. Since the current ulcerative colitis drugs display limited benefits and adverse side effects, potential therapeutic and/or prophylactic role of gilaburu can be evaluated in ulcerative colitis.

Folic Acid-Modified Long-Circulating Liposomes Loaded with Sulfasalazine For Targeted Induction of Ferroptosis in Melanoma.

Melanoma is a malignant tumor that originates from melanocytes. The incidence of melanoma is increasing worldwide, partially because of its insensitivity to radiotherapy or chemotherapy. Therefore, effective treatments for melanoma are urgently required. In this study, we employed folic acid-modified sulfasalazine long-circulating liposomes (FA-SSZ-Lips) to precisely target drug delivery to melanoma cells, eliciting ferroptosis effectively. The synthesized FA-SSZ-Lips were characterized as small spheres of a double-layer membrane, a particle size of 110.1 nm, and a ζ-potential of -22.8 ± 0.66 mV. FA-SSZ-Lips are effective drug carriers with SSZ-loading ratio and SSZ release rate of 6.2 ± 0.10%, and 72.63 ± 1.40%, respectively. The liposomes enhanced SSZ solubility, and the folic acid modifications increased the liposome targeting to melanoma cells. Compared with SSZ alone, FA-SSZ-Lips more strongly inhibited B16F10 cell growth, significantly disrupted the intracellular redox balance, and induced ferroptosis. After treatment, considerable differences were observed in the tumor volumes between FA-SSZ-Lips and phosphate-buffered saline control groups. The tumor growth-inhibition value of the FA-SSZ-Lips group reached 70.09%. Thus, FA-SSZ-Lips exhibited favorable antitumor effects in vitro and in vivo and are a promising strategy for melanoma treatment.

Comparative treatment of Sulfasalazine+Ezetimibe combination and Sulfasalazine in a rat model with induced colitis.

Ulcerative colitis is a chronic inflammatory disease with high mortality and morbidity worldwide. It causes inflammation in the lining of the colon, resulting in several symptoms that negatively impact the quality of life. Unfortunately, there is currently no known cure for this condition. Therefore, it is crucial to explore alternative treatment approaches. This research aimed to investigate the anti-inflammatory and antioxidative effects of a combination therapy involving Sulfasalazine+Ezetimibe compared to Sulfasalazine alone in a rat model of ulcerative colitis. Forty adult rats were divided into four groups for this study. The groups consisted of a control group (negative control), an acetic acid group (positive control), an acetic acid+Sulfasalazine (100 mg/kg per day) group, and an acetic acid+Sulfasalazine (50 mg/kg)+Ezetimibe (5 mg/kg) group. Rats were treated for one week, and colitis was induced by administering 2 ml of 4% (v/v) acetic acid inter-rectally. After sacrifice, the colonic tissue homogenate was analyzed for several markers, including proinflammatory cytokines (TNF-α, IL-1ß, NF-κB), oxidative stress markers (malondialdehyde, myeloperoxidase), and adhesive molecule markers (E-selectin, ICAM-1). Sulfasalazine and the combination of Sulfasalazine+Ezetimibe significantly reduced the colonic levels of TNF-α, IL-1ß, NF-κB, MDA, and E-selectin in the homogenate. However, the combination therapy of Sulfasalazine and Ezetimibe demonstrated a superior effect.

Sulfasalazine, a potent cystine-glutamate transporter inhibitor, enhances osteogenic differentiation of canine adipose-derived stem cells.

Cystine-glutamate transporter (xCT) is a plasma membrane transporter that imports cystine and indirectly contributes to the oxidative stress resistance associated with increased intracellular glutathione levels. Canine adipose-derived stem cells (CADSCs) include an xCT-positive subpopulation and show relatively low expression of osteogenic markers during in vitro osteogenic differentiation. Sulfasalazine (SSZ), a drug used to treat rheumatoid arthritis, suppresses xCT expression in cancer cells. In this study, we found that the SSZ treatment at 100 µM significantly suppressed xCT mRNA expression in CADSCs but did not significantly affect cell proliferation under the same conditions. Additionally, this treatment decreased the intracellular glutathione concentration. During in vitro osteogenic differentiation, the SSZ treatment at 50 µM and 100 µM significantly increased alizarin red staining and its quantification, as well as the concentration-dependent osteogenic differentiation markers (BMP1 and SPP) mRNA expression. Our results suggested that SSZ enhances the osteogenic differentiation potential of CADSCs and can potentially exhibit a superior therapeutic profile in canine bone regenerative medicine.

3,6-Epidioxy-1,10-bisaboladiene and sulfasalazine synergistically induce ferroptosis-like cell death in human breast cancer cell lines.

3,6-Epidioxy-1,10-bisaboladiene (EDBD) is an endoperoxide compound isolated from edible wild plants that induces iron-dependent ferroptosis-like cell death in HL-60 cells by decreasing the expression of GPX4 and glutathione. In contrast, sulfasalazine (SSZ), a clinically used anti-inflammatory drug, induces ferroptosis through the system xc-. In this study, we investigated the synergistic effects of these 2 compounds on 3 human breast cancer cell lines (HBC-5, MCF-7, and MDA-MB-231). EDBD-induced cell death was relieved by the lipid peroxidation inhibitor ferrostatin-1 and the iron chelator deferoxamine mesylate (DFOM), indicating that EDBD induced ferroptosis-like cell death. Moreover, cotreatment with EDBD and SSZ synergistically induced cell death in all 3 cell lines. Because the cytotoxicity of the cotreatment was inhibited by DFOM and ferrostatin-1, the combination of EDBD and SSZ synergistically induced ferroptosis. Collectively, EDBD enhanced the effects of SSZ as a clinical anti-inflammatory and anticancer drug candidate.

Inhibition of xCT by sulfasalazine alleviates the depression-like behavior of adult male mice subjected to maternal separation stress.

Maternal separation (MS) can induce emotional disorders. Our previous study reported that MS resulted in depression-like behavior. In this study, we aimed to explore the role of xCT in depression-like behavior in adult mice subjected to MS stress. Pups were divided into the control group, the control + sulfasalazine (SSZ, 75 mg/kg/day, i.p.) group, the MS group, and the MS+SSZ group. After MS, all pups were raised until PD60. Then, the depression-like behavior was detected by the novelty suppressed feeding (NSF) test, the forced swimming test (FST), and the tail suspension test (TST). The synaptic plasticity was examined by electrophysiological recordings and molecular biotechnology. The data showed that, compared with the control group, the mice in the MS group presented depression-like behavior, impairment of long-term potentiation (LTP), a reduction in the number of astrocytes, and activation of the microglia. Moreover, the expression of xCT was increased in the prefrontal cortex of MS mice, the EAAT2 and the Group Ⅱ metabotropic glutamate receptors (mGluR2/3) were decreased, and the level of pro-inflammatory factors was increased in the prefrontal cortex. After the administration with SSZ, the depression-like behavior and the impairment of LTP were alleviated, the number of astrocytes was increased, and the microglial activation was inhibited. Moreover, the levels of EAAT2 and mGluR2/3 were ameliorated, the over-activation of the microglia was mitigated, and the levels of glutamate and pro-inflammatory factors were decreased. In conclusion, the inhibition of xCT by SSZ could alleviate depression-like behavior partly via modulating the homeostasis of the glutamate system and dampening neuroinflammation.

Genistein and/or sulfasalazine ameliorate acetic acid-induced ulcerative colitis in rats via modulating INF-γ/JAK1/STAT1/IRF-1, TLR-4/NF-κB/IL-6, and JAK2/STAT3/COX-2 crosstalk.

Ulcerative Colitis (UC) is a chronic idiopathic inflammatory bowel disease in which the colon's lining becomes inflamed. Exploring herbal remedies that can recover mucosal damage is becoming popular in UC. The study aims to investigate the probable colo-protective effect of a natural isoflavone, genistein (GEN), and/or a drug, sulfasalazine (SZ), against acetic acid (AA)-induced UC in rats, in addition to exploring the possible underlying mechanisms. UC was induced by the intrarectal installation of 1-2 ml of 5% diluted AA for 24 h. Ulcerated rats were allocated into the disease group and three treated groups, with SZ (100 mg/kg), GEN (100 mg/kg), and their combination for 14 days, besides the control groups. The anti-colitic efficacy of GEN and/or SZ was evidenced by hindering the AA-induced weight loss, colon edema, and macroscopic scores, besides reduced disease activity index and colon weight/length ratio. Furthermore, treatments attenuated the colon histopathological injury scores, increased the number of goblet cells, and lessened fibrosis. Both treatments reduced the up-regulation of INF-γ/JAK1/STAT1 and INF-γ /TLR-4/ NF-κB signaling pathways and modulated the IRF-1/iNOS/NO and IL-6/JAK2/STAT3/COX-2 pathways and consequently, reduced the levels of TNF-α and IL-1ß. Moreover, both treatments diminished oxidative stress, which appeared by reducing the MPO level and elevating the SOD activity, and hindered apoptosis; proved by the decreased immunohistochemical expression of caspase-3. The current findings offer novel insights into the protective effects of GEN and suggest a superior benefit of combining GEN with SZ, over either drug alone, in the UC management.

Mechanism of Cell Death by Combined Treatment with an xCT Inhibitor and Paclitaxel: An Alternative Therapeutic Strategy for Patients with Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian carcinoma that responds poorly to chemotherapy. Glutathione (GSH) is a primary antioxidant, which protects cells against reactive oxygen species (ROS). High levels of GSH are related to chemotherapeutic resistance. The glutamine/cystine transporter xCT is essential for intracellular GSH synthesis. However, whether xCT inhibition can overcome the resistance to chemotherapeutic agents in OCCC remains unclear. This study demonstrated that combined treatment with paclitaxel (PTX) and the xCT inhibitor sulfasalazine (SAS) significantly enhanced cytotoxicity more than the individual drugs did in OCCC cells. Treatment with PTX and SAS induced apoptosis more effectively than did individual drug treatments in the cells with significant generation of ROS. Moreover, combined treatment with PTX and SAS induced ferroptosis in the cells with low expression of glutathione peroxidase (GPx4), high levels of intracellular iron and significant lipid ROS accumulation. Therefore, our findings provide valuable information that the xCT inhibitor might be a promising therapeutic target for drug-resistant OCCC. The strategy of combined administration of PTX and SAS can potentially be used to treat OCCC and help to develop novel therapeutic methods.

Sulfasalazine inhibits esophageal cancer cell proliferation by mediating ferroptosis.

This study aimed to explore the potential mechanism by which sulfasalazine (SAS) inhibits esophageal cancer cell proliferation. A cell counting kit-8 (CCK-8) assay was used to detect the effect of SAS (0, 1, 2, and 4 mM) on the proliferation of TE-1 cells. Subsequently, TE-1 cells were divided into control group, SAS group, SAS + ferrostatin-1 (ferroptosis inhibitor) group, and SAS + Z-VAD (OH)-FMK (apoptosis inhibitor) group, and cell proliferation was measured using a CCK-8 assay. Real-time quantitative polymerase chain reaction and western blotting were used to determine the expression of solute carrier family member 7 11 (SLC7A11, also called xCT), glutathione peroxidase 4 (GPX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) in TE-1 cells. Measurement of ferroptosis in TE-1 cells was achieved by flow cytometry. Compared with the control group (0 mM SAS), the proliferation of TE-1 cells was significantly inhibited by different concentrations of SAS for different time lengths, and 4 mM SAS treatment for 48 h could obtain the maximum inhibition rate (53.9%). In addition, SAS treatment caused a significant decrease in the mRNA and protein expression of xCT and GPX4, and a significant increase in ACSL4 expression in TE-1 cells treated with SAS. Flow cytometry results showed that the ferroptosis level was significantly increased after SAS treatment. However, the activation of ferroptosis by SAS was partially eliminated by treatment with ferrostatin-1 or Z-VAD (OH)-FMK. In conclusion, SAS inhibits the proliferation of esophageal carcinoma cells by activating the ferroptosis pathway.

Phytochemical analysis of Origanum majorana L. extract and investigation of its antioxidant, anti-inflammatory and immunomodulatory effects against experimentally induced colitis downregulating Th17 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Origanum majorana L. is a member of the Lamiaceae family and is commonly used in Egyptian cuisine as a seasoning and flavor enhancer. It is also recognized as a well-known traditional medicine in Egypt and is widely used for treating abdominal colic due to its antispasmodic properties. However, the protective effects of Origanum majorana L. against ulcerative colitis and its underlying mechanisms remain unclear. AIM OF THE STUDY: This study aimed to identify the biologically active components present in methanol extracts of Origanum majorana L. using gas chromatography/mass spectrometry (GC/MS). Additionally, it aimed to investigate the therapeutic effects of these extracts on acetic acid-induced ulcerative colitis and elucidate the potential mechanisms involved. MATERIALS AND METHODS: We conducted a GC-MS analysis of the methanolic extract obtained from Origanum majorana L. Thirty-two male rats were included in the study and divided into four experimental groups, with eight rats in each group: sham, UC, UC + O. majorana, and UC sulfasalazine. After euthanizing the rats, colon tissue samples were collected for gross and microscopic examinations, assessment of oxidative stress, and molecular evaluation. GC-MS analysis identified 15 components in the extracts. Pretreatment with O. majorana L. extract and sulfasalazine significantly improved the disease activity index (DAI) and resulted in notable improvements in macroscopic and microscopic colon findings. Additionally, both treatments demonstrated preventive effects against colonic oxidative damage by reducing the levels of malondialdehyde (MDA) and increasing the levels of the antioxidant systems superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), which operate through the Nrf2/HO-1 signaling pathway. Moreover, these treatments downregulated the colonic inflammatory cascade by inhibiting NFκB, TNFα, IL-1ß, IL6, IL23, IL17, COX-2, and iNOS, subsequently leading to downregulation of the JAK2/STAT3 signaling pathway and a decrease in the Th17 cell response. Furthermore, a reduction in the number of apoptotic epithelial cells that expressed caspase-3 was observed. CONCLUSION: pretreatment with O. majorana L. extract significantly ameliorated acetic acid-induced ulcerative colitis. This effect could be attributed to the protective, antioxidant, anti-inflammatory, and anti-apoptotic properties of the extract.

Medroxyprogesterone acetate-resistant endometrial cancer cells are susceptible to ferroptosis inducers.

AIMS: Medroxyprogesterone acetate (MPA) is the most common fertility-sparing treatment in patients with early-stage endometrial cancer. If MPA treatment fails, hysterectomy is recommended. Thus, there is an urgent need for novel treatment approaches for MPA-resistant endometrial cancer patients who wish to preserve their fertility. Ferroptosis is a recently discovered type of regulated cell death caused by the excessive accumulation of reactive oxygen species (ROS), followed by aberrant lipid peroxidation. Recent studies have shown that inducing ferroptosis is a potential therapeutic strategy for cancer. However, the role of ferroptosis in endometrial cancer treatment remains to be discussed. We therefore investigated the effects of ferroptosis inducers on MPA-resistant endometrial cancer cells. MAIN METHODS: The levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), the main mediators of ferroptosis, were examined. Cell viability was evaluated after treatment with the ferroptosis inducers sulfasalazine, erastin, or RSL3. The degree of intracellular oxidative stress after treatment with these drugs was evaluated by the glutathione level, ROS level, ferrous iron level, lipid peroxidation and changes in mitochondrial morphology. The effect of ferroptosis inducers in vivo was also examined. KEY FINDINGS: The expression of SLC7A11 and GPX4 in MPA-resistant ECC-1 cells decreased in comparison to parental ECC-1 cells. Sulfasalazine, erastin, and RSL3 significantly reduced cell viability and increased intracellular oxidative stress in MPA-resistant ECC-1 cells. Ferroptosis inducers also suppressed in vivo tumor growth more effectively in MPA-resistant ECC-1. SIGNIFICANCE: Treatment with ferroptosis inducers could be a novel therapeutic approach for MPA-resistant endometrial cancer.