Cyclin-dependent kinase inhibitor 1A inhibits pyroptosis to enhance human lung adenocarcinoma cell radioresistance by promoting DNA repair.

Purpose: One of the best anticancer treatments available is radiotherapy, which can be used either alone or in conjunction with other forms of treatment including chemotherapy and surgery. Nevertheless, a number of biochemical and physiological processes that react to ionizing radiation might provide tumor cells radioresistance, which makes radiotherapy ineffective. It has been found that CDKN1A regulates DNA damage repair, which contributes to tumor radioresistance. However, the precise mechanism is still unknown. Therefore, this study aimed to explore the mechanisms underlying CDKN1A-enhanced radioresistance in tumor cells. Methods: Cells were irradiated with 4 Gy after CDKN1A overexpression or knockdown. CDKN1A expression was measured using real-time PCR, cell viability was evaluated using cell counting kit-8 and colony formation assays, and cytotoxicity was assessed using a lactate dehydrogenase assay. Pyroptosis in cells was analyzed using caspase-1 activity assay, enzyme-linked immunosorbent assay, and flow cytometry. Inflammation activation was detected through a co-immunoprecipitation assay. Activation of pyroptosis-related proteins was analyzed using immunohistochemistry, Western blot, and immunofluorescence. Tumor radioresistance in vivo was evaluated in a mouse xenograft model. Results: Radiotherapy upregulated CDKN1A expression, which promoted lung adenocarcinoma cell survival. CDKN1A influenced radiation-induced pyroptosis in A549, which mainly depended on inhibiting the activation of the AIM2 inflammasome by promoting DNA repair. Additionally, CDKN1A upregulation enhanced A549 xenograft tumor radioresistance by inhibiting radiation-induced pyroptosis in vivo. Conclusions: CDKN1A inhibits pyroptosis to enhance the radioresistance of lung adenocarcinoma cells by promoting DNA repair. This study may serve as a reference for developing novel targeted therapies against cancer.

8-Oxoguanine DNA glycosylase 1 selectively modulates ROS-responsive NF-κB targets through recruitment of MSK1 and phosphorylation of RelA/p65 at Ser276.

Nuclear factor kappa B (NF-κB) activity is regulated by various posttranslational modifications, of which Ser276 phosphorylation of RelA/p65 is particularly impacted by reactive oxygen species (ROS). This modification is responsible for selective upregulation of a subset of NF-κB targets; however, the precise mechanism remains elusive. ROS have the ability to modify cellular molecules including DNA. One of the most common oxidation products is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is repaired by the 8-oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair pathway. Recently, a new function of OGG1 has been uncovered. OGG1 binds to 8-oxoGua, facilitating the occupancy of NF-κB at promoters and enhancing transcription of pro-inflammatory cytokines and chemokines. In the present study, we demonstrated that an interaction between DNA-bound OGG1 and mitogen-and stress-activated kinase 1 is crucial for RelA/p65 Ser276 phosphorylation. ROS scavenging or OGG1 depletion/inhibition hindered the interaction between mitogen-and stress-activated kinase 1 and RelA/p65, thereby decreasing the level of phospho-Ser276 and leading to significantly lowered expression of ROS-responsive cytokine/chemokine genes, but not that of Nfkbis. Blockade of OGG1 binding to DNA also prevented promoter recruitment of RelA/p65, Pol II, and p-RNAP II in a gene-specific manner. Collectively, the data presented offer new insights into how ROS signaling dictates NF-κB phosphorylation codes and how the promoter-situated substrate-bound OGG1 is exploited by aerobic mammalian cells for timely transcriptional activation of ROS-responsive genes.

Quercetin alleviates lipopolysaccharide­induced acute lung injury by inhibiting ferroptosis via the Sirt1/Nrf2/Gpx4 pathway.

Acute lung injury (ALI) causes high morbidity and mortality rates in critically ill patients, and there are currently no effective therapeutic drugs. Ferroptosis is a newly discovered mode of regulated cell death that contributes to the progression of ALI. Quercetin possesses anti­inflammatory and antioxidant properties. However, whether quercetin can protect against lipopolysaccharide (LPS)­induced ALI by inhibiting ferroptosis and its underlying mechanisms remains unclear. The present study evaluated the protective effects of quercetin and underlying molecular mechanisms in LPS­induced ALI by establishing an ALI mouse model and an alveolar epithelial cell injury model via treatment of the mice or alveolar epithelial cells with LPS. Mouse lung injury was assessed by evaluating the histological lung injury score, bronchoalveolar lavage fluid cell count and inflammatory cytokine levels; alveolar epithelial cell injury was assessed by Cell counting kit­8, lactate dehydrogenase and EDU assays; and ferroptosis was assessed by detecting the changes in the levels of malondialdehyde, glutathione, iron, glutathione peroxidase 4 (Gpx4) and 4­hydroxynonenal in vivo and vitro. The present study indicated that quercetin effectively ameliorated LPS­induced ALI in the mouse model by reducing histopathological changes, proinflammatory cytokine release and reactive oxygen species generation and inhibiting ferroptosis. Quercetin significantly decreased ferroptosis and improved the proliferative ability of LPS­treated alveolar epithelial cells. Additionally, it was demonstrated that quercetin markedly enhanced the alveolar epithelial barrier, as evidenced by the upregulation of tight junction protein expression both in vivo and in vitro. Mechanistically, quercetin effectively activated the sirtuin 1 (Sirt1)/nuclear factor erythroid 2­related factor 2 (Nrf2)/Gpx4 signaling pathway, and targeted in vivo inhibition or in vitro knockdown of Sirt1 significantly reduced the anti­ferroptotic functions of quercetin. In conclusion, the results demonstrated that quercetin exerts its therapeutic effects against LPS­induced ALI by inhibiting ferroptosis via the activation of the Sirt1/Nrf2/Gpx4 signaling pathway.

Autophagy induced by PP121 alleviates MSU crystal-induced acute gouty arthritis via inhibition of the NLRP3 inflammasome.

Acute gouty arthritis (AGA) is a frequent self-limiting inflammatory condition produced by the deposition of monosodium urate (MSU) crystals in the joints and periarticular tissues of patients with hyperuricemia. However, no effective interventional measures currently exist for AGA. Pyroptosis, a kind of pro-inflammatory programmed cell death, plays a crucial role in MSU crystal-induced inflammation and represents a potential treatment target for AGA. Therefore, we determined the therapeutic benefits and mechanism of PP121, a pyroptosis-related compound, on AGA. First, we injected an MSU crystal solution intra-articularly into the left foot pad of C57BL/6 mice to create an AGA mouse model. Subsequent treatment with PP121 substantially decreased tissue damage, pro-inflammatory cytokine release, and inflammatory cell infiltration caused by MSU crystals in the ankle joint. Consistent with these observations, the beneficial effects of PP121 on AGA were cancelled in Beclin1+/-(Becn1+/-) mice. Furthermore, after PP121 treatment, super-resolution microscopy revealed a strong relationship between lysosome-connected membrane protein/light chain 3 positive vesicles and the nucleotide-binding domain of leucine-rich family pyrin domain-containing 3 (NLPR3), demonstrating that PP121 promotes phagocytosis of the NLPR3 inflammasome. In summary, PP121-mediated autophagy can improve degradation of the NLRR3 inflammasome in AGA, which suggests the therapeutic potential of PP121 in AGA.

Dipeptidase­2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin.

Lung cancer accounts for the highest percentage of cancer morbidity and mortality worldwide, and lung adenocarcinoma (LUAD) is the most prevalent subtype. Although numerous therapies have been developed for lung cancer, patient prognosis is limited by tumor metastasis and more effective treatment targets are urgently required. In the present study, gene expression profiles were extracted from the Gene Expression Omnibus database and mRNA expression data were downloaded from The Cancer Genome Atlas database. In addition, TIMER 2.0 database was used to analyze the expression of genes in normal and multiple tumor tissues. Protein expression was confirmed using the Human Protein Atlas database and LUAD cell lines, sphere formation assay, western blotting, and a xenograft mouse model were used to confirm the bioinformatics analysis. Dipeptidase­2 (DPEP2) expression was significantly decreased in LUAD and was negatively associated with prognosis. DPEP2 overexpression substantially inhibited epithelial­mesenchymal transition (EMT) as well as LUAD cell metastasis, and limited the expression of the cancer stem cell transformation markers, CD44 and CD133. In addition, DPEP2 improved LUAD sensitivity to cisplatin by inhibiting EMT; this was verified in vitro and in vivo. These data indicated that DPEP2 upregulates E­cadherin, thereby regulating cell migration, cancer stem cell transformation, and cisplatin resistance, ultimately affecting the survival of patients with LUAD. Overall, the findings of the present suggest that DPEP2 is important in the development of LUAD and can be used both as a prognostic marker and a target for future therapeutic research.

CC-115 Mediates GSDME-Dependent Pyroptosis in Lung Adenocarcinoma Through the Akt/Bax Pathway.

Chemotherapeutic agents remain the first-line treatment for solid tumors, including lung cancer, but chemotherapy resistance is hampering global efforts to treat this disease. CC-115 is a novel antitumoral compound used in phase I clinical trials. However, it is unclear whether CC-115 is effective against lung adenocarcinoma (LUAD). In the present study, we found that CC-115 induced lytic cell death in A549 and H1650 tumor cells via swelling of cells and formation of large bubbles on the plasma membrane that closely resembled those typical of pyroptosis, a type of programmed cell death linked to chemotherapy. We demonstrated that CC-115 exerts antitumor effects in LUAD through gasdermin E (GSDME)-mediated pyroptosis by acting as a dual inhibitor of DNA-PK and mTOR. CC-115 can inhibit Akt phosphorylation, impairing its inhibitory effect on Bax, thereby inducing pyroptosis via the Bax-mitochondrial intrinsic pathway. CC-115-induced pyroptosis was abrogated by treatment with the Akt activator SC79 or by depletion of Bax. Importantly, CC-115 significantly upregulated the expression of Bax and GSDME-N in a xenograft mouse model, with a reduction in tumor size. Our results revealed that CC-115 suppresses tumor growth by inducing GSDME-mediated pyroptosis through the Akt/Bax-mitochondrial intrinsic pathway, indicating CC-115 as a promising therapeutic agent for LUAD.

Novel recombinant protein flagellin A N/C attenuates experimental autoimmune encephalomyelitis by suppressing the ROS/NF-κB/NLRP3 signaling pathway.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease characterized by demyelination and neurodegeneration, for which traditional treatment offers limited relief. Microglial/macrophage modulation plays a critical role in the pathogenesis of MS. Oxygen free radical accumulation can induce axonal and nerve cell damage, and further promote MS development. We created a new recombinant protein based on flagellin from Legionella pneumophila named flagellin A with linked C- and N-terminal ends (FLaAN/C), which is an independent intellectual property of our team. We previously showed that FLaAN/C might mitigate radiation-induced damage by inhibiting inflammatory responses and oxidative stress. However, whether FLaAN/C protects against MS remains unknown. Here, we investigated the anti-inflammatory effects of FLaAN/C on mice with experimental autoimmune encephalomyelitis (EAE) induced by oligodendrocyte glycoprotein peptide 35-55 (MOG35-55). The mice were injected intraperitoneally with FLaAN/C after the onset of clinical symptoms, then clinical behavior scores and changes in body weight were recorded daily. The spinal lumbar spine in model mice was enlarged and accompanied by inflammatory cell infiltration and demyelination that were reversed by FLaAN/C. FLaAN/C also induced microglia/macrophages to generate less pro-inflammatory (CD86, iNOS, and TNF-α), and more anti-inflammatory (CD206, IL-10, and Arginase-1) cytokines. These findings suggesting that FLaAN/C promoted microglial/macrophages polarization from the inflammatory M1 to the anti-inflammatory M2 phenotype. Moreover, FLaAN/C inhibited release of the inflammatory cytokines, TNF-α, IL-8, IL-6, IL-17, and IFN-γ. These results indicated that the anti-inflammatory effect of FLaAN/C was associated with the inhibited generation of reactive oxygen species. FLaAN/C downregulated the expression of phosphorylated NF-κB-p65 and prevented downstream NLRP3 inflammasome-mediated pyroptosis. Collectively, these results indicated that FLaAN/C prevents pyroptosis by inhibiting the ROS/NF-κB/NLRP3 signaling pathway, and promotes the microglial/macrophage M1/M2 polarization that significantly alleviated inflammation in mouse models of EAE. Our findings suggested that FLaAN/C could be a promising candidate for MS therapy.

Increased susceptibility of irradiated mice to Aspergillus fumigatus infection via NLRP3/GSDMD pathway in pulmonary bronchial epithelia.

BACKGROUND: Aspergillus fumigatus infection is difficult to diagnose clinically and can develop into invasive pulmonary aspergillosis, which has a high fatality rate. The incidence of Aspergillus fumigatus infection has increased die to widespread application of radiotherapy technology. However, knowledge regarding A. fumigatus infection following radiation exposure is limited, and the underlying mechanism remains unclear. In this study, we established a mouse model to explore the effect of radiation on A. fumigatus infection and the associated mechanisms. METHODS: In this study, a mouse model of A. fumigatus infection after radiation was established by irradiating with 5 Gy on the chest and instilling 5 × 107/ml Aspergillus fumigatus conidia into trachea after 24 h to explore the effect and study its function and mechanism. Mice were compared among the following groups: normal controls (CON), radiation only (RA), infection only (Af), and radiation + infection (RA + Af). Staining analyses were used to detect infection and damage in lung tissues. Changes in protein and mRNA levels of pyroptosis-related molecules were assessed by western blot analysis and quantitative reverse transcription polymerase chain reaction, respectively. Protein concentrations in the serum and alveolar lavage fluid were also measured. An immunofluorescence colocalization analysis was performed to confirm that NLRP3 inflammasomes activated pyroptosis. RESULTS: Radiation destroyed the pulmonary epithelial barrier and significantly increased the pulmonary fungal burden of A. fumigatus. The active end of caspase-1 and gasdermin D (GSDMD) were highly expressed even after infection. Release of interleukin-18 (IL-18) and interleukin-1ß (IL-1ß) provided further evidence of pyroptosis. NLRP3 knockout inhibited pyroptosis, which effectively attenuated damage to the pulmonary epithelial barrier and reduced the burden of A. fumigatus. CONCLUSIONS: Our findings indicated that the activation of NLRP3 inflammasomes following radiation exposure increased susceptibility to A. fumigatus infection. Due to pyroptosis in lung epithelial cells, it resulted in the destruction of the lung epithelial barrier and further damage to lung tissue. Moreover, we found that NLRP3 knockout effectively inhibited the pyroptosis and reducing susceptibility to A. fumigatus infection and further lung damage. Overall, our results suggest that NLRP3/GSDMD pathway mediated-pyroptosis in the lungs may be a key event in this process and provide new insights into the underlying mechanism of infection. Video abstract.

Obacunone alleviates ferroptosis during lipopolysaccharide-induced acute lung injury by upregulating Nrf2-dependent antioxidant responses.

BACKGROUND: Acute lung injury (ALI) has received considerable attention in the field of intensive care as it is associated with a high mortality rate. Obacunone (OB), widely found in citrus fruits, is a natural bioactive compound with anti-inflammatory and antioxidant activities. However, it is not clear whether OB protects against lipopolysaccharide (LPS)-induced ALI. Therefore, in this study, we aimed to evaluate the protective effects of OB and the potential mechanisms against LPS-induced ALI and BEAS-2B cell injury. METHODS: We established a model of BEAS-2B cell injury and a mouse model of ALI by treating with LPS. Samples of in vitro model were subjected to cell death, Cell Counting Kit-8, and lactate dehydrogenase (LDH) release assays. The total number of cells and neutrophils, protein content, and levels of IL-6, TNF-α, and IL-1ß were determined in bronchoalveolar lavage fluid (BALF). Glutathione, reactive oxygen species, and malondialdehyde levels were determined in lung tissue. Additionally, immunohistochemical analysis, immunofluorescence, western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay were conducted to examine the effects of OB. Furthermore, mice were treated with an Nrf2 inhibitor (ML385) to verify its role in ferroptosis. Data were analyzed using one-way analysis of variance or paired t-tests. RESULTS: Compared with the LPS group, OB effectively alleviated LPS-induced ALI by decreasing lung wet/dry weight ratio, reactive oxygen species and malondialdehyde production, and superoxide dismutase and glutathione consumption in vivo. In addition, OB significantly alleviated lung histopathological injury, reduced inflammatory cytokine secretion and Fe2+ and 4-HNE levels, and upregulated GPX4, SLC7A11, and Nrf2 expression. Mechanistically, OB activated Nrf2 by inhibiting Nrf2 ubiquitinated proteasome degradation. ML385 reversed the protective effects of OB against LPS-induced ALI. CONCLUSION: Overall, OB alleviates LPS-induced ALI, making it a potential novel protective agent against LPS-induced ALI.

CircNEIL3 mediates pyroptosis to influence lung adenocarcinoma radiotherapy by upregulating PIF1 through miR-1184 inhibition.

Circular RNAs (circRNAs) belong to an abundant category of non-coding RNAs that are stable and specific, and thus have great potential in cancer treatment. However, little is known about the role of circRNAs during radiotherapy in lung adenocarcinoma (LUAD). Here, we established the expression profiles of 1,875 dysregulated circRNAs in non-irradiated and irradiated A549 cells and identified circNEIL3 as a significantly downregulated circRNA in A549 cells treated with 0, 2, or 4 Gy of radiation, respectively. Functional assays demonstrated that circNEIL3 knockdown promoted radiation-induced cell pyroptosis, whereas circNEIL3 overexpression had the opposite effects. Importantly, the effects of circNEIL3 overexpression on inhibiting pyroptosis were reversed by PIF1 knockdown. Mechanistically, circNEIL3-mediated pyroptosis was achieved through directly binding to miR-1184 as a sponge, thereby releasing the inhibition of miR-1184 on PIF1, which ultimately induces DNA damage and triggers AIM2 inflammasome activation. In vivo, circNEIL3 knockdown significantly enhanced the efficacy of radiotherapy as evidenced by decreases in tumor volume and weight. Collectively, the circNEIL3/miR-1184/PIF1 axis that mediate pyroptosis induction may be a novel, promising therapeutic strategy for the clinical treatment of lung cancer.

NVP-AUY922 alleviates radiation-induced lung injury via inhibition of autophagy-dependent ferroptosis.

Radiation-induced lung injury (RILI) is a common complication of radiotherapy for which no effective interventions are available. NVP-AUY922, a resorcinylic isoxazole amide drug, exhibits anti-inflammatory, immunomodulatory, and therapeutic effects against various types of cancers. In this study, we explore the role and underlying mechanisms of NVP-AUY922 in the treatment of RILI. We established a model of BEAS-2B cell injury and a mouse model of RILI. Cell proliferation, death, gross weight, and survival rates of mice, and histological parameters were assessed. Additionally, inflammation-related indices and indicators related to ferroptosis were evaluated. Furthermore, immunofluorescence and co-immunoprecipitation were used to determine the interaction between GPX4, LAMP-2A, and HSC70. NVP-AUY922 significantly ameliorated radiation-induced lung tissue damage, inflammatory cell infiltration, proinflammatory cytokine release, and lung epithelial BEAS-2B cell damage. NVP-AUY922 markedly limited the activation of ferroptosis, which is involved in RILI. Mechanistically, NVP-AUY922 prevented chaperone-mediated autophagy of the GPX4 pathway in vitro and in vivo, and the autophagy inhibitor Baf-A1 significantly increased the level of GPX4 and alleviated lung inflammation. NVP-AUY922 can alleviate RILI by inhibiting chaperone-mediated lysosomal degradation of GPX4, demonstrating its potential as a novel protective agent against RILI.

TBHQ attenuates ferroptosis against 5-fluorouracil-induced intestinal epithelial cell injury and intestinal mucositis via activation of Nrf2.

BACKGROUND: Intestinal mucositis is a common side effect of chemotherapy and radiotherapy. Very few drugs can efficiently ameliorate it. Tertiary butylhydroquinone (TBHQ) is a widely used food preservative with known immunomodulatory activity. Whether it has an effect on intestinal mucositis remains unknown. In this study, we investigated the role and mechanism of action of TBHQ on 5-fluorouracil-induced (5-FU-induced) human intestinal epithelial cell (HIEC) injury and intestinal mucositis in mice. METHODS: We established a cell model of HIEC injury and a mouse model of intestinal mucositis via treatment with 5-FU. Cell death, Cell Counting Kit-8, and lactate dehydrogenase (LDH) release were assessed for the HIECs. Diarrhea, body weight, intestinal length, mucosal damage, and the levels of IL-6, TNF-α, IL-1ß, glutathione, reactive oxygen species, and malondialdehyde were determined for the mice. Additionally, we performed immunohistochemical analysis, immunofluorescence, western blotting, quantitative real-time PCR, and ELISA to examine the effects of TBHQ. Finally, HIECs were transfected with an Nrf2 gene silencer to verify its role in ferroptosis. All data were analyzed using one-way analysis of variance or paired t-tests. RESULTS: TBHQ markedly decreased LDH release and cell death and improved the proliferative ability of 5-FU-treated HIECs. The TBHQ-treated mice showed reduced weight loss, a lower diarrhea score, and longer colons than the 5-FU-treated mice. The in vivo expressions of IL-1ß, IL-6, and TNF-α were suppressed by TBHQ treatment. Ferroptosis was shown to be involved in 5-FU-induced intestinal mucositis, and TBHQ markedly hampered its activation. Mechanistically, TBHQ activated Nrf2 effectively and selective Nrf2 knockdown significantly reduced the anti-ferroptotic functions of TBHQ in 5-FU-treated HIECs. CONCLUSIONS: TBHQ attenuates ferroptosis in 5-FU-induced intestinal mucositis, making it a potential novel protective agent against intestinal mucositis.

AZD8055 ameliorates experimental autoimmune encephalomyelitis via the mTOR/ROS/NLRP3 pathway.

AIMS: Experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), is characterized by immune-mediated demyelination and neurodegeneration. NOD-like receptor protein 3 (NLRP3) inflammasome activation aggravates spinal cord inflammation in EAE. Autophagy is associated with alleviation of systemic inflammation, including that encountered in EAE. However, the effects of autophagy on NLRP3 in EAE are still unclear. Here, we evaluated the effects of the autophagy activator AZD8055 on EAE. METHODS: EAE model mice were established, histological examination was performed to assess the degree of inflammatory cell infiltration and demyelination. And the levels of autophagy and NLRP3-mediated pyroptosis in spinal cords were assessed. Western blotting and immunofluorescence analyses were performed to evaluate protein expression and localization. RESULTS: AZD8055 significantly enhanced autophagy in the spinal cords of EAE model mice, coupled with decreased abnormal clinical behavior scores and increased body weights. The degree of inflammatory cell infiltration and demyelination was mild in AZD8055-treated EAE model mice.Meanwhile, the pathway of ROS/NLRP3 was downregulated, and LC3 and NLRP3 were colocalized. CONCLUSIONS: AZD8055 ameliorated EAE through anti-inflammatory and anti-pyroptosis effects via the mammalian target of mTOR/ROS/NLRP3 pathway. These findings provide insights into the interactions between autophagy and pyroptosis and may facilitate the development of novel treatments for MS.

TGF-ß1-mediated exosomal lnc-MMP2-2 increases blood-brain barrier permeability via the miRNA-1207-5p/EPB41L5 axis to promote non-small cell lung cancer brain metastasis.

Brain metastases remain a major problem in patients with advanced non-small cell lung cancer (NSCLC). The permeability of the blood-brain barrier (BBB) is highly increased during lung cancer brain metastasis; however, the underlying mechanism remains largely unknown. We previously found that lnc-MMP2-2 is highly enriched in tumor growth factor (TGF)-ß1-mediated exosomes and regulates the migration of lung cancer cells. This study aimed to explore the role of exosomal lnc-MMP2-2 in the regulation of BBB and NSCLC brain metastasis. Here, using endothelial monolayers and mouse models, we found that TGF-ß1-mediated NSCLC-derived exosomes efficiently destroyed tight junctions and the integrity of these natural barriers. Overexpression of lnc-MMP2-2 in human brain microvascular endothelial cells increased vascular permeability in endothelial monolayers, whereas inhibition of lnc-MMP2-2 alleviated these effects. Furthermore, lnc-MMP2-2 knockdown markedly reduced NSCLC brain metastasis in vivo. Mechanistically, through luciferase reporter assays, RNA pull-down assay, and Ago2 RNA immunoprecipitation assay, we showed that lnc-MMP2-2 served as a microRNA sponge or a competing endogenous RNA for miR-1207-5p and consequently modulated the derepression of EPB41L5. In conclusion, TGF-ß1-mediated exosomal lnc-MMP2-2 increases BBB permeability to promote NSCLC brain metastasis. Thus, exosomal lnc-MMP2-2 may be a potential biomarker and therapeutic target against lung cancer brain metastasis.

miR-324-3p reverses cisplatin resistance by inducing GPX4-mediated ferroptosis in lung adenocarcinoma cell line A549.

PURPOSE: MicroRNAs act as crucial regulators of a diverse range of biological processes, including chemoresistance. Our study aimed to investigate the effect of miR-324-3p on lung adenocarcinoma cell line A549 resistant to cis-diamminedichloroplatinum II (DDP, aka cisplatin). METHODS: The miR-324-3p expression levels in cisplatin-sensitive A549（A549） and cisplatin-resistant A549 (A549/DDP) cells were determined by qRT-PCR assay. Cell proliferation was determined with the commercial kit CCK-8 and colony formation assay, whereas cell death was analyzed using flow cytometry. The target gene of miR-324-3p was identified and validated with the luciferase reporter and western blot assays. The role of miR-324-3p in modulating cisplatin resistance was evaluated in vitro. RESULTS: The expression of miR-324-3p was found to be significantly downregulated in the A549/DDP cells. Conversely, miR-324-3p overexpression reversed cisplatin resistance in the cells. With regard to the possible mechanism underlying this phenomenon, we identified the glutathione peroxidase 4 (GPX4) gene as the direct target of miR-324-3p, where overexpression of the gene reversed the miR-324-3p effect of sensitizing the A549/DDP cells to cisplatin. Furthermore, the GPX4 inhibitor RSL3 could mimic the effect of miR-324-3p upregulation in increasing the sensitivity of the cisplatin-resistant cells to the drug. Significantly, miR-324-3p enhanced cisplatin-induced ferroptosis in the A549/DDP cells. CONCLUSION: Our findings revealed the role of the miR-324-3p-GPX4 signaling axis in A549/DDP cells and how the targeting of this axis could be a potential strategy for reversing cisplatin resistance in human non small cell lung cancer (NSCLC).

Autophagy Induced by Micheliolide Alleviates Acute Irradiation-Induced Intestinal Injury via Inhibition of the NLRP3 Inflammasome.

Radiation-induced enteropathy (RIE) is one of the most common and fatal complications of abdominal radiotherapy, with no effective interventions available. Pyroptosis, a form of proinflammatory regulated cell death, was recently found to play a vital role in radiation-induced inflammation and may represent a novel therapeutic target for RIE. To investigate this, we found that micheliolide (MCL) exerted anti-radiation effects in vitro. Therefore, we investigated both the therapeutic effects of MCL in RIE and the possible mechanisms by which it may be therapeutic. We developed a mouse model of RIE by exposing C57BL/6J mice to abdominal irradiation. MCL treatment significantly ameliorated radiation-induced intestinal tissue damage, inflammatory cell infiltration, and proinflammatory cytokine release. In agreement with these observations, the beneficial effects of MCL treatment in RIE were abolished in Becn1 +/- mice. Furthermore, super-resolution microscopy revealed a close association between NLR pyrin domain three and lysosome-associated membrane protein/light chain 3-positive vesicles following MCL treatment, suggesting that MCL facilitates phagocytosis of the NLR pyrin domain three inflammasome. In summary, MCL-mediated induction of autophagy can ameliorate RIE by NLR pyrin domain three inflammasome degradation and identify MCL as a novel therapy for RIE.

Bixin Attenuates Experimental Autoimmune Encephalomyelitis by Suppressing TXNIP/NLRP3 Inflammasome Activity and Activating NRF2 Signaling.

Multiple sclerosis (MS), an autoimmune and degenerative disease, is characterized by demyelination and chronic neuroinflammation. Bixin is a carotenoid isolated from the seeds of Bixa orellana that exhibits various potent pharmacological activities, including antioxidant, anti-inflammatory, and anti-tumor properties. However, the effects of bixin on MS have not yet been examined. To evaluate the effects and underlying molecular mechanisms of bixin on MS, experimental autoimmune encephalomyelitis (EAE) was established in C57BL/6 mice, which were treated via intragastric administration of bixin solutions. To evaluate the molecular mechanisms of bixin, quantitative reverse-transcription PCR, western blot, immunohistochemistry, flow cytometry, and enzyme-linked immunosorbent assay analyses were performed. We found that bixin significantly improved the symptoms and pathology in EAE mice, reduced the release of inflammatory cytokines TNF-α, IL-6, IL-8, IL-17, and IFN-γ, and increased the expression of the anti-inflammatory cytokine IL-10. And bixin reduced the proportion of Th1 and Th17 cells in the spleen and CNS, and suppressed microglia aggregation, and TXNIP/NLRP3 inflammasome activity by scavenging excessive reactive oxygen species (ROS) in EAE mice. Furthermore, bixin inhibited inflammation and oxidative stress via activating nuclear factor erythroid 2-related factor 2 (NRF2), and its downstream genes in EAE mice, meanwhile, these effects were suppressed upon treatment with an NRF2 inhibitor, ML385. Bixin prevented neuroinflammation and demyelination in EAE mice primarily by scavenging ROS through activation of the NRF2 signaling pathway. Taken together, our results indicate that bixin is a promising therapeutic candidate for treatment of MS.

Amifostine ameliorates induction of experimental autoimmune encephalomyelitis: Effect on reactive oxygen species/NLRP3 pathway.

Multiple sclerosis (MS) is an autoimmune disease for which conventional treatments have limited efficacy or side effects. Free radicals are primarily involved in blood-brain barrier disruption and induce neuronal and axonal damage, thus promoting the development of MS. Amifostine, a radioprotective drug used as a cytoprotective agent, attenuates oxidative stress and improves radiation damage by acting as a direct scavenger of reactive oxygen and nitrogen species. The aim of this study was to evaluate the effects of amifostine on MS in a mouse model of experimental autoimmune encephalomyelitis (EAE), which was developed by immunizing C57BL/6 mice with myelin oligodendrocyte glycoprotein and pertussis toxin. EAE mice received intraperitoneal injections of amifostine prior to onset of clinical symptoms and were monitored up to day 15 post induction. We observed abnormal clinical behavioral scores and a decrease in body weight. Histological analysis showed severe inflammatory infiltration and demyelination in the brain and spinal cord lumbar enlargements where significant upregulation of the mRNA expression of the pro-inflammatory cytokines interleukin-6 and interleukin-8, downregulation of the anti-inflammatory cytokine interleukin-10, and obvious microgliosis were also observed. Amifostine treatment potently reversed these abnormal changes. The anti-inflammatory effect of amifostine was associated with the inhibition of reactive oxygen species generation. Furthermore, the expression of proteins involved in the NLRP3 signaling pathway and pyroptosis was decreased. In conclusion, our study showed that amifostine ameliorates induction of experimental autoimmune encephalomyelitis via anti-inflammatory and anti-pyroptosis effects, providing further insights into the use of amifostine for the treatment of MS.

RNAseq profiling of circRNA expression in radiation-treated A549 cells and bioinformatics analysis of radiation-related circRNA-miRNA networks.

With the development of new biochemical and computational methods, circular RNAs (circRNAs) have been identified as microRNA sponges. circRNAs are associated with many diseases, particularly cancer. The present study aimed to investigate the expression profile of circRNAs in irradiated A549 lung cancer cells using high-throughput sequencing. Bioinformatics analyses were used to examine the potential functions of circRNAs. RNA sequencing data demonstrated that 1,875 circRNA targets were differentially expressed in A549 cells in response to irradiation. A total of 30 circRNAs were upregulated and 37 circRNAs were downregulated significantly in irradiation-treated A549 cells (fold change ≥2.0; P<0.05). The top 5 upregulated and downregulated circRNAs were successfully validated by reverse transcription-quantitative PCR. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that differentially expressed circRNAs might be pivotal in biological irradiation responses to irradiation. circRNA-microRNA co-expression networks highlighted the biological significance of circRNA_0002174 and circRNA_0036627, which require further study. In conclusion, the present study is, to the best of the authors' knowledge, the first to describe the differentially expressed profile of circRNAs in response to irradiation in A549 cells. These results provide a new perspective to elucidate insight into the molecular mechanisms by which A549 cells respond to radiation, and a basis for a more in-depth analysis of the potential application of circRNAs in the treatment of lung cancer therapy.