ROS Inhibitor Research Articles

Chemical and cell antioxidant activity of amaranth flour and beverage after simulated gastrointestinal digestion. Role of peptides

The potential of peptides generated by simulated gastrointestinal digestion (SGID) of two products derived from Amaranthus manteggazianus seeds, flour (F) and beverage (B), to exert peroxyl scavenging activity (ORAC) and antioxidant action on intestinal cells was studied. B was prepared by solubilisation of seed proteins, with the addition of gums and the application of a pasteurization treatment. The gastrointestinal digests FD and BD showed some differences in the peptide/polypeptide composition. The SGID produced increased ORAC activity for both samples, with some differences in the ORAC of the whole digests BD and FD and of some gel filtration fractions. Bioaccessible fractions (FDdbs and BDdbs) were obtained after treatment with cholestyramine resin to remove bile salts due to their cytotoxicity and oxidative effect. BDdbs presented a greater ORAC potency (IC50: 0.05 ± 0.01 and 0.008 ± 0.004 mg protein/ml for FDdbs and BDdbs, respectively). These fractions showed low cytotoxicity values (measured by LDH release) and produced high intracellular ROS inhibition (around 80 %), increased the SOD activity and the GSH content, with no effect on GPx activity in Caco2-TC7 cells exposed to H2O2. Several fractions with MM < 2.2 kDa presented also these cellular actions; fractions from FD induced higher increases in GSH concentration. Amaranth flour and a processed matrix like the beverage are shown as sources of bioactive peptides with potential cell antioxidant activity.

Lagopsis supina ameliorates myocardial ischemia injury by regulating angiogenesis, thrombosis, inflammation, and energy metabolism through VEGF, ROS and HMGB1 signaling pathways in rats

BackgroundLagopsis supina (Steph. ex. Willd.) Ikonn.-Gal. is an important traditional Chinese medicine used to treat various ailments. However, its impact on myocardial ischemia (MI) injury remains unknown. PurposeThis research aimed to reveal the therapeutic effect, potential mechanism, and metabolomics of L. supina against MI injury in rats. MethodsThe therapeutic effects of the ethanolic extract of L. supina (LS) and its four fractions (LSA∼D) on a left anterior descending (LAD) artery occlusion-induced MI model rat were explored. The pharmacodynamics including myocardial infraction area, myocardial tissue pathology and apoptosis, and serum biochemical parameters (CK, CK-MB, CTn-T, SOD, ET-1, NO, eNOS, VEGF, TXB2, 6-keto-PGF1α, TNF-α, IL-6, and CRP) were evaluated. The 24 related protein expressions were detected using western blotting assay. Simultaneously, the qualitative and quantitative analyses of microporous adsorption resin with 30% (LSC) and 60% (LSD) aqueous ethanol fractions were performed using UHPLC-MS and HPLC. Moreover, the serum metabolomics analysis of rats was profiled using UHPLC-MS. ResultsLS exerted remarkable alleviating effect on MI in rats. Importantly, LSC and LSD, two effective fractions of LS, significantly reduced myocardial infraction area, alleviated myocardial tissue pathology and apoptosis, regulated serum biochemical parameters. Furthermore, LSC and LSD markedly up-regulated the levels of VEGF-A, VEGFR-2, PKC, Bcl-2, Nrf2, HO-1, and thrombin, as well as prominently down-regulated the protein expression of Notch 1, p-PI3K, p-PI3K/PI3K, p-Akt, p-Akt/Akt, Bax, cleaved-caspase-3, cleaved-caspase-3/caspase-3, vWF, p-Erk, p-Erk/Erk, HMGB1, p-p38, p-p38/p38, p-p65, and p-p65/p65. A total of 26 candidate biomarkers were significantly regulated by LSC and LSD and they are mainly involved in amino acid metabolism, glycerophospholipid metabolism, and sphingolipid metabolism. Finally, phenylethanols and flavonoids may be major bio-constituents of LSC and LSD against MI. ConclusionsThis work, for the first time, demonstrated that L. supina had a significant therapeutic effect on MI in rats. Additionally, LSC and LSD, two bio-fractions from L. supina, exerted their potential to ameliorate MI injury by promoting angiogenesis, inhibiting thrombosis, blocking inflammation, and facilitating energy metabolism through promotion of VEGF pathway, as well as suppression of ROS and HMGB1 pathways in rats. These findings suggest that LSC and LSD hold promise as potential therapeutic agents for MI injury in clinical application.

Amorphous silica nanoparticles cause abnormal cytokinesis and multinucleation through dysfunction of the centralspindlin complex and microfilaments

BackgroundWith the large-scale production and application of amorphous silica nanoparticles (aSiNPs), its adverse health effects are more worthy of our attention. Our previous research has demonstrated for the first time that aSiNPs induced cytokinesis failure, which resulted in abnormally high incidences of multinucleation in vitro, but the underlying mechanisms remain unclear. Therefore, the purpose of this study was firstly to explore whether aSiNPs induced multinucleation in vivo, and secondly to investigate the underlying mechanism of how aSiNPs caused abnormal cytokinesis and multinucleation.MethodsMale ICR mice with intratracheal instillation of aSiNPs were used as an experimental model in vivo. Human hepatic cell line (L-02) was introduced for further mechanism study in vitro.ResultsIn vivo, histopathological results showed that the rate of multinucleation was significantly increased in the liver and lung tissue after aSiNPs treatment. In vitro, immunofluorescence results manifested that aSiNPs directly caused microfilaments aggregation. Following mechanism studies indicated that aSiNPs increased ROS levels. The accumulation of ROS further inhibited the PI3k 110β/Aurora B pathway, leading to a decrease in the expression of centralspindlin subunits MKLP1 and CYK4 as well as downstream cytokines regulation related proteins Ect2, Cep55, CHMP2A and RhoA. Meanwhile, the particles caused abnormal co-localization of the key mitotic regulatory kinase Aurora B and the centralspindlin complex by inhibiting the PI3k 110β/Aurora B pathway. PI3K activator IGF increased the phosphorylation level of Aurora B and improved the relative ratio of the centralspindlin cluster. And ROS inhibitors NAC reduced the ratio of multinucleation, alleviated the PI3k 110β/Aurora B pathway inhibition, and then increased the expression of MKLP1, CYK4 and cytokinesis-related proteins, whilst NAC restored the clustering of the centralspindlin.ConclusionThis study demonstrated that aSiNPs led to multinucleation formation both in vivo and in vitro. ASiNPs exposure caused microfilaments aggregation and inhibited the PI3k 110β/Aurora B pathway through excessive ROS, which then hindered the centralspindlin cluster as well as restrained the expression of centralspindlin subunits and cytokinesis-related proteins, which ultimately resulted in cytokinesis failure and the formation of multinucleation.

Abstract P3001: Regulation Of Perivascular Adipose Tissue By Follistatin In Hypertension

Follistatin lowers BP and improves resistance artery health in spontaneously hypertensive rats (SHR), a model of essential HTN. Functionally, follistatin improves smooth muscle contraction and endothelium-dependent relaxation. Structurally, increased medial collagen was inhibited. Vascular ROS were also reduced. Perivascular adipose tissue (PVAT) regulates vascular tone via release of vasoactive substances. Oxidative stress in PVAT, as in HTN, induces release of contractile agents. Transition from white to brown PVAT is associated with decreased risk of CVD. We analyze effects of follistatin on PVAT and its regulation of vessel function in SHR and normotensive Wistar Kyoto (WKY) rats. White and brown PVAT were dissected from treated rats; arteries were isolated from untreated donors. PVAT was introduced near strain-matched donor arteries and assessed for ROS levels and contractility using DHE and wire myography, respectively. In some vessels, endothelium was denuded to assess its contribution to contraction. Follistatin reduced ROS, hypercontractility, and improved relaxation in a PVAT-dependent manner. Brown PVAT induced significant anti-contractile and antioxidant effects. Elevated expression of adipose tissue browning markers and decreased adipocyte area with follistatin, support PVAT browning effects. Endothelium removal results in augmented vessel constriction, effects reduced by follistatin. Follistatin improves SHR smooth muscle tone in a PVAT-dependent manner, likely through the inhibition of vascular ROS and browning of PVAT. Future work will profile PVAT effects of follistatin through unbiased proteomic analysis. Although most vessels are surrounded by PVAT, studies of PVAT-vessel interaction in HTN are few. By characterizing PVAT dysfunction, we aim to outline specific PVAT-mediated pathways involved in lowering BP and improving vascular function and structure. Results will provide new insights into PVAT-targeted therapy for essential HTN, allowing potential translation of benefits to obesity and other diseases exhibiting PVAT dysfunction.

Injectable Bioadhesive Hydrogels Scavenging ROS and Restoring Mucosal Barrier for Enhanced Ulcerative Colitis Therapy.

Despite the progress in the therapy of ulcerative colitis (UC), long-lasting UC remission can hardly be achieved in the majority of UC patients. The key pathological characteristics of UC include an impaired mucosal barrier and local inflammatory infiltration. Thus, a two-pronged approach aiming at repairing damaged mucosal barrier and scavenging inflammatory mediators simultaneously might hold great potential for long-term remission of UC. A rectal formulation can directly offer preferential and effective drug delivery to inflamed colon. However, regular intestinal peristalsis and frequent diarrhea in UC might cause transient drug retention. Therefore, a bioadhesive hydrogel with strong interaction with intestinal mucosa might be preferable for rectal administration to prolong drug retention. Here, we designed a bioadhesive hydrogel formed by the cross-linking of sulfhydryl chondroitin sulfate and polydopamine (CS-PDA). The presence of PDA would ensure the mucosa-adhesive behavior, and the addition of CS in the hydrogel network was expected to achieve the restoration of the intestinal epithelial barrier. To scavenge the key player (excessive reactive oxygen species, ROS) in inflamed colon, sodium ferulic (SF), a potent ROS inhibitor, was incorporated into the CS-PDA hydrogel. After rectal administration, the SF-loaded CS-PDA hydrogel could adhere to the colonic mucosa to allow prolonged drug retention. Subsequently, sustained SF release could be achieved to persistently scavenge ROS in inflammatory areas. Meanwhile, the presence of CS would promote the restoration of the mucosal barrier. Ultimately, scavenging ROS and restoring the mucosal barrier could be simultaneously achieved via this SF-loaded bioadhesive hydrogel scaffold. Our two-pronged approach might provide new insight for effective UC treatment.

Ginsenoside Re blocks Bay k-8644-induced neurotoxicity via attenuating mitochondrial dysfunction and PKCδ activation in the hippocampus of mice: Involvement of antioxidant potential.

Although the anticonvulsant effects of ginsenosides are recognized, little is known about their effects on the convulsive behaviors induced by the activation of L-type Ca2+ channels. Here, we investigated whether ginsenoside Re (GRe) modulates excitotoxicity induced by the L-type Ca2+ channel activator Bay k-8644. GRe significantly attenuated Bay k-8644-induced convulsive behaviors and hippocampal oxidative stress in mice. GRe-mediated antioxidant potential was more pronounced in the mitochondrial fraction than cytosolic fraction. As L-type Ca2+ channels are thought to be targets of protein kinase C (PKC), we investigated the role of PKC under excitotoxic conditions. GRe attenuated Bay k-8644-induced mitochondrial dysfunction, PKCδ activation, and neuronal loss. The PKCδ inhibition and neuroprotection mediated by GRe were comparable to those by the ROS inhibitor N-acetylcysteine, the mitochondrial protectant cyclosporin A, the microglial inhibitor minocycline, or the PKCδ inhibitor rottlerin. Consistently, the GRe-mediated PKCδ inhibition and neuroprotection were counteracted by the mitochondrial toxin 3-nitropropionic acid or the PKC activator bryostatin-1. GRe treatment did not have additional effects on PKCδ gene knockout-mediated neuroprotection, suggesting that PKCδ is a molecular target of GRe. Collectively, our results suggest that GRe-mediated anticonvulsive/neuroprotective effects require the attenuation of mitochondrial dysfunction and altered redox status and inactivation of PKCδ.

Ginsenoside Rg1 attenuates diabetic vascular endothelial dysfunction by inhibiting the calpain-1/ROS/PKC-β axis

AimsVascular endothelial dysfunction (VED) is the onset event of cardiovascular complications in type 2 diabetes mellitus. Ginsenoside Rg1 (Rg1) can improve the cardiovascular system, but its mechanism in diabetic vascular endothelial dysfunction has received little attention. Main methodsMale calpain-1-knockout and wild-type C57BL/6 J mice were intraperitoneally injected with streptozotocin and treated with Rg1 (10 and 20 mg/kg) for 8 weeks. Human aortic endothelial cells (HAECs) were incubated with high glucose (HG) and were pretreated with Rg1 (10, 20 μM), MDL-28170 (calpain-1 inhibitor), LY-333531 (PKC-β inhibitor), NAC (ROS inhibitor) and calpain-1 overexpression. Then, factors related to mitochondrial dysfunction, oxidative stress and VED were measured. Key findingsThe administration of Rg1 and calpain-1 knockout ameliorated diabetic mitochondrial dysfunction, oxidative stress and VED and inhibited the calpain-1/ROS/PKC-β axis. LY-333531 and NAC treatment restored destructive endothelium-dependent vasodilation in mice with diabetes, while pyrogallol (ROS agonist), PMA (PKC-β agonist) or L-NAME (eNOS inhibitor) treatment abrogated the protective effect of Rg1 against diabetic endothelial dysfunction. The administration of Rg1, MDL-28170, LY-333531 and NAC improved mitochondrial dysfunction, oxidative stress and VED, whereas the overexpression of calpain-1 amplified mitochondrial dysfunction, oxidative stress and VED and further upregulated the expression of PKC-β in HAECs exposed to HG. Overexpression of calpain-1 abrogated the protective effect of Rg1 against HG-induced oxidative stress and VED. SignificanceThese findings reveal that Rg1 can protect against VED by suppressing the calpain-1/ROS/PKC-β axis and alleviating the development of mitochondrial dysfunction and oxidative stress.

Bilobetin induces apoptosis in human hepatocellular carcinoma cells via ROS level elevation and inhibition of CYP2J2

Bilobetin is a biflavonoid isolated from the leaves of Ginkgo biloba. Bilobetin displays several biological effects, however, the activities of bilobetin against cancer have been solely demonstrated. Thus, the aim of this study is investigating the apoptotic effects of and anticancer mechanism of bilobetin in Huh7 and HepG2 cells, both of human hepatocellular carcinoma (HCC) cell lines. MTT, cell counting, and colony formation assay showed that anti-proliferation effect of bilobetin. Cell cycle analysis revealed that bilobetin induces increase of population of the sub G1 phase. Annexin V/PI staining and TUNEL assay showed that bilobetin treatment promotes apoptotic cell death and DNA fragmentation. Bilobetin also induces ROS elevation and DNA damage in HCC cells. Western blot analysis elucidated that bilobetin displays apoptotic signaling pathway in HCC cells via upregulating cleaved PARP, cleaved caspase3 and Bax and downregulating CYP2J2. Bilobetin inhibited CYP2J2-catalyzed terfenadine and ebastine hydroxylase activities with IC50 values of 0.81 and 2.21 μM, respectively. Transfection of CYP2J2 siRNA increased the anticancer effect of bilobetin in HCC cells through the inhibition of CYP2J2 expression. Taken together, this study suggests that bilobetin induces apoptosis against HCC cells via ROS level elevation and inhibition of CYP2J2.

Cathepsin K inhibition induces Raptor destabilization and mitochondrial dysfunction via Syk/SHP2/Src/OTUB1 axis-mediated signaling

The Raptor signaling pathway is a critical point of intervention in the invasion and progression of cancer. The non-receptor tyrosine kinase Src-mediated phosphorylation of OTUB1-Y26 plays a critical role in Raptor stabilization, whereas cathepsin K inhibitor (odanacatib; ODN) and knockdown (siRNA) induce Raptor destabilization. However, the mechanisms involved in cathepsin K inhibition-induced OTUB1-Y26 phosphorylation in Raptor stabilization have not been yet elucidated. This study showed that cathepsin K inhibition activates SHP2, a tyrosine phosphatase, that dephosphorylates OTUB1 and destabilizes Raptor, whereas SHP2 deletion and pharmacological inhibition increase OTUB1-Y26 phosphorylation and Raptor expression. SHP2 deletion also led to the inhibition of ODN-induced mitochondrial ROS, fusion, and dysfunction. Furthermore, cathepsin K inhibition phosphorylated spleen tyrosine kinase (Syk) at Y525 and Y526, resulting in the SHP2-mediated dephosphorylation of OTUB1-Y26. Collectively, our findings identified Syk not only as an upstream tyrosine kinase required for SHP2 activation but also showed a critical mechanism that regulates ODN-induced Raptor downregulation and mitochondrial dysfunction. In conclusion, Syk/SHP2/Src/OTUB1 axis-mediated signaling can act as a therapeutic target in cancer management.

Perfluorooctanoic acid-induced cell death via the dual roles of ROS-MAPK/ERK signaling in ameloblast-lineage cells

Perfluorooctanoic acid (PFOA) is an artificial fluorinated organic compound that has generated increased public attention due to its potential health hazards. Unsafe levels of PFOA exposure can affect reproduction, growth and development. During tooth enamel development (amelogenesis), environmental factors including fluoride can cause enamel hypoplasia. However, the effects of PFOA on ameloblasts and tooth enamel formation remain largely unknown. In the present study we demonstrate several PFOA-mediated cell death pathways (necrosis/necroptosis, and apoptosis) and assess the roles of ROS-MAPK/ERK signaling in PFOA-mediated cell death in mouse ameloblast-lineage cells (ALC).ALC cells were treated with PFOA. Cell proliferation and viability were analyzed by MTT assays and colony formation assays, respectively. PFOA suppressed cell proliferation and viability in a dose dependent manner. PFOA induced both necrosis (PI-positive cells) and apoptosis (cleaved-caspase-3, γH2AX and TUNEL-positive cells). PFOA significantly increased ROS production and up-regulated phosphor-(p)-ERK. Addition of ROS inhibitor N-acetyl cysteine (NAC) suppressed p-ERK and decreased necrosis, and increased cell viability compared to PFOA alone, whereas NAC did not change apoptosis. This suggests that PFOA-mediated necrosis was induced by ROS-MAPK/ERK signaling, but apoptosis was not associated with ROS. Addition of MAPK/ERK inhibitor PD98059 suppressed necrosis and increased cell viability compared to PFOA alone. Intriguingly, PD98059 augmented PFOA-mediated apoptosis. This suggests that p-ERK promoted necrosis but suppressed apoptosis. Addition of the necroptosis inhibitor Necrostatin-1 restored cell viability compared to PFOA alone, while pan-caspase inhibitor Z-VAD did not mitigate PFOA-mediated cell death. These results suggest that 1) PFOA-mediated cell death was mainly caused by necrosis/necroptosis by ROS-MAPK/ERK signaling rather than apoptosis, 2) MAPK/ERK signaling plays the dual roles (promoting necrosis and suppressing apoptosis) under PFOA treatment. This is the initial report to indicate that PFOA could be considered as a possible causative factor for cryptogenic enamel malformation. Further studies are required to elucidate the mechanisms of PFOA-mediated adverse effects on amelogenesis.

Necroptosis signaling and mitochondrial dysfunction cross-talking facilitate cell death mediated by chelerythrine in glioma.

Glioma is the most common primary malignant brain tumor with poor survival and limited therapeutic options. Chelerythrine (CHE), a natural benzophenanthridine alkaloid, has been reported to exhibit the anti-tumor effects in a variety of cancer cells. However, the molecular target and the signaling process of CHE in glioma remain elusive. Here we investigated the underlying mechanisms of CHE in glioma cell lines and glioma xenograft mice model. Our results found that CHE-induced cell death is associated with RIP1/RIP3-dependent necroptosis rather than apoptotic cell death in glioma cells at the early time. Mechanism investigation revealed the cross-talking between necroptosis and mitochondria dysfunction that CHE triggered generation of mitochondrial ROS, mitochondrial depolarization, reduction of ATP level and mitochondrial fragmentation, which was the important trigger for RIP1-dependent necroptosis activation. Meanwhile, PINK1 and parkin-dependent mitophagy promoted clearance of impaired mitochondria in CHE-incubated glioma cells, and inhibition of mitophagy with CQ selectively enhanced CHE-induced necroptosis. Furthermore, early cytosolic calcium from the influx of extracellular Ca2+ induced by CHE acted as important "priming signals" for impairment of mitochondrial dysfunction and necroptosis. Suppression of mitochondrial ROS contributed to interrupting positive feedback between mitochondrial damage and RIPK1/RIPK3 necrosome. Lastly, subcutaneous tumor growth in U87 xenograft was suppressed by CHE without significant body weight loss and multi-organ toxicities. In summary, the present study helped to elucidate necroptosis was induced by CHE via mtROS-mediated formation of the RIP1-RIP3-Drp1 complex that promoted Drp1 mitochondrial translocation to enhance necroptosis. Our findings indicated that CHE could potentially be further developed as a novel therapeutic strategy for treatment of glioma.

Metformin inhibits ovarian granular cell pyroptosis through the miR-670-3p/NOX2/ROS pathway

Recent studies have demonstrated that ovarian granular cells (OGCs) pyroptosis is present in the ovaries of polycystic ovary syndrome (PCOS) mice and that NLRP3 activation destroys follicular functions. Metformin has been shown to protect against PCOS by reducing insulin resistance in women, whereas its role in OGC pyroptosis is unknown. This study aimed to investigate the impact of metformin on OGC pyroptosis and the underlying mechanisms. The results showed that treating a human granulosa-like tumor cell line (KGN) with metformin significantly decreased LPS-induced expression of miR-670-3p, NOX2, NLRP3, ASC, cleaved caspase-1, and GSDMD-N. Cellular caspase-1 activity; ROS production; oxidative stress; and the secretion of IL-1β, IL-6, IL-18, and TNF-α were also diminished. These effects were amplified by adding N-acetyl-L-cysteine (NAC), a pharmacological inhibitor of ROS. In contrast, metformin's anti-pyroptosis and anti-inflammatory effects were robustly ameliorated by NOX2 overexpression in KGN cells. Moreover, bioinformatic analyses, RT-PCR, and Western blotting showed that miR-670-3p could directly bind to the NOX2 (encoded by the CYBB gene in humans) 3'UTR and decrease NOX2 expression. Metformin-induced suppression of NOX2 expression, ROS production, oxidative stress, and pyroptosis was significantly alleviated by transfection with the miR-670-3p inhibitor. These findings suggest that metformin inhibits KGN cell pyroptosis via the miR-670-3p/NOX2/ROS pathway.

Tricyclic antidepressants induce liver inflammation by targeting NLRP3 inflammasome activation

BackgroundIdiosyncratic drug-induced liver injury (IDILI) is common in hepatology practices and, in some cases, lethal. Increasing evidence show that tricyclic antidepressants (TCAs) can induce IDILI in clinical applications but the underlying mechanisms are still poorly understood.MethodsWe assessed the specificity of several TCAs for NLRP3 inflammasome via MCC950 (a selective NLRP3 inhibitor) pretreatment and Nlrp3 knockout (Nlrp3−/−) BMDMs. Meanwhile, the role of NLRP3 inflammasome in the TCA nortriptyline-induced hepatotoxicity was demonstrated in Nlrp3−/− mice.ResultsWe reported here that nortriptyline, a common TCA, induced idiosyncratic hepatotoxicity in a NLRP3 inflammasome-dependent manner in mildly inflammatory states. In parallel in vitro studies, nortriptyline triggered the inflammasome activation, which was completely blocked by Nlrp3 deficiency or MCC950 pretreatment. Furthermore, nortriptyline treatment led to mitochondrial damage and subsequent mitochondrial reactive oxygen species (mtROS) production resulting in aberrant activation of the NLRP3 inflammasome; a selective mitochondrial ROS inhibitor pretreatment dramatically abrogated nortriptyline-triggered the NLRP3 inflammasome activation. Notably, exposure to other TCAs also induced aberrant activation of the NLRP3 inflammasome by triggering upstream signaling events.ConclusionCollectively, our findings revealed that the NLRP3 inflammasome may act as a crucial target for TCA agents and suggested that the core structures of TCAs may contribute to the aberrant activation of NLRP3 inflammasome induced by them, an important factor involved in the pathogenesis of TCA-induced liver injury.DBhggTBWtuReWsEtfqqAGmVideo

Astragaloside IV Attenuates High-Glucose-Induced Impairment in Diabetic Nephropathy by Increasing Klotho Expression via the NF-κB/NLRP3 Axis.

Deficiencies in klotho are implicated in various kidney dysfunctions including diabetic nephropathy (DN) related to inflammatory responses. Klotho is closely related to inflammatory responses and is a potential target for ameliorating kidney failure. Pyroptosis, an inflammatory form of programmed cell death, is reported to take part in DN pathogenesis recently. This study is aimed at exploring whether and how klotho inhibited podocyte pyroptosis and whether astragaloside IV (AS-IV) protect podocyte through the regulation of klotho. SD rat model of DN and conditionally immortalized mouse podocytes exposed to high glucose were treated with AS-IV. Biochemical assays and morphological examination, cell viability assay, cell transfection, phalloidin staining, ELISA, LDH release assay, SOD and MDA detection, MMP assay, ROS level detection, flow cytometry analysis, TUNEL staining assay, PI/Hoechst 33342 staining, immunofluorescence assay, and western blot were performed to elucidate podocyte pyroptosis and to observe the renal morphology. The treatment of AS-IV can improve renal function and protect podocytes exposed to high glucose. Klotho was decreased, and AS-IV increased klotho levels in serum and kidney tissue of DN rats as well as podocytes exposed to high glucose. AS-IV can inhibit DN glomeruli pyroptosis in vivo. In vitro, overexpressed klotho and treatment with AS-IV inhibited pyroptosis of podocytes cultured in high glucose. Klotho knockdown promoted podocyte pyroptosis, and treatment with AS-IV reversed this effect. Furthermore, the overexpression of klotho and AS-IV reduces oxidative stress levels and inhibited NF-κB activation and NLRP3-mediated podocytes' pyroptosis which was abolished by klotho knockdown. In addition, both the ROS inhibitor NAC and the NF-κB pathway inhibitor PDTC can inhibit NLRP3 inflammasome activation. NLRP3 inhibitor MCC950 can inhibit pyroptosis of podocytes exposed to high glucose. Altogether, our results demonstrate that the protective effect of AS-IV in upregulating klotho expression in diabetes-induced podocyte injury is associated with the inhibition of NLRP3-mediated pyroptosis via the NF-κB signaling pathway.

Influence of Pore Size and Surface Functionalization of Mesoporous Silica Nanoparticles on the Solubility and Antioxidant Activity of Confined Coenzyme Q10.

Coenzyme Q10 is a potent antioxidant that plays an important role in the maintenance of various biochemical pathways of the body and has a wide range of therapeutic applications. However, it has low aqueous solubility and oral bioavailability. Mesoporous silica nanoparticles (MCM-41 and SBA-15 types) exhibiting varying pore sizes and modified with phosphonate and amino groups were used to study the influence of pore structure and surface chemistry on the solubility, in vitro release profile, and intracellular ROS inhibition activity of coenzyme Q10. The particles were thoroughly characterized to confirm the morphology, size, pore profile, functionalization, and drug loading. Surface modification with phosphonate functional groups was found to have the strongest impact on the solubility enhancement of coenzyme Q10 when compared to that of pristine and amino-modified particles. Phosphonate-modified MCM-41 nanoparticles (i.e., MCM-41-PO3) induced significantly higher coenzyme Q10 solubility than the other particles studied. Furthermore, MCM-41-PO3 led to a twofold decrease in ROS generation in human chondrocyte cells (C28/I2), compared to the free drug in a DMSO/DMEM mixture. The results confirmed the significant contribution of small pore size and negative surface charge of MSNs that enable coenzyme Q10 confinement to allow enhanced drug solubility and antioxidant activity.

The genomic regions and candidate genes associated with drought tolerance and yield-related traits in foxtail millet: an integrative meta-analysis approach

Drought stress is one of the most significant limiting factors limiting crop productions. Foxtail millet (Setaria italica) is among the most drought-tolerant crop plants, with a high degree of collinearity with other staple cereals. The present study used a meta-analysis approach to identify genomic regions and candidate genes associated with drought tolerance and yield-related traits in foxtail millet. A meta-analysis employing all 448 collected original quantitative trait loci (QTL) identified 41 meta-QTL (MQTL) on the nine foxtail millet chromosomes. The confidence interval (CI) of the identified MQTL was determined to be 0.31–14.47 cM (5.23 cM average), which was 3.5 times narrower than the mean CI of the original QTL. Based on the available RNA-seq and microarray data, 1631 differentially expressed genes (DEGs) were detected in 41 MQTL. Furthermore, through synteny analysis, 8, 4, and 2 ortho-MQTL were recognized within co-linear regions of foxtail millet with rice (Oryza sativa), barley (Hordeum vulgare), and maize (Zea mays), respectively. To detect the most significant genome regions involved in the genetic control of drought tolerance and yield maintenance in foxtail millet, 10 MQTL with physical intervals of less than 1 Mb and seven hotspot regions with a high QTL-overview index were identified. Several candidate genes involved in foxtail millet sensing and signaling, transcription regulation, ROS inhibition, and adaptation to abiotic stress were detected by seeking drought-responsive genes in MQTL regions with a CI < 1 Mb. We hope that the achieved results would aid in developing new high-yielding drought-tolerant genotypes.

In Vitro and In Vivo Evaluation of Synergistic Effects of Everolimus in Combination with Antifungal Agents on Exophiala dermatitidis.

To investigate the combined function of the novel oral mTOR inhibitor, everolimus, with antifungal agents and their potential mechanisms against Exophiala dermatitidis, the CLSI microliquid-based dilution method M38-A2, chequerboard technique, and disk diffusion testing were performed. The efficacy of everolimus was evaluated in combination with itraconazole, voriconazole, posaconazole, and amphotericin B against 16 clinically isolated strains of E. dermatitidis. The synergistic effect was determined by measuring the MIC and fractional inhibitory concentration index. Dihydrorhodamine 123 was used for the quantification of ROS levels. The differences in the expression of antifungal susceptibility-associated genes were analyzed following different types of treatment. Galleria mellonella was used as the in vivo model. While everolimus alone showed minimal antifungal effects, combinations with itraconazole, voriconazole, posaconazole, or amphotericin B resulted in synergy in 13/16 (81.25%), 2/16 (12.5%), 14/16 (87.75%), and 5/16 (31.25%) of isolates, respectively. The disk diffusion assay revealed that the combination of everolimus and antifungal drugs showed no significant increase in the inhibition zones compared with the single agent, but no antagonistic effects were observed. Combination of everolimus and antifungal agents resulted in increased ROS activity (everolimus + posaconazole versus posaconazole [P < 0.05], everolimus + amphotericin B versus amphotericin B [P < 0.002]). Simultaneously, compared to mono-treatment, the combination of everolimus + itraconazole suppressed the expression of MDR2 (P < 0.05) and the combination of everolimus + amphotericin B suppressed the expression of MDR3 (P < 0.05) and CDR1B (P < 0.02). In vivo, combinations of everolimus and antifungal agents improved survival rates, particularly the combination of everolimus + amphotericin B (P < 0.05). In summary, the in vivo and in vitro experiments performed in our study suggest that the combination of everolimus with azoles or amphotericin B can have synergistic effects against E. dermatitidis, potentially due to the induction of ROS activity and inhibition of efflux pumps, providing a promising new approach for the treatment of E. dermatitidis infections. IMPORTANCE Cancer patients with E. dermatitidis infection have high mortality if untreated. Clinically, the conventional treatment of E. dermatitidis is poor due to the long-term use of antifungal drugs. In this study, we have for the first time investigated the interaction and action mechanism of everolimus combined with itraconazole, voriconazole, posaconazole, and amphotericin B on E. dermatitidis in vitro and in vivo, which provided new ideas and direction for further exploring the mechanism of drug combination and clinical treatment of E. dermatitidis.

Hexavalent chromium disrupts the skin barrier by targeting ROS-mediated mitochondrial pathway apoptosis in keratinocytes

Hexavalent chromium (Cr(VI)), a toxic heavy metal, is ubiquitous in daily life. Exposure to this toxic substance in occupational settings can cause dermatitis and cancer. As the body's largest organ, the skin plays a crucial role in protecting the organism against external aggressions. While previous studies have focused on the effects of Cr(VI) on skin inflammation, this study investigates the potential toxicity of Cr(VI) from the skin barrier and integrity perspective. The in vivo results of this study showed that mice exposed to Cr(VI) experienced skin deterioration and hemorrhaging, as well as a reduction in the thickness of the collagen fiber layer. TUNEL and Occludin staining results revealed that Cr(VI)'s toxicity primarily targeted keratinocytes. Experiments in vitro demonstrated that Cr(VI) treatment decreased the activity of HaCaT cells, altered cell morphology, and increased LDH secretion. Further research revealed that Cr(VI) could modify membrane permeability, impair membrane integrity, and reduce the protein expression of ZO-1 and Occludin. In addition, it was discovered that Cr(VI) promoted cell apoptosis and inhibited AKT activation. However, the addition of a caspase inhibitor and an AKT activator prevented Cr(VI)-induced injury to the cell membrane barrier, indicating that apoptosis plays a crucial role in this process. The addition of three apoptotic pathway inhibitors, confirmed that Cr(VI) damaged the cell barrier through ROS-mediated mitochondrial pathway apoptosis. Moreover, the use of a ROS inhibitor significantly reduced Cr(VI)-induced apoptosis and cell barrier injury. In conclusion, this study provides an experimental foundation for the treatment of skin injury caused by Cr(VI).

DcR3 suppresses LPS-Induced Aggresome-like induced structures in Macrophages via inhibition of ROS and p38 MAPK

Abstract Decoy receptor 3 (DcR3), also known as tumor necrosis factor receptor (TNFR) superfamily member 6b (TNFRSF6B), is a pleiotropic factor which modulates cell functions via decoy and non-decoy actions. DcR3 has been shown its anti-apoptotic and anti-inflammatory effects on cancers and inflammatory diseases. Toll-like receptors (TLRs), which are the members of pattern recognition receptors (PRRs), trigger inflammatory response against invading pathogens. However, it remains unclear whether DcR3 plays a role in TLR4-induced inflammatory response. In this study, we demonstrated that in bone marrow-derived macrophages (BMDMs) DcR3 did not affect lipopolysaccharide (LPS)-induced COX-2, iNOS, NLRP3 and pro-IL-1b protein expression. It has been reported that aggresome-like induced structures (ALIS) are formed in response to LPS in macrophages. ALIS, which containing ubiquitinated protein aggregates, are stress-induced response involved in MHC class I antigen presentation. Our findings illustrated that DcR3 can decrease LPS-induced ALIS formation via inhibiting cellular ROS production and p38 MAPK phosphorylation. In addition to LPS, ZnPP-, bafilomycin A1- and MG132-induced ubiquitinated protein expression were reduced by DcR3. Confirming the notion that ALIS is controlled by autophagy, we found that rapamycin can decrease LPS-induced ALIS formation, while bafilomycin A1 can increase LC3-II accumulation with similar extent in WT and DcR3 macrophages. Nevertheless, DcR3 expression does not change p62 and HO-1 expression induced by LPS. Taken together, despite the fact that DcR3 does not alter LPS-induced inflammatory response, our data suggest a novel role of DcR3 in suppression of ALIS formation. MOST 111-2320-B-002-028

Environmental toxin chlorpyrifos induces liver injury by activating P53-mediated ferroptosis via GSDMD-mtROS

AimWe investigate the mechanism whereby chlorpyrifos (CHI), an environmental toxin, causes liver injury by inducing ferroptosis in hepatocytes. MethodsThe toxic dose (LD50 = 50 μM) of CHI for inducing AML12 injury in normal mouse hepatocytes was determined, and the ferroptosis-related indices were measured, including the levels of SOD, MDA and GSH-Px, as well as the cellular content of iron ions. JC-1 and DCFH-DA assays were employed to detect the mtROS levels, the levels of mitochondrial proteins (GSDMD, NT-GSDMD), as well as the cellular levels of ferroptosis-related proteins (P53, GPX4, MDM2, SLC7A11). We knocked out the GSDMD and P53 in AML12 and observed the CHI-induced ferroptosis of ALM12 after applying YGC063, an ROS inhibitor. In animal experiments, we explored the effect of CHI on liver injury by using conditional GSDMD-knockout mice (C57BL/6 N-GSDMDem1(flox)Cya) and ferroptosis inhibitor Fer-1. Small molecule-protein docking and Pull-down assay were employed to verify the association between CHI and GSDMD. ResultsWe found that CHI could induce ferroptosis of AML12. CHI promoted the cleavage of GSDMD, leading to upregulation of mitochondrial NT-GSDMD expression, as well as ROS levels. P53 activation promoted the ferroptosis. Knock out of GSDMD and P53 could inhibit the CHI-induced ferroptosis, and YGC063 could also inhibit ferroptosis. In mice experiments, GSDMD knockout or Fer-1 intervention could significantly inhibit the CHI-induced liver injury. CHI promoted the cleavage of GSDMD by binding to its SER234 site. ConclusionCHI can bind to GSDMD to promote its cleavage, while NT-GSDMD can open mitochondrial membrane to promote the mtROS release. Cytoplasmic upregulation of ROS levels can facilitate the P53-mediated ferroptosis. GSDMD-mtROS is the primary mechanism whereby CHI induces ferroptosis in hepatocytes.