Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Design and synthesis of visible light-activatable photocaged peroxides for optical control of ROS-mediated cellular signaling.

We designed and synthesized two novel photocaged peroxide compounds, N5TBHP and N6TBHP, featuring nitrogen-containing fused ring coumarin skeletons. Notably, a tetrahydroquinoline fused coumarin derivative, N6TBHP demonstrated significantly higher photocleavage efficiency under visible light at 455 nm compared to N5TBHP, which contains an indoline fused coumarin. This process effectively releases the oxidative stress inducer tert-butylhydroperoxide (TBHP). Additionally, N6TBHP exhibits high resistance to glutathione (GSH), and its UV spectral analysis suggests enhanced intracellular stability due to reduced reactivity with GSH through self-assembly. Furthermore, N6TBHP can release an optimal amount of TBHP into cells under visible light irradiation with minimal cell damage. These properties position N6TBHP as a promising tool for advancing research in intracellular redox signaling.

Biphasic dose-response and effects of near-infrared photobiomodulation on erythrocytes susceptibility to oxidative stress in vitro.

The effect of simultaneous application of tert-butyl hydroperoxide (tBHP) and polychromatic near-infrared (NIR) radiation on bovine blood was examined to determine whether NIR light decreases the susceptibility of red blood cells (RBCs) to oxidative stress. The study assessed various exposure methods, wavelength ranges, and optical filtering types. Continuous NIR exposure revealed a biphasic response in cell-free hemoglobin changes, with antioxidative effects observed at low fluences and detrimental effects at higher fluences. Optimal exposure duration was identified between 60 s and 15 min. Protective effects were also tested across wavelengths in the range of 750-1100 nm, with all of them reducing hemolysis, notably at 750 nm, 875 nm, and 900 nm. Comparing broadband NIR and far-red light (750 nm) showed no significant difference in hemolysis reduction. Pulse-dosed NIR irradiation allowed safe increases in radiation dose, effectively limiting hemolysis at higher doses where continuous exposure was harmful. These findings highlight NIR photobiomodulation's potential in protecting RBCs from oxidative stress and will be helpful in the effective design of novel medical therapeutic devices.

Baicalein attenuates oxidative damage in mice haematopoietic cells through regulation of PDGFRß.

Platelet-derived growth factor receptor ß (PDGFRß) plays a crucial role in murine haematopoiesis. Baicalein (BAI), a naturally occurring flavonoid, can alleviate disease damage through anti-oxidative, anti-apoptotic, and anti-inflammatory mechanisms. However, whether BAI attenuates oxidative damage in murine haematopoietic cells by PDGFRß remains unexplored. In this study, we utilized a tert-butyl hydroperoxide (TBHP)-induced BaF3 cell injury model and an ionising radiation (IR)-induced mice injury model to investigate the impact of the presence or absence of PDGFRß on the pharmacological effects of BAI. In addition, the BAI-PDGFRß interaction was characterized by molecular docking and dynamics simulations. The results show that a specific concentration of BAI led to increased cell viability, reduced reactive oxygen species (ROS) content, upregulated nuclear factor erythroid 2-related factor 2 (NRF2) expression, and its downstream target genes heme oxygenase 1 (HO-1) and NAD(P)H Quinone Dehydrogenase 1 (NQO1), and activated protein kinase B (AKT) pathway in cells expressing PDGFRß plasmid and experiencing damage. Similarly, BAI elevated lineage-Sca1+cKIT+ (LSK) cell proportion, promoted haematopoietic restoration, enhanced NRF2-mediated antioxidant response in PDGFRß+/+ mice. However, despite BAI usage, PDGFRß knockout mice (PDGFRß-/-) showed lower LSK proportion and less antioxidant capacity than the total body irradiation (TBI) group. Furthermore, we demonstrated an interaction between BAI and PDGFRß at the molecular level. Collectively, our results indicate that BAI attenuates oxidative stress injury and helps promote haematopoietic cell recovery through regulation of PDGFRß.

Dihydroethidium-derived fluorescence in electrically stressed cells indicates intracellular microenvironment modifications independent of ROS.

Intracellular reactive oxygen species (ROS) generation is widely suggested as a trigger for biological consequences of electric field exposures, such as those in electroporation applications. ROS are linked with membrane barrier function degradation, genetic damage, and complex events like immunological cell death. Dihydroethidium (DHE) is commonly used to monitor ROS in cells. DHE is linked to intracellular ROS by a primary oxidation product, Ethidium (Eth+), that shows increased fluorescence upon binding to polynucleotides. We observed changes in DHE-derived fluorescence in Chinese hamster ovary (CHO) cells post 300-ns electric pulse exposures, comparing them to tert-butyl-hydroperoxide (t-BHP) induced oxidative stress. Immediate intracellular fluorescence changes were noted in both cases, but with distinct localization patterns. After electrical stress, cytosolic DHE-derived fluorescence intensity decreases, and nucleolar intensity increases. Conversely, t-BHP exposure increases DHE-derived fluorescence uniformly across the cell. Surprisingly, fluorescence patterns after electrical stress in Eth+-loaded cells is identical to those in DHE-loaded cells, in kinetics and localization patterns. These findings indicate that DHE-derived fluorescence changes after pulsed electric field stress are not due to intracellular ROS generation leading to DHE oxidation, but rather indicate stress-induced intracellular microenvironment alterations affecting Eth+ fluorescence.

Antioxidant and anti-inflammatory function of Eupatorium adenophora Spreng leaves (EASL) on human intestinal Caco-2 cells treated with tert-butyl hydroperoxide.

Chronic non-communicable diseases (CNCDs) pose a significant public health challenge. Addressing this issue, there has been a notable breakthrough in the prevention and mitigation of NCDs through the use of antioxidants and anti-inflammatory agents. In this study, we aim to explore the effectiveness of Eupatorium adenophora Spreng leaves (EASL) as an antioxidant and anti-inflammatory agent, and its potential applications. To construct a cellular model of oxidative damage and inflammation, Caco-2 cells were treated with tert-butyl hydroperoxide (t-BHP). The biocompatibility of EASL-AE with Caco-2 cells was assessed using the MTT assay, while compatibility was further verified by measuring LDH release and the protective effect against oxidative damage was also assessed using the MTT assay. Additionally, we measured intracellular oxidative stress indicators such as ROS and 8-OHdG, as well as inflammatory pathway signalling protein NFκB and inflammatory factors TNF-α and IL-1ß using ELISA, to evaluate the antioxidant and anti-inflammatory capacity of EASL-AE. The scavenging capacity of EASL-AE against free radicals was determined through the DPPH Assay and ABTS Assay. Furthermore, we measured the total phenolic, total flavonoid, and total polysaccharide contents using common chemical methods. The chemical composition of EASL-AE was analyzed using the LC-MS/MS technique. Our findings demonstrate that EASL-AE is biocompatible with Caco-2 cells and non-toxic at experimental levels. Moreover, EASL-AE exhibits a significant protective effect on Caco-2 cells subjected to oxidative damage. The antioxidant effect of EASL-AE involves the scavenging of intracellular ROS, while its anti-inflammatory effect is achieved by down-regulation of the NFκB pathway. Which in turn reduces the release of inflammatory factors TNF-α and IL-1ß. Through LC-MS/MS analysis, we identified 222 compounds in EASL-AE, among which gentianic acid, procaine and L-tyrosine were the compounds with high antioxidant capacity and may be the effective constituent for EASL-AE with antioxidant activity. These results suggest that EASL-AE is a natural and high-quality antioxidant and anti-inflammatory biomaterial that warrants further investigation. It holds great potential for applications in healthcare and other related fields.

HSP70 protects PC12 cells against TBHP-induced apoptosis and oxidative stress by activating the Nrf2/HO-1 signaling pathway.

HSP70 exhibits neuroprotective, antioxidant, and anti-apoptotic properties, which are crucial in preventing spinal cord injury (SCI) induced by oxidative stress and apoptosis. In this study, we assessed the potential protective effects and underlying mechanisms of HSP70 on tert-butyl hydroperoxide (TBHP)-damaged PC12 cells in an in vitro model of SCI. To establish the model, PC12 cells were subjected to oxidative damage induced by TBHP, followed by overexpression of HSP70. Cell viability was assessed using the CCK8 kit, intracellular reactive oxygen species level was evaluated using a commercial kit, cell apoptosis was detected using the Annexin V-APC/7-ADD Apoptosis Detection Kit, and the oxidative stress level was determined using SOD and MDA assay kits. Western blot analysis was used to detect the expression levels of Bax, cleaved caspase-3, and Bcl-2 proteins. Furthermore, immunofluorescence staining and Western bolt were used to detect the expression levels of proteins associated with the Nrf2/HO-1 signaling pathway. We found that HSP70 overexpression reduced apoptosis and oxidative stress in TBHP-induced PC12 cells. Furthermore, it activated the Nrf2/HO-1 signaling pathway. In addition, the Nrf2 inhibitor ML385 attenuated the protective effects of HSP70 on TBHP-induced PC12 cells. In conclusion, HSP70 can partially alleviate TBHP-induced apoptosis and oxidative stress in PC12 cells by promoting the Nrf2/HO-1 signaling pathway.

Quantifying redox transcription factor dynamics as a tool to investigate redox signalling.

A critical feature of the cellular antioxidant response is the induction of gene expression by redox-sensitive transcription factors. In many cells, activating these transcription factors is a dynamic process involving multiple redox steps, but it is unclear how these dynamics should be measured. Here, we show how the dynamic profile of the Schizosaccharomyces pombe Pap1 transcription factor is quantifiable by three parameters: signal amplitude, signal time and signal duration. In response to increasing hydrogen peroxide concentrations, the Pap1 amplitude decreased while the signal time and duration showed saturable increases. In co-response plots, these parameters showed a complex, non-linear relationship to the mRNA levels of four Pap1-regulated genes. We also demonstrate that hydrogen peroxide and tert-butyl hydroperoxide trigger quantifiably distinct Pap1 activation profiles and transcriptional responses. Based on these findings, we propose that different oxidants and oxidant concentrations modulate the Pap1 dynamic profile, leading to specific transcriptional responses. We further show how the effect of combination and pre-exposure stresses on Pap1 activation dynamics can be quantified using this approach. This method is therefore a valuable addition to the redox signalling toolbox that may illuminate the role of dynamics in determining appropriate responses to oxidative stress.

Dimethylarginine Dimethylaminohydrolase - 1 expression is increased under tBHP-induced oxidative stress regulates nitric oxide production in PCa cells attenuates mitochondrial ROS-mediated apoptosis.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression is frequently elevated in different cancers including prostate cancer (PCa) and enhances nitric oxide (NO) production in tumor cells by metabolising endogenous nitric oxide synthase (NOS) inhibitors. DDAH1 protects the PCa cells from cell death and promotes survival. In this study, we have investigated the cytoprotective role of DDAH1 and determined the mechanism of DDAH1 in protecting the cells in tumor microenvironment. Proteomic analysis of PCa cells with stable overexpression of DDAH1 has identified that oxidative stress-related activity is altered. Oxidative stress promotes cancer cell proliferation, survival and causes chemoresistance. A known inducer of oxidative stress, tert-Butyl Hydroperoxide (tBHP) treatment to PCa cells led to elevated DDAH1 level that is actively involved in protecting the PCa cells from oxidative stress induced cell damage. In PC3-DDAH1- cells, tBHP treatment led to higher mROS levels indicating that the loss of DDAH1 increases the oxidative stress and eventually leads to cell death. Under oxidative stress, nuclear Nrf2 controlled by SIRT1 positively regulates DDAH1 expression in PC3 cells. In PC3-DDAH1+ cells, tBHP induced DNA damage is well tolerated compared to wild-type cells while PC3-DDAH1- became sensitive to tBHP. In PC3 cells, tBHPexposure has increased the production of NO and GSH which may be acting as an antioxidant defence to overcome oxidative stress. Furthermore, in tBHP treated PCa cells, DDAH1 is controlling the expression of Bcl2, active PARP and caspase 3. Taken together, these results confirm that DDAH1 is involved in the antioxidant defence system and promotes cell survival.

Treatment of porcine ovarian follicles with tert-butyl hydroperoxide as an ovarian senescence model in vitro.

Ovarian aging is the main reason of female reproductive problems. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing the reproductive performance. Follicles were divided into five groups for in vitro culture based on the duration of stimulation with tert-butyl hydroperoxide (t-BHP)-control group and groups 1 h, 2 h, 6 h, and 12 h. The results revealed that the ratio of progesterone (P4) to estradiol (E2) was increased after 24 and 36 h of follicle culture, shifting follicles toward atresia (P < 0.05). Stimulated by 200 µM t-BHP, follicles showed progressive aging phenotype. Senescence-associated ß-galactosidase staining (SA-ß-Gal) showed a significant increase in the number of positive cells (P < 0.05). Reactive oxygen species were also significantly upregulated (P < 0.05). t-BHP treatment for 6 h induced significant increases in Caspase 3, P53, and Foxo1 mRNA and protein levels (P < 0.05) and significant decreases in SOD mRNA and protein levels (P < 0.05). Transcriptome sequencing analysis of the follicles showed that the aged and treatment groups were clustered together in hierarchical clustering. Correlation analysis indicated significant changes at the transcriptome level in the treatment groups versus the control group. The common differentially expressed genes in the treatment groups were enriched in three growth-factor signaling pathways associated with cell proliferation and apoptosis (P53, mTOR, and MAPK). In conclusion, induction of follicular senescence by treatment with 200 µM t-BHP for 6 h is an effective in vitro model to simulate ovarian senescence in sows.

Synthesis and photochemical properties of caged peroxides for photocontrol of cellular oxidative stress.

We developed a caged hydroperoxide, BhcTBHP, releasing prooxidant TBHP under blue light irradiation. MitoTBHP with triphenylphosphonium at position 7 triggered selective oxidative stress and membrane depolarization in mitochondria upon photoirradiation. This study presents a powerful tool for studying redox signaling and oxidative stress in living cells.

Ginkgo biloba extracts protect human retinal Müller glial cells from t-BHP induced oxidative damage by activating the AMPK-Nrf2-NQO-1 axis.

OBJECTIVES: Retinal Müller glial cell loss is almost involved in all retinal diseases, especially diabetic retinopathy (DR). Oxidative stress significantly contributes to the development of Müller glial cell loss. Ginkgo biloba extracts (GBE) have been reported to possess antioxidant property, beneficial in treating human retinal diseases. However, little is known about its role in Müller glial cells. This study investigated the protective effect of GBE (prepared from ginkgo biloba dropping pills) in human Müller glial cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress and its underlying molecular mechanism. METHODS: MIO-M1 cells were pretreated with or without GBE prior to the exposure to t-BHP-induced oxidative stress. Cell viability, cell death profile and lipid peroxidation were subsequently assessed. Protein expression of the key anti-oxidative signalling factors were investigated. KEY FINDINGS: We showed that GBE can effectively protect human MIO-M1 cells from t-BHP-induced oxidative injury by improving cell viability, reducing intracellular ROS accumulation and suppressing lipid peroxidation, which effect is likely mediated through activating AMPK-Nrf2-NQO-1 antioxidant respondent axis. CONCLUSIONS: Our study is the first to reveal the great potentials of GBE in protecting human retinal Müller glial cell loss against oxidative stress. GBE might be used to prevent human retinal diseases particularly DR.

Hydrogen sulfide protects retinal pigment epithelium cells against ferroptosis through the AMPK- and p62-dependent non-canonical NRF2-KEAP1 pathway.

Oxidative stress-induced ferroptosis of retinal pigment epithelium (RPE) cells contributes to retinal degenerative diseases. The antioxidant molecule hydrogen sulfide (H2S) regulates oxidative stress response, but its effect on the ferroptosis of RPE cells is unclear. In this study, sodium hydrosulfide (NaHS) was used as an exogenous H2S donor to intervene tert-butyl hydroperoxide (t-BHP)-induced ferroptosis of APRE-19 cells. We found that NaHS pretreatment attenuates t-BHP-induced oxidative stress and ferroptosis. Analysis of mRNA-sequencing coupled with FerrDb database identified nuclear factor erythroid-2-related factor 2 (NRF2) as a primary target for the cytoprotective role of H2S. NRF2 inhibitor ML385 reverses the effects of H2S on ferroptosis. Biochemical analysis revealed that H2S stabilizes NRF2. H2S decreases the interaction between NRF2 and KEAP1, but enhances the interaction between KEAP1 and p62. These results suggest that H2S activates the non-canonical NRF2-KEAP1 pathway. Further study demonstrated that H2S stimulates AMPK to interact and phosphorylate p62. Additionally, inhibiting AMPK or knocking down p62 blocks the effects of H2S. We speculate that targeting the non-canonical NRF2-KEAP1 pathway by H2S-based drug may benefit the treatment of retinal degenerative diseases.

Chantriolides F-P, Highly Oxidized Withanolides with Hepatoprotective Activity from Tacca chantrieri.

Eleven highly oxidized withanolides, chantriolides F-P (1-11), together with six known analogues (12-17), were isolated from the rhizomes of Tacca chantrieri. Their structures were established on the basis of comprehensive spectroscopic data analysis and comparison with published NMR data, and their absolute configurations were further confirmed by experimental ECD data and single crystal X-ray diffraction analysis. The structures of compounds 5-8 contained a chlorine atom substituted at C-3. Compounds 1 and 12 are a pair of epimers isomerized at C-24 and C-25, while compounds 9 and 16 are isomerized at C-1, C-7, C-24, and C-25. Next, the hepatoprotective effect of all the isolates was evaluated on tert-butyl hydroperoxide (t-BHP)-injured AML12 hepatocytes. Compounds 5-11 and 16 significantly enhanced cell viability. Compound 8 decreased reactive oxygen species accumulation and increased glutathione level in t-BHP injured AML12 hepatocytes through promoting nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2).

Diphenyl Diselenide Alleviates Tert-Butyl Hydrogen Peroxide-Induced Oxidative Stress and Lipopolysaccharide-Induced Inflammation in Rat Glomerular Mesangial Cells.

Hyperglycemia, oxidative stress, and inflammation play key roles in the onset and development of diabetic complications such as diabetic nephropathy (DN). Diphenyl diselenide (DPDS) is a stable and simple organic selenium compound with anti-hyperglycemic, anti-inflammatory, and anti-oxidative activities. Nevertheless, in vitro, the role and molecular mechanism of DPDS on DN remains unknown. Therefore, we investigated the effects of DPDS on tert-butyl hydrogen peroxide (t-BHP)-induced oxidative stress and lipopolysaccharide (LPS)-induced inflammation in rat glomerular mesangial (HBZY-1) cells and explored the underlying mechanisms. DPDS attenuated t-BHP-induced cytotoxicity, concurrent with decreased intracellular ROS and MDA contents and increased SOD activity and GSH content. Moreover, DPDS augmented the protein and mRNA expression of Nrf2, HO-1, NQO1, and GCLC in t-BHP-stimulated HBZY-1 cells. In addition, DPDS suppressed LPS-induced elevations of intracellular content and mRNA expression of interleukin (IL)-6, IL-1ß and TNF-α. Furthermore, LPS-induced NFκB activation and high phosphorylation of JNK and ERK1/2 were markedly suppressed by DPDS in HBZY-1 cells. In summary, these data demonstrated that DPDS improves t-BHP-induced oxidative stress by activating the Nrf2/Keap1 pathway, and also improves LPS-induced inflammation via inhibition of the NFκB/MAPK pathways in HBZY-1 cells, suggesting that DPDS has the potential to be developed as a candidate for the prevention and treatment of DN.

Single cell RNA-seq analysis identifies ferroptotic chondrocyte cluster and reveals TRPV1 as an anti-ferroptotic target in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease primarily characterized by cartilage destruction. The aim of this study was to investigate the role, molecular characteristics and potential therapeutic target of chondrocyte ferroptosis in the pathogenesis of OA. METHODS: The expression of ferroptotic hallmarks (iron and lipid peroxidation accumulation, glutathione deletion) were analyzed in paired intact and damaged cartilages from OA patients. Single cell RNA sequencing (scRNA-seq) analysis was performed on 17,638 chondrocytes to verify the presence, investigate the molecular signatures and unveil the potential therapeutic target of ferroptotic chondrocyte cluster in human OA cartilages. Destabilization of medial meniscus (DMM)-induced OA model and tert-butyl hydroperoxide (TBHP)-treated primary mouse chondrocytes and human cartilage explants were used to evaluate the protective effect of pharmacologically activated transient receptor potential vanilloid 1 (TRPV1). The downstream molecular mechanisms of TRPV1 was further investigated in glutathione peroxidase 4 (Gpx4) heterozygous genetic deletion mice (Gpx4+/-). FINDINGS: The concentrations of iron and lipid peroxidation and the expression of ferroptotic drivers in the damaged areas of human OA cartilages were significantly higher than those in the intact cartilage. scRNA-seq analysis revealed a chondrocyte cluster characterized by preferentially expressed ferroptotic hallmarks and genes, namely ferroptotic chondrocyte cluster. Comprehensive gene set variation analysis revealed TRPV1 as an anti-ferroptotic target in human OA cartilage. Pharmacological activation of TRPV1 significantly abrogated cartilage degeneration by protecting chondrocytes from ferroptosis. Mechanistically, TRPV1 promoted the expression of GPX4, and its anti-ferroptotic role was largely mitigated in the OA model of Gpx4+/- mice. INTERPRETATION: TRPV1 activation protects chondrocytes from ferroptosis and ameliorates OA progression by upregulating GPX4. FUNDING: National Key R&D Program of China (2018YFC1105904), Key Program of NSFC (81730067), National Science Foundation of China (81772335, 81941009, 81802196), Natural Science Foundation of Jiangsu Province, China (BK20180127), Jiangsu Provincial Key Medical Talent Foundation, Six Talent Peaks Project of Jiangsu Province (WSW-079).

Safranal Treatment Induces Sirt1 Expression and Inhibits Endoplasmic Reticulum Stress in Mouse Chondrocytes and Alleviates Osteoarthritis Progression in a Mouse Model.

Osteoarthritis (OA) is an age-related degenerative disease. Oxidative stress (OS) modulates OA pathogenesis by enhancing chondrocyte apoptosis and extracellular matrix (ECM) degeneration via activation of the endoplasmic reticulum (ER) stress. Prior studies revealed that safranal plays a critical role in multiple diseases treatments, but there are no reports on its effect on OA. Therefore, investigating the effect of safranal on OA is needed. As a compound that can lead excessive reactive oxygen species (ROS) accumulation, tert-butyl hydroperoxide (TBHP) was used to induce OS and OS-mediated endoplasmic reticulum (ER) stress for imitating OA in vitro. Besides, the bilateral medial meniscus was removed to induce joint instability and excessive friction of the joint surface to establish destabilization of medial meniscus for imitating the initiation and progression of OA in vivo. We, next, conducted Western blot and RT-PCR analyses to identify biomarkers of the underlying signaling pathway. Our results demonstrated that 30 µM safranal strongly upregulated Sirt1 expression, suppressed TBHP-mediated ER stress, and, in turn, prevented chondrocyte apoptosis and ECM degeneration. Furthermore, compared with the other two classic signaling pathways of ER stress, safranal can inhibit the PERK-eIF2α-CHOP axis at the lower concentration (5 and 15 µM). In vivo, using Safranin O staining, X-ray, immunofluorescence (IF), and immunohistochemical (IHC) staining, we demonstrated that OA progression can be postponed with intraperitoneal injection of 90 and 180 mg/kg safranal in an OA mouse model. Taken together, our analyses revealed that safranal can potentially prevent OA development.

Persistent red blood cells retain their ability to move in microcapillaries under high levels of oxidative stress.

Oxidative stress is one of the key factors that leads to red blood cells (RBCs) aging, and impairs their biomechanics and oxygen delivery. It occurs during numerous pathological processes and causes anaemia, one of the most frequent side effects of cancer chemotherapy. Here, we used microfluidics to simulate the microcirculation of RBCs under oxidative stress induced by tert-Butyl hydroperoxide. Oxidative stress was expected to make RBCs more rigid, which would lead to decrease their transit velocity in microfluidic channels. However, single-cell tracking combined with cytological and AFM studies reveals cell heterogeneity, which increases with the level of oxidative stress. The data indicates that the built-in antioxidant defence system has a limit exceeding which haemoglobin oxidation, membrane, and cytoskeleton transformation occurs. It leads to cell swelling, increased stiffness and adhesion, resulting in a decrease in the transit velocity in microcapillaries. However, even at high levels of oxidative stress, there are persistent cells in the population with an undisturbed biophysical phenotype that retain the ability to move in microcapillaries. Developed microfluidic analysis can be used to determine RBCs' antioxidant capacity for the minimization of anaemia during cancer chemotherapy.

Synthesis and in†Vivo Evaluation of Triphenylphosphonium Conjugated Trimetazidine with Enhanced Cardioprotection and Ability to Restore Mitochondrial Function.

Trimetazidine exhibits great therapeutic potential in cardiovascular diseases and mitochondria-mediated cardioprotection by trimetazidine has been widely reported. In this study, to enhance its cardioprotection, the triphenylphosphonium-based modification of trimetazidine was conducted to deliver it specifically to mitochondria. Fifteen triphenylphosphonium (TPP) conjugated trimetazidine analogs were designed and synthesized. Their protective effects were evaluated in vivo using a tert-butyl hydroperoxide (t-BHP) induced zebrafish injury model. Structure-activity relationship correlations revealed the best way to couple the TPP moiety to trimetazidine, and led to a new conjugate (18a) with enhanced therapeutic properties. Compared to trimetazidine, 18a effectively protects against heart injury in the zebrafish model at a much lower concentration. Further study in t-BHP treated zebrafish and H9c2 cells demonstrated that 18a protects against cardiomyocyte death and damage by inhibiting excessive production of ROS, maintaining mitochondrial morphology, and preventing mitochondrial dysfunction. Consequently, 18a can be regarded as a potential therapeutic agent for cardioprotection.

Hepatoprotective effect of Sophora moorcroftiana (Benth.) Benth.Ex baker seeds in vivo and in vitro.

The leguminosae of Sophora moorcroftiana (Benth.) Benth.ex Baker is a drought-resistant endemic Sophora shrub species from the Qinghai-Tibet Plateau, and its seeds have hepatoprotective effects. To study the effect of S. moorcroftiana seeds on liver injury and the molecular mechanism underlying the beneficial effects, liquid chromatography-mass spectrometry was used to detect the main active components in the ethanol extract of S. moorcroftiana seeds (SM). Male mice were divided into six groups (n = 8): normal control (NC), CCl4, SM (50, 100, 200 mg/kg), and dimethyl diphenyl bicarboxylate (150 mg/kg) groups. Mice were treated as indicated (once/day, orally) for 14 days, and CCl4 (2 mL/kg) was administered intraperitoneally. The serum and liver of mice were used for biochemical assays. To explore the underlying mechanism, HepG2 cells were treated with SM, stimulated with tert-butyl hydroperoxide (t-BHP, 50 µM), and analyzed by Western blotting. The major active compounds of SM were alkaloids including 22 compounds. Serum alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) decreased in the SM (200 mg/kg) group. SM can activate the expression of pregnane X receptor (PXR) and downstream molecules cytochrome P4503A11 enzyme (CYP3A11), UDP glucuronosyltransferase 1 family polypeptide A 1 (UGT1A1), and inhibit the multidrug resistance protein 2 (MRP2). In addition, SM improved cell viability in t-BHP-induced HepG2 cells (64% to 83%) and decreased the activation of the mitogen-activated protein kinase (MAPK) pathway. The main compounds in SM were alkaloids. SM showed hepatoprotective effects possibly mediated by the suppression of oxidative stress through the MAPK pathway.