Exosomes generated from mesenchymal stem cells (MSCs) are thought to be a unique therapeutic strategy for several autoimmune deficiency illnesses. The purpose of this study was to elucidate the protective effects of human umbilical cord mesenchymal stem cell -derived exosomes (hUCMSC-Exo) on CD4 + T cells dysfunction during graft -versus -host disease (GVHD) and to identify the underlying processes involved. Here, we showed that hUCMSC-Exo treatment can effectively attenuate GVHD injury by alleviating redox metabolism disorders and inflammatory cytokine bursts in CD4 + T cells. Furthermore, hUCMSC-Exo ameliorate ER stress and ATF6/CHOP signaling -mediated apoptosis in CD4 + T cells and promote the development of CD4 + IL-10 + T cells during GVHD. Moreover, downregulating miR-16-5p in hUCMSC-Exo impaired their ability to prevent CD4 + T cells apoptosis and weakened their ability to promote the differentiation of CD4 + IL-10 + T cells. Collectively, the obtained data suggested that hUCMSC-Exo suppress ATF6/CHOP signaling -mediated ER stress and apoptosis in CD4 + T cells, enhance the differentiation of CD4 + IL-10 + T cells, and reverse the imbalance of immune homeostasis in the GVHD process by transferring miR-16-5p. Our study provided further evidence that GVHD patients can benefit from hUCMSC-Exo-mediated therapy.

High-altitude hypoxia may disturb the metabolic modulation and function of both adipose tissue and liver. The endoplasmic reticulum (ER) is a crucial organelle in lipid metabolism and ER stress is closely correlated with lipid metabolism dysfunction. The aim of this study is to elucidate whether the inhibition of ER stress could alleviate hypoxia-induced white adipose tissue (WAT) lipolysis and liver lipid accumulation-mediated hepatic injury. A rat model of high-altitude hypoxia (5500 m) was established using hypobaric chamber. The response of ER stress and lipolysis-related pathways were analyzed in WAT under hypoxia exposure with or without 4-phenylbutyric acid (PBA) treatment. Liver lipid accumulation, liver injury, and apoptosis were evaluated. Hypoxia evoked significant ER stress in WAT, evidenced by increased GRP78, CHOP, and phosphorylation of IRE1 alpha, PERK. Moreover, Lipolysis in perirenal WAT significantly increased under hypoxia, accompanied with increased phosphorylation of hormone-sensitive lipase (HSL) and perilipin. Treatment with 4-PBA, inhibitor of ER stress, effectively attenuated hypoxia-induced lipolysis via cAMP-PKA-HSL/perilipin pathway. In addition, 4-PBA treatment significantly inhibited the increase in fatty acid transporters (CD36, FABP1, FABP4) and ameliorated liver FFA accumulation. 4-PBA treatment significantly attenuated liver injury and apoptosis, which is likely resulting from decreased liver lipid accumulation. Our results highlight the importance of ER stress in hypoxia-induced WAT lipolysis and liver lipid accumulation.

Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Hydroxysafflor yellow A (HSYA), a natural compound isolated from Carthamus tinctorius L., has been found to possess anti-inflammatory and antioxidant properties. However, the protective effects and potential mechanism of HSYA on I/R-induced AKI remains unclear. In the present study, the in vitro hypoxia/reoxygenation (H/R) and in vivo renal I/R models were employed to investigate the renal protective effects and molecular mechanisms of HSYA on I/R-induced AKI. The present results indicated that HSYA pretreatment significantly ameliorated renal damage and dysfunction in the I/R injury mice via enhancing the antioxidant capacity and suppressing the oxidative stress injury, inflammatory response, and apoptosis. Mechanistic studies showed that HSYA could upregulate Akt/GSK-3β/Fyn-Nrf2 axis-mediated antioxidant gene expression both in vitro and in vivo. Moreover, HSYA-mediated improvement in antioxidant, anti-inflammatory, and anti-apoptotic effects in H/R-treated HK-2 cells was abrogated by Akt inhibitor LY294002 supplementation. In summary, the present results demonstrated that HSYA attenuated kidney oxidative stress, inflammation response, and apoptosis induced by I/R, at least in part, via activating the Akt/GSK-3β/Fyn-Nrf2 axis pathway. These findings provided evidence that HSYA may be applied as a potential therapeutic agent in the treatment of I/R induced AKI.

Mesenchymal stem cells (MSCs)-derived exosomes were considered a novel therapeutic approach in many aging-related diseases. This study aimed to clarify the protective effects of human placenta MSCs-derived exosomes (hPMSC-Exo) in aging-related CD4+ T cell senescence and identified the underlying mechanisms using a D-gal induced mouse aging model. Senescent T cells were detected SA-β-gal stain. The degree of DNA damage was evaluated by detecting the level of 8-OH-dG. The superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities were measured. The expression of aging-related proteins and senescence-associated secretory phenotype (SASP) were detected by Western blot and RT-PCR. We found that hPMSC-Exo treatment markedly decreased oxidative stress damage (ROS and 8-OH-dG), SA-β-gal positive cell number, aging-related protein expression (p53 and γ-H2AX), and SASP expression (IL-6 and OPN) in senescent CD4+ T cells. Additionally, hPMSC-Exo containing miR-21 effectively downregulated the expression of PTEN, increased p-PI3K and p-AKT expression, and Nrf2 nuclear translocation and the expression of downstream target genes (NQO1 and HO-1) in senescent CD4+ T cells. Furthermore, in vitro studies uncovered that hPMSC-Exo attenuated CD4+ T cell senescence by improving the PTEN/PI3K-Nrf2 axis by using the PTEN inhibitor bpV (HOpic). We also validated that PTEN was a target of miR-21 by using a luciferase reporter assay. Collectively, the obtained results suggested that hPMSC-Exo attenuates CD4+ T cells senescence via carrying miRNA-21 and activating PTEN/PI3K-Nrf2 axis mediated exogenous antioxidant defenses.

BACKGROUND:Recently, nanocatalyst-induced endoplasmic reticulum (ER) stress for cancer therapy has been attracting considerable attention. However, cancer cells are often able to overcome ER stress-induced death by activating the unfolded protein response (UPR), making nanocatalytic monotherapy a poor defense against cancer progression.PURPOSE:In this study, to improve the nanocatalytic treatment efficacy, a phase change material (PCM) was used to encapsulate the upstream ER stress initiator, iron oxide nanoparticles (Fe3O4 NPs), and the downstream UPR modulator, PR-619. Subsequently, the tumor-homing peptide tLyP-1 was coupled to it to form tLyP-1/PR-619/Fe3O4@PCM (tPF@PCM) theranostic platform.MATERIALS AND METHODS:tPF@PCM was synthesized using nanoprecipitation and resolidification methods followed by the EDC/NHS cross-linking method. The targeting capacity of tPF@PCM was evaluated in vitro and in vivo using flow cytometry and magnetic resonance imaging, respectively. The therapeutic efficacy of tPF@PCM was investigated in a renal cell carcinoma mouse model. Moreover, we explored the synergistic anti-tumor mechanism by examining the intracellular reactive oxygen species (ROS), aggregated proteins, ER stress response levels, and type of cell death.RESULTS:tPF@PCM had excellent tumor-targeting properties and exhibited satisfactory photothermal-enhanced tumor inhibition efficacy both in vitro and in vivo. Specifically, the phase transition temperature (45 °C) maintained using 808 nm laser irradiation significantly increased the release and catalytic activity of the peroxidase mimic Fe3O4 NPs. This strongly catalyzed the generation of hydroxyl radicals (•OH) via the Fenton reaction in the acidic tumor microenvironment. The redox imbalance subsequently resulted in an increase in the level of damaged proteins in the ER and initiated ER stress. Moreover, the pan-deubiquitinase inhibitor PR-619 blocked the "adaptive" UPR-mediated degradation of these damaged proteins, exacerbating the ER burden. Consequently, irremediable ER stress activated the "terminal" UPR, leading to apoptosis in cancer cells.CONCLUSION:This ER stress-exacerbating strategy effectively suppresses tumorigenesis, offering novel directions for advances in the treatment of conventional therapy-resistant cancers.

AbstractHigh‐altitude hypoxia‐induced oxidative stress and inflammation played an essential role in the incidence and development of liver injury. Salidroside (Sal), a phenylpropanoid glycoside extracted from the plant Rhodiola rosea, has recently demonstrated antioxidant, anti‐inflammatory, and antihypoxia properties. Herein, we hypothesized that salidroside may alleviate hypoxia‐induced liver injury via antioxidant and antiinflammatory‐related pathways. A high‐altitude hypoxia animal model was established using hypobaric chamber. Male SD rats were randomly divided into the control group, hypoxia group, control +Sal group, and hypoxia +Sal group. Salidroside treatment significantly inhibited hypoxia‐induced increases of serum and hepatic pro‐inflammatory cytokines release, hepatic ROS production and MDA contents; attenuated hypoxia‐induced decrease of hepatic SOD, CAT, and GSH‐Px activities. Furthermore, salidroside treatment also potentiated the activation of Nrf2‐mediated anti‐oxidant pathway, as indicated by upregulation of n‐Nrf2 and its downstream HO‐1 and NQO‐1. In vitro study found that blocking the Nrf2 pathway using specific inhibitor ML385 significantly reversed the protective effect of salidroside on hypoxia‐induced liver oxidative stress. In addition, salidroside treatment significantly inhibited hepatic pro‐inflammatory cytokines release via JAK2/STAT3‐mediated pathway. Taken together, our findings suggested that salidroside protected against hypoxia‐induced hepatic oxidative stress and inflammation via Nrf2 and JAK2/STAT3 signaling pathways.

Red blood cells (RBCs) are susceptible to sustained free radical damage during circulation, while the changes of antioxidant capacity and regulatory mechanism of RBCs under different oxygen gradients remain unclear. Here, we investigated the changes of oxidative damage and antioxidant capacity of RBCs in different oxygen gradients and identified the underlying mechanisms using an in vitro model of the hypoxanthine/xanthine oxidase (HX/XO) system. In the present study, we reported that the hypoxic RBCs showed much higher oxidative stress injury and lower antioxidant capacity compared with normoxic RBCs. In addition, we found that the disturbance of the recycling process, but not de novo synthesis of glutathione (GSH), accounted for the significantly decreased antioxidant capacity of hypoxic RBCs compared to normoxic RBCs. We further elucidated the underlying molecular mechanism by which oxidative phosphorylation of Band 3 blocked the hexose monophosphate pathway (HMP) and decreased NADPH production aggravating the dysfunction of GSH synthesis in hypoxic RBCs under oxidative conditions.

Background: Mesenchymal stem cells (MSCs) was considered as regenerative therapeutic approach in both acute and chronic diseases. However, whether MSCs regulate the antioxidant metabolism of CD4 + T cells and weaken immunosenescence remains unclear. Here, we reported the protective effects of hPMSCs in aging-related CD4 + T cell senescence and identified the underlying mechanisms using a D-gal induced mouse aging model. Methods: In vivo study, 40 male C57BL/6 mice (8 weeks) were randomly divided into four groups: control group, D-gal group, hPMSC group and PBS group. In in vitro experiment, human naive CD4 + T (CD4CD45RA) cells were prepared using a naive CD4 + T cell isolation kit II and pretreated with the Akt inhibitor LY294002 and Nrf2 inhibitor ML385. Then, isolated naive CD4 + T cell were cocultured with hPMSCs for 72 h in the absence or presence of anti-CD3/CD28 Dynabeads and IL-2 as a mitogenic stimulus. Intracellular ROS changes were detected by flow cytometry. The activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase were measured by colorimetric analysis. The senescent T cells were detected SA-β-gal stain. The expression of aging related proteins were detected by Western blotting, RT-PCR and confocal microscopy. Results: We found that hPMSC treatment markedly decreased the ROS level, SA-β-gal positive cells number, senescence-associated secretory phenotype (IL-6 and OPN) expression and aging-related protein (P16 and P21) expression in senescent CD4 + T cells. Furthermore, hPMSC treatment effectively upregulated Nrf2 nuclear translocation and the expression of downstream target genes (HO-1, CAT, GCLC and NQO1) in senescent CD4 + T cells. Moreover, in vitro studies revealed that hPMSCs attenuated CD4 + T cell senescence by upregulating the Akt/GSK-3β/Fyn pathway to activate Nrf2 functions. Conversely, the antioxidant effects of hPMSCs were blocked by the Akt inhibitor LY294002 and Nrf2 inhibitor ML385 in senescent CD4 + T cells. Conclusions: Our results indicate that hPMSCs attenuate D-gal induced CD4 + T cell senescence by activating Nrf2-mediated antioxidant defenses and that upregulation of Nrf2 by hPMSCs is regulated via the Akt/GSK-3β/Fyn pathway.

Chitosan oligosaccharide (COS) is the depolymerized product of chitosan possessing various biological activities and protective effects against inflammation and oxidative injury. The aim of the present study was to investigate the antioxidant effects of COS supplements on aging-related liver dysfunction. We found that COS treatment significantly attenuated elevated liver function biomarkers and oxidative stress biomarkers and decreased antioxidative enzyme activities in liver tissues in D-galactose (D-gal)-treated mice. Furthermore, COS treatment significantly upregulated the expression of Nrf2 and its downstream target genes HO-1, NQO1, and CAT. Moreover, in vitro experiments showed that COS treatment played a vital role in protecting H2O2-exposed L02 cells against oxidative stress by activating Nrf2 antioxidant signaling. These data indicate that COS could protect against D-gal-induced hepatic aging by activating Nrf2 antioxidant signaling, which may provide novel applications for the prevention and treatment of aging-related hepatic dysfunction.

Endoplasmic reticulum (ER) stress and subsequent apoptosis played vital role in liver injury and dysfunction. The aim of this study was to investigate the protective effect and mechanism of salidroside on hypoxia induced liver injury both in vivo and in vitro. Male SD rats were exposed to hypobaric chamber to simulate high altitude hypoxia model. High altitude hypoxia led to significant liver injury and apoptosis, increased the expression levels of p-JNK, BAX and ER stress markers. Salidroside treatment significantly inhibited hypoxia induced ER stress by decreasing the protein expression of glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated inositol-requiring enzyme 1α (p-IRE1α). In addition, salidroside treatment also restrained the ER stress-mediated apoptotic pathway, as indicated by decreased pro-apoptotic proteins p-JNK, TRAF2, BAX, and cleaved caspase 9 and caspase 12, as well as upregulation of Bcl-2. Furthermore, in vitro study found that blocking IRE1α pathway using specific inhibitor STF-083010 subsequently reversed the protective effect of salidroside on liver apoptosis. Taken together, our findings revealed that salidroside exerts protective effects against hypoxia induced liver injury through inhibiting ER stress mediated apoptosis via IRE1α/JNK pathway.

The aim of the present study was to explore the effect of exhaustive running exercise in the oxygen release capacity of rat erythrocytes. Rats were divided into sedentary control, moderate running exercise, and exhaustive running exercise groups. The thermodynamic and kinetic properties of the erythrocyte oxygen release process of the different groups were tested. We also determined the degree of band-3 oxidation and phosphorylation, anion transport activity, and carbonic anhydrase isoform II activity. Biochemical studies suggested that exhaustive running significantly increased oxidative injury parameters in thiobarbituric acid reactive substances and methaemoglobin levels. Furthermore, exhaustive running significantly decreased anion transport activity and carbonic anhydrase isoform II activity. Thermodynamic analysis indicated that erythrocytes oxygen release ability also significantly increased due to elevated 2,3-DPG level after exhaustive running. Kinetic analysis indicated that exhaustive running resulted in significantly decreased T-50 value. We presented evidence that exhaustive running remarkably impacted thermodynamic and kinetic properties of RBC oxygen release. In addition, changes in 2,3-DPG levels and band-3 oxidation and phosphorylation could be the driving force for exhaustive-running-induced alterations in erythrocyte oxygen release thermodynamic and kinetic properties.

Background/Aims: Although red blood cells (RBCs) transfusions can be lifesaving, they are not without risk. RBCs storage is associated with the abnormal metabolism of glutathione (GSH), which may increase the risk of the oxidative damage of RBCs after transfusion. The responsible mechanisms remain unknown. Methods: We determined the L-cysteine efflux and influx by evaluating the changes of free -SH concentrations in stored RBCs. The glutamate cysteine ligase (GCL) activities and protein content in stored RBCs was determined by fluorescence assay and western blotting. In addition, the glucose metabolism enzyme activity of RBCs was measured by spectrophotometric assay under in vitro incubation conditions. Results: We found that both L-cysteine transport and GCL activity significantly declined, thereby inducing the dysfunction of GSH synthesis during blood storage, which could be attenuated by ATP supplement and DTT treatment. In addition, the glycometabolic enzyme (G6PDH, HK, PK and LDH) activity significantly decreased after 6 weeks storage. Oxidant stress-induced dysfunction in glucose metabolism was the driving force for decreased GSH synthesis during storage. Conclusion: These experimental findings reflect an underlying molecular mechanism that oxidant stress induced glucose metabolism dysfunction contribute to decreased GSH synthesis in stored RBCs.