SOD1 Protein Research Articles

Computational insight into in silico analysis and molecular dynamics simulation of the dimer interface residues of ALS-linked hSOD1 forms in apo/holo states: a combined experimental and bioinformatic perspective.

The aggregation of misfolded SOD1 proteins in neurodegenerative illnesses is a key pathological hallmark in amyotrophic lateral sclerosis (ALS). SOD1 is stabilized and enzymatically activated after binding to Cu/Zn and forming intramolecular disulfide. SOD1 aggregation/oligomerization is triggered by the dissociation of Cu and/or Zn ions. Therefore, we compared the possible effects of ALS-associated point mutations of the holo/apo forms of WT/I149T/V148G SOD1 variants located at the dimer interface to determine structural characterization using spectroscopic methods, computational approaches as well as molecular dynamics (MD) simulations. Predictive results of computational analysis of single-nucleotide polymorphisms (SNPs) suggested that mutant SOD1 has a deleterious effect on activity and structure destabilization. MD data analysis indicated that changes in flexibility, stability, hydrophobicity of the protein as well as increased intramolecular interactions of apo-SOD1 were more than holo-SOD1. Furthermore, a decrease in enzymatic activity in apo-SOD1 was observed compared to holo-SOD1. Comparative intrinsic and ANS fluorescence results of holo/apo-WT-hSOD1 and mutants indicated structural alterations in the local environment of tryptophan residue and hydrophobic patches, respectively. Experimental and MD data supported that substitution effect and metal deficiency of mutants (apo forms) in the dimer interface may promote the tendency to protein mis-folding and aggregation, consequently disrupting the dimer-monomer equilibrium and increased propensity to dissociation dimer into SOD-monomer ultimately leading to loss of stability and function. Overall, data analysis of apo/holo SOD1 forms on protein structure and function using computational and experimental studies will contribute to a better understanding of ALS pathogenicity.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

DIA-MS Based Proteomics Combined with RNA-Seq Data to Unveil the Mitochondrial Dysfunction in Human Glioblastoma

Mitochondrial dysfunctions underlie the pathogenesis in glioblastoma multiforme (GBM). Comprehensive proteomic profiling of mitochondria-specific changes in human GBM is still insufficient. This study carried out a DIA-MS based proteomic analysis on the mitochondria isolated from human primary GBM and peritumoral tissue (as paired control), and further compared those findings with the transcriptomic datasets. A total of 538 mitochondrion-specific proteins were rigorously confirmed, among which 190 differentially expressed proteins were identified. Co-regulations of the mitochondrial dysfunction pathway networks were observed, including significant up-regulations of mitochondrial translation and apoptosis, as well as down-regulations of OXPHOS and mitochondrial dynamics. Proteins related to FA, AA metabolism and ROS also showed significant variations. Most of these alterations were consistent in trend when compared the proteomics findings with the RNA-Seq datasets, while the changes at protein levels appeared to be more dramatic. Potentially key proteins in GBM were identified, including up-regulated pro-apoptotic protein CASP3, BAX, fatty acid oxidation enzymes CPT1A, CPT2, ACADM, serine-glycine enzymes SHMT2, GATM, ROS-related protein SOD2, GPX1, and CAT; and down-regulated dynamin-related protein MFN1, MFN2, OPA1, and OXPHOS components; and also several differentially expressed ALDH isoforms. This study systematically profiled the mitochondrial dysfunctions by combining proteomic findings and mRNA datasets, which would be a valuable resource to the community for further thorough analyses.

Efficacy of oligodendrocyte precursor cells as delivery vehicles for single-chain variable fragment to misfolded SOD1 in ALS rat model

Superoxide dismutase1 (SOD 1) mutation is a leading cause of familial amyotrophic lateral sclerosis (ALS). Growing evidence suggests that antibody therapy against misfolded SOD1 protein can be therapeutic. However, the therapeutic effects are limited, partly because of the delivery system. Therefore, we investigated the efficacy of oligodendrocyte precursor cells (OPCs) as a drug delivery vehicle of single-chain variable fragments (scFv). Using a Borna disease virus vector that is pharmacologically removable and episomally replicable in the recipient cells, we successfully transformed wild-type OPCs to secrete scFv of a novel monoclonal antibody (D3-1), specific for misfolded SOD1. Single intrathecal injection of OPCs scFvD3-1, but not OPCs alone, significantly delayed disease onset and prolonged the lifespan of ALS rat models expressing SOD1 H46R . The effect of OPC scFvD3-1 surpassed that of a 1month intrathecal infusion of full-length D3-1 antibody alone. scFv-secreting OPCs suppressed neuronal loss and gliosis, reduced levels of misfolded SOD1 in the spinal cord, and suppressed the transcription of inflammatory genes, including Olr1, an oxidized low-density lipoprotein receptor 1. The use of OPCs as a delivery vehicle for therapeutic antibodies is a new option for ALS in which misfolded protein and oligodendrocyte dysfunction are implicated in the pathogenesis.

Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

BackgroundZearalenone (ZEA) widely exists in moldy grains, which seriously destroys the fertility of females. Isorhamnetin, a natural flavonoid, has extensive of pharmacological activities. However, the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.MethodsOocytes were treated with different concentrations of ZEA (3, 5, 8 and 10 μmol/L) and isorhamnetin (5, 10, 20 and 30 μmol/L) for 44 h at 39 ℃. ZEA (5 μmol/L) and isorhamnetin (10 μmol/L) were selected for subsequent studies. Polar body exclusion rate, apoptosis rate and apoptosis related proteins, ROS levels and SOD2 protein, mitochondrial membrane potential and distribution, endoplasmic reticulum distribution and proteins expression, and PI3K, Akt and p-Akt proteins expression of oocytes were detected. In addition, the effect of PI3K antagonist (LY294002) on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.ResultsOur findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes. Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes. Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production. Moreover, isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA. Changing the expression of endoplasmic reticulum stress-related marker proteins (CHOP, GRP78) and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress. Mechanistically, isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.ConclusionsCollectively, these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway, which enhances meiotic maturation and mitochondrial function, and inhibits early apoptosis, oxidative stress and endoplasmic reticulum stress in porcine oocytes. Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.Graphical A possible mechanism by which isorhamnetin protected porcine oocytes from ZEA-induced damage. Isorhamnetin inhibited meiosis arrest and apoptosis of porcine oocytes induced by ZEA through the PI3K/Akt signaling pathway. Moreover, isorhamnetin repaired ZEA-induced oocyte damage by alleviating oxidative stress, mitochondrial dysfunction and ER stress.

Sequence adaptation in various environments across organisms; similar yet different superoxide dismutase.

Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that attack the host immune system and killed millions of people annually. The body first defense mechanism is through phagocytosis where an outburst of radical oxygen species (ROS) is released and potentially kill the bacterium. M. tuberculosis superoxide dismutase (MtSOD) survives the phagocytosis and suppresses the host immune system by converting reactive oxygen species to H2O2 and O2. SOD proteins are important for cell survival to prevent the buildup of reactive ROS in the cell that may cause damage to DNA, RNA, proteins, and even cell death. MtSOD (oxidized) is homologous to human SOD1 (hSOD1, reduced) which contains both zinc and copper metals along with the disulfide bond. Though, MtSOD lacks the zinc binding site which proposed to play a role in stability, dimerization, and function of SOD. Utilizing Circular Dichroism, an apparent correlation of MtSOD stabilization with the copper and disulfide bond as more stable secondary structure is observed for oxidized apoprotein compared to the reduced apo and oxidized holoprotein. To further investigate the novel role of zinc in SOD (ZnMtSOD), a mutation of zinc binding site consists of T119H, A123H, T192K (modeled from hSOD1) is introduced in the wild type MtSOD. With the low sequence identity of 19.7% but similar in protein structures, the thermodynamic and kinetics results are compared for hSOD1, ZnMtSOD, and wt MtSOD.

Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting both upper and lower motor neurons (MNs) with large unmet medical needs. Multiple pathological mechanisms are considered to contribute to the progression of ALS, including neuronal oxidative stress and mitochondrial dysfunction. Honokiol (HNK) has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke, Alzheimer's disease and Parkinson's disease. Here we found that honokiol also exhibited protective effects in ALS disease models both invitro and invivo. Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins (SOD1-G93A cells for short). Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione (GSH) synthesis and activating the nuclear factor erythroid 2-related factor 2 (NRF2)-antioxidant response element (ARE) pathway. Also, honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells. Importantly, honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function. The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice. Overall, honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.

Modulating Morphological and Redox/Glycative Alterations in the PCOS Uterus: Effects of Carnitines in PCOS Mice

(1) Background: Polycystic ovarian syndrome (PCOS) is a common and multifactorial disease affecting reproductive-age women. Although PCOS ovarian and metabolic features have received extensive research, uterine dysfunction has been poorly investigated. This research aims to investigate morphological and molecular alterations in the PCOS uterus and search for modulating effects of different carnitine formulations. (2) Methods: CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg L-carnitine (LC) and 0.20 mg acetyl-L-carnitine (ALC) in the presence or absence of 0.08 mg propionyl-L-carnitine (PLC). Uterine horns from the four groups were subjected to histology, immunohistochemistry and immunoblotting analyses to evaluate their morphology, collagen deposition, autophagy and steroidogenesis. Oxidative-/methylglyoxal (MG)-dependent damage was investigated along with the effects on the mitochondria, SIRT1, SOD2, RAGE and GLO1 proteins. (3) Results: The PCOS uterus suffers from tissue and oxidative alterations associated with MG-AGE accumulation. LC-ALC administration alleviated PCOS uterine tissue alterations and molecular damage. The presence of PLC prevented fibrosis and maintained mitochondria content. (4) Conclusions: The present results provide evidence for oxidative and glycative damage as the main factors contributing to PCOS uterine alterations and include the uterus in the spectrum of action of carnitines on the PCOS phenotype.

LncRNA SNHG16 regulates RAS and NF-κB pathway-mediated NLRP3 inflammasome activation to aggravate diabetes nephropathy through stabilizing TLR4.

LncRNA SNHG16 and Toll-like receptor-4 (TLR4) participate in diabetes nephropathy. This study investigated whether SNHG16 regulates diabetic renal injury (DRI) via TLR4 and its related mechanism. Diabetic mice and high glucose (HG)-induced HRMCs were used to examine the expressions of SNHG16 and TLR4. The SNHG16 expression, cytokines, reactive oxygen species, MDA, SOD, GSH, and fibrosis-related proteins were evaluated in HG-induced HRMCs transfected with sh-NC or sh-SHNG16. RNA immunoprecipitation and RNA pull-down determined the interaction between SNHG16 and EIF4A3 or TLR4 and EIF4A3. We used HG-treated HRMCs or diabetic mice to investigate the roles of TLR4 or SNHG16 in renal injuries. Both SNHG16 and TLR4 were upregulated in diabetic conditions. HG increased serum Scr and BUN, led to significant fibrosis, increased inflammation- and renal fibrosis-related proteins in mice, and increased ROS, MDA, and decreased SOD and GSH in HRMCs. SNHG16 silencing diminished HG-upregulated SNHG16, decreased HG-increased cytokines secretion, ROS, MDA, and fibrosis but increased SOD and GSH. RIP and RNA pull-down confirmed that SNHG16 recruits EIF4A3 to stabilize TLR4 mRNA. TLR4 knockdown alleviated HG-induced renal injuries by suppressing RAS and NF-κB-mediated activation of NLRP3 inflammasomes. SNHG16 knockdown alleviated HG-induced renal injuries in HG-induced HRMCs or diabetic mice. Interestingly, TLR4 overexpression reversed the effects of SNHG16 knockdown. Mechanistically, SNHG16 knockdown alleviated HG-induced renal injuries by suppressing TLR4. SNHG16 accelerated HG-induced renal injuries via recruiting EIF4A3 to enhance the stabilization of TLR4 mRNA. The SNGHG16/ELF4A3/TLR4 axis might be a novel target for treating DRI.

Fibroblast Growth Factor 9 Inhibited Apoptosis in Random Flap via the ERK1/2-Nrf2 Pathway to Improve Tissue Survival.

The application of random pattern skin flaps is limited in plastic surgery reconstruction due to necrosis. Fibroblast growth factor 9 (FGF9) was reported to exert a protective effect against myocardial damage and cerebral ischemia injury, but the impact of FGF9 in random flap survival is still unclear. In this study, we used a mouse model of random flaps to verify that FGF9 can directly increase flap survival area and blood flow intensity by promoting angiogenesis. In total, 84 male C57BL/6 mice weighing between 22 and 25 g were randomly divided into three groups (n = 28 each group). After skin flap operation, one group served as a control, a treatment group received FGF9, and a treatment group received FGF9+U0126. All flap samples were incised on postoperative day 7. Our results showed that flap survival was significantly increased in the FGF9 group compared with that in the control group. This protective function was restrained by U0126. The results of histopathology, laser Doppler, and fluorescent staining all showed significant increases in capillary count, collagen deposition, and angiogenesis. FGF9 also significantly increased the expression of antioxidant stress proteins SOD1, eNOS, HO-1, vascular marker proteins CD31, VE cadherin, and pericyte marker protein PDGFRβ. Western blot showed that the phosphorylation degree of ERK1/2 increased after FGF9 treatment, and the expression of Nrf2, a downstream factor, was u-regulated. Western blot and immunofluorescence results of apoptosis-related proteins cleaved caspase-3, BAX, and Bcl2 showed that FGF9 inhibited apoptosis. ERK inhibitor U01926 reduced the beneficial effects of FGF9 on skin flap survival, including promoting angiogenesis, and showing antiapoptosis and antioxidative stress activities. Exogenous FGF9 stimulates angiogenesis of random flap and survival of tissue. the impact of FGF9 is closely linked to the prevention of oxidative stress mediated by ERK1/2-Nrf2. In the function of FGF9 in promoting effective angiogenesis, there may be a close interaction in the FGF9-FGFR-PDGFR-ERK-VE cadherin pathway. In particular, PDGFR and VE cadherin may interact.

Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression.

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease.

Azilsartan Modulates HMGB1/NF-κB/p38/ERK1/2/JNK and Apoptosis Pathways during Renal Ischemia Reperfusion Injury

Renal ischemia/reperfusion (IR) injury is characterized by an unexpected impairment of blood flow to the kidney. Azilsartan is an angiotensin receptor blocker that is approved for the management of hypertension. The present study aimed to investigate, on molecular basics, the nephroprotective activity of azilsartan on renal IR injury in rats. Rats were assigned into four groups: (1) Sham group, (2) Azilsartan group, (3) IR group, and (4) IR/Azilsartan-treated group. Histological examination and renal function were evaluated. Levels of KIM-1, HMGB1, caspase 3, GPX, SOD, NF-κB, and p53 proteins were investigated using ELISA. mRNA levels of IL-1β, IL6, IL10, TNF-α, NF-κB, p53, and bax were assessed by qRT-PCR. Expression of p38, JNK, and ERK1/2 proteins was investigated by Western blotting. IR injury resulted in tissue damage, elevation of creatinine, BUN, KIM-1, HMGB1, caspase 3, NF-κB, and p53 levels, decreasing GPX and SOD activities, and up-regulation of NF-κB, IL-1β, IL6, TNF-α, p53, and bax genes. Furthermore, it up-regulated the expression of phosphorylated/total ratio of p38, ERK1/2, and JNK proteins. Interestingly, treatment of the injured rats with azilsartan significantly alleviated IR injury-induced histopathological and biochemical changes. It reduced the creatinine, BUN, KIM-1, HMGB1, caspase-3, NF-κB, and p53 levels, elevated GPX and SOD activities, down-regulated the expression of NF-κB, IL-1β, IL6, TNF-α, p53, and bax genes, and up-regulated IL10 gene expression. Furthermore, it decreased the phosphorylated/total ratio of p38, ERK1/2, and JNK proteins. Azilsartan exhibited nephroprotective activity in IR-injured rats via its antioxidant effect, suppression of inflammation, attenuation of apoptosis, and inhibition of HMGB1/NF-κB/p38/ERK1/2/JNK signaling pathway.

STRUCTURAL ANALYSIS OF ZnO/GO NANOCOMPOSITE AND ITS IMPACT ON MUTATED AND NON-MUTATED SOD1 PROTEINS THROUGH DOCKING STUDIES

The interaction of nanocomposite (ZnO/GO) with biomolecules like Protein SOD1 (which is related to neurodegenerative disorders) as a result of many physiological changes in that protein leads to the study of various neurodegenerative disorders. In this work, we investigate the mutated and non-mutated SOD1 proteins (which are responsible for many neurodegenerative diseases) that were suggested to interact with the ZnO-GO composite, both compounds having extraordinary bioactive performances against neurodegenerative disorders. Morphological studies of ZnO-GO nanocomposite were studied by using innovative spectroscopy techniques such as XRD (X-ray diffraction), SAED (selected area electron diffraction), EDX, and TEM (transmission electron microscopy) methods. Impacts on SOD1 proteins, both mutated and non-mutated, such as binding energy, bonding and non-bonding interaction, and docking score studies, were analyzed using docking software.

Transcriptome Analysis of a Wild Eggplant Germplasm M239 in Response to Verticillium dahliae Infection

In this study, wild eggplant germplasm No. M239, which is highly susceptible to Verticillium wilt, was used as the experimental material. The physiological and biochemical indices (SOD, PAL, MDA and soluble protein) of M239 roots were measured at different times (0, 12, 24, 36, 48, 60 and 72 h) post inoculation with Verticillium dahliae, and the key time points for the M239 response to Verticillium wilt infection were screened. Then, RNA-Seq technology was used to screen the differentially expressed genes (DEGs) in M239 roots at 0, 12 and 48 h post-inoculation (hpi). The transcriptional results of M239 were also compared with those resistance genes from some reported wild relative Solanum species (S. sisymbriifolium and S. aculeatissimum). Then some DEGs were chosen for validation by qRT–PCR. The results showed that 12 and 48 hpi were the turning points in the changes in all physiological and biochemical indices. A total of 6,783 DEGs were identified by RNA-Seq, including 6,141 DEGs (3,046 upregulated and 3,095 downregulated) at the M_12 h vs. M_0 h, 1,903 DEGs (792 upregulated and 1,111 downregulated) at M_48 h vs. M_12 h, and 1,261 DEGs that appeared simultaneously in both stages. KEGG enrichment analysis showed that there were 5 metabolic pathways enriched from DEGs, which were mostly related to primary metabolism, such as glycolysis, amino acid and ribosome biogenesis. Compared with the NCBI non-redundant protein (NR) database, one Ve2 homologous gene and 8 PR protein-related genes were screened. Transcription factor analysis showed that there were a large number of DEGs, such as MYB, AP2-EREBP, bHLH, NAC and Orphans in the two stages. Compared with the reported Verticillium wilt-resistant wild eggplant species, it was found that there were fewer genes and enriched metabolic pathways in the M239 response to Verticillium wilt infection, and it also lacked the response of some known key resistance genes. These results proved that the above resistance genes and metabolic pathways played a key role in the wild eggplant response to V. dahliae infection.

Eucommia ulmoides male flower as a remarkable edible floral resource exerts lifespan/healthspan-promoting effects on Caenorhabditis elegans.

Natural products, especially phytochemicals, can effectively improve the health of various model organisms and ultimately prolong their lifespan. As an emerging resource of plant-based food, edible flowers have potential anti-aging effects. Here, we showed that twelve out of 30 drug-food homologous flowers' extracts significantly extended the lifespan of C. elegans, and the Eucommia ulmoides male flower was screened out by comparing centrally. The lifespan of C. elegans increased by 18.61% under the treatment of 100 μg mL-1 floral extract (EUFE). Interestingly, this effect was attenuated when EUFE was administered late or at higher concentrations. Significantly, EUFE improved health indicators that decline with aging including pharyngeal pumping, mobility, muscle morphology, and lipofuscin accumulation. EUFE also enhanced the resistance of C. elegans to oxidative/heat stress. The longevity-extending effect of EUFE was dependent on transcription factor DAF-16 and mitochondrial function. Moreover, EUFE triggered the nuclear translocation of DAF-16 and promoted downstream LGG-1 and SOD3 protein expression. In body-wall muscles, EUFE stimulated mitochondrial fission and mitophagy to mitigate age-related mitochondrial impairments. The transcriptional checkpoints of daf-16, drp-1, eat-3, lgg-1, and dct-1 further showed that EUFE regulated DAF-16 signaling and mitochondrial homeostasis. Finally, the interpretation of the EUFE components by correlation analysis, UHPLC-QE-MS, and verification experiments showed that aucubin, geniposide, and asperuloside are the main active compounds. We revealed the excellent lifespan/healthspan-promoting efficacy of EUFE and highlighted that edible flowers are worthy of further investigation as anti-aging dietary resources. Meanwhile, related mechanisms enriched the hypothesis that mitochondria might be involved in the healthspan modulation of longevity pathways.

Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons.

ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.

Tandem mass tag-based quantitative proteomic analysis of effects of multiple sevoflurane exposures on the cerebral cortex of neonatal and adult mice.

Sevoflurane is the most commonly used general anesthetic in pediatric surgery, but it has the potential to be neurotoxic. Previous research found that long-term or multiple sevoflurane exposures could cause cognitive deficits in newborn mice but not adult mice, whereas short-term or single inhalations had little effect on cognitive function at both ages. The mechanisms behind these effects, however, are unclear. In the current study, 6- and 60-day-old C57bl mice in the sevoflurane groups were given 3% sevoflurane plus 60% oxygen for three consecutive days, each lasting 2 hours, while those in the control group only got 60% oxygen. The cortex tissues were harvested on the 8th or 62nd day. The tandem mass tags (TMT)pro-based quantitative proteomics combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, Golgi staining, and western blotting analysis were applied to analyze the influences of multiple sevoflurane anesthesia on the cerebral cortex in mice with various ages. The Morris water maze (MWM) test was performed from postnatal day (P)30 to P36 or P84 to P90 after control or multiple sevoflurane treatment. Sevoflurane anesthesia affected spatial learning and memory and diminished dendritic spines primarily in newborn mice, whereas mature animals exhibited no significant alterations. A total of 6247 proteins were measured using the combined quantitative proteomics methods of TMTpro-labeled and LC-MS/MS, 443 of which were associated to the age-dependent neurotoxic mechanism of repeated sevoflurane anesthesia. Furthermore, western blotting research revealed that sevoflurane-induced brain damage in newborn mice may be mediated by increasing the levels of protein expression of CHGB, PTEN, MAP2c, or decreasing the level of SOD2 protein expression. Our findings would help to further the mechanistic study of age-dependent anesthetic neurotoxicity and contribute to seek for effective protection in the developing brain under general anesthesia.

Interactive Effects of Swimming High-Intensity Interval Training and Resveratrol Supplementation Improve Mitochondrial Protein Levels in the Hippocampus of Aged Rats.

Mitochondrial dysfunction and increased oxidative stress cause damage to cells which can lead to the aging process and age-related diseases. Antioxidants such as resveratrol and high-intensity exercise can benefit oxidative damage prevention. This study is aimed at evaluating the effects of swimming high-intensity interval training and resveratrol on mitochondrial metabolism key proteins, SIRT5, SOD1, and PDH-E1α, and the level of NAD+ as a cofactor in the deacetylation process in aged rat hippocampus. Forty-five male Wistar rats, aged 20 months, were randomly divided into five groups: control (C), Swimming High-Intensity Interval Training (HIIT) (S-HIIT), Swimming HIIT with resveratrol supplementation (S-HIIT-R), resveratrol supplementation (R), and solvent of resveratrol supplementation (SR). S-HIIT and resveratrol groups performed the exercise and received resveratrol (10 mg/kg/day, gavage) for six weeks. Western blot analysis was performed to determine the protein level in the hippocampus. The amount of SIRT5 and SOD1 proteins in the hippocampus increased. S-HIIT with resveratrol or resveratrol alone increased the PDH-E1α level significantly. The amount of NAD+ was analyzed by assay kit that was reduced in S-HIIT, S-HIIT-R, and SR groups compared to controls. The results showed that resveratrol and S-HIIT attenuated the age-related brain changes by increasing the expression of SOD1 and SIRT5 and reducing the level of NAD+ in the hippocampus. Considering these findings, S-HIIT and resveratrol supplementation could be proposed as strategies to attenuate age-related brain changes. Resveratrol alone and exercise through the regulation of crucial proteins and cofactors can influence mitochondrial metabolism and oxidative stress in the hippocampus of aged rats.

Medium-chain triglycerides reduce glomerulosclerosis and induce expression of redox genes in NSY mice with diabetic nephropathy

BackgroundNephropathy is a type of complication of type 2 diabetes, and medium chain triglycerides (MCTs) potentially improve metabolic abnormalities. This study examined whether MCT intake inhibits nephropathy development in a type 2 diabetic mouse model. MethodsSix-week-old control non-diabetic ICR mice and type 2 diabetic Nagoya Shibata Yasuda (NSY) mice were fed a diet enriched with long-chain saturated/mono-unsaturated triglycerides, including palmitic-, stearic-, and oleic acids (C16/C18-rich group). NSY mice were fed a diet containing medium-chain triglycerides with either caprylic acid (C8-rich group) or capric acid (C10-rich group). Feeding was performed for 20 weeks. Plasma biomarker levels were measured using ELISA or enzymatic methods. Kidney pathology was evaluated using periodic acid–Schiff staining, and the expression of redox genes and proteins were determined using qRT-PCR and western blotting. ResultsThe C16/C18-rich group of NSY mice exhibited expansion of the mesangial region in the kidney. Plasma cystatin C and creatinine concentrations were not different between ICR mice and C16/C18-rich group of NSY mice and between C16/C18-rich and C8- or C10-rich groups of NSY mice. The C8- and C10-rich groups had suppressed mesangial region than the C16/C18-rich groups. The mRNA expression of Sod1, Prdx1, and Gpx2, and SOD1 protein levels were higher in the C8- and/or C10-rich groups than in the C16/C18-rich group of NSY mice. ConclusionsMCT intake in NSY mice at the stage without increased markers indicating glomerular filtration decrease, reduces glomerulosclerosis, with induction of redox gene expression, in the kidney.

Green rooibos extract attenuates high glucose induced oxidative stress in a human derived (HepG2) liver cell line

• Oxidative stress biomarkers are attenuated by REE in a glucotoxic HepG2 cell model. • REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose. • REE activates NRF2 antioxidant pathway in HepG2 cells exposed to high glucose. The exposure of hepatocytes to high concentrations of glucose results in a glucotoxic environment and ensuing hepatocellular injury, often due to oxidative stress. The popularly consumed herbal tea, Rooibos ( Aspalathus linearis ), demonstrates pleiotropic pharmacological and antioxidant activities. Our study investigated the molecular events regulating the antioxidant effects of a Rooibos ethanolic extract (REE) in a glucotoxic HepG2 liver cell model. It is hypothesized that REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose to attenuate glucotoxic damage. Glucotoxicity was induced by treating the cells with 25 mM glucose. For this study, colorimetric, luminometric and western blot analyses were used to determine the potential of the REE to reduce biomarkers associated with oxidative damage and its potential to enhance the intracellular antioxidant defense system. The REE was found to be hepatoprotective as evidenced by reduced levels of oxidative damage biomarkers (MDA and protein carbonylation) and enhanced antioxidant capacity under glucotoxic conditions as evidenced by elevated GSH (reduced form). We also observed decreased intracellular hydrogen peroxide levels and caspase activity in the REE treated cells and further showed that the REE enhanced SOD2, CAT, and GPx1 protein expression. These effects were dependent on the transcriptional activity of NRF2. Collectively, our results show that the REE enhances the cellular antioxidant defense capacity and may provide a relevant supportive therapeutic option for treating diabetic complications associated with oxidative stress.