METTL3/YTHDF2 m6A axis mediates the progression of diabetic nephropathy through epigenetically suppressing PINK1 and mitophagy.

AIMS: This research aimed to investigate the specific mechanism of methyltransferase like 3 (METTL3) in the progression of diabetic kidney disease (DKD). MATERIALS AND METHODS: The model of diabetic kidney disease was established with HK-2 cells and mice in vitro and in vivo. The N6 methyladenosine (m6A) contents in the cells and tissues were detected with a commercial kit and the m6A levels of PTEN induced putative kinase 1 (PINK2) were detected with a MeRIP kit. The mRNA and protein levels were determined with RT-qPCR and western blot. The ROS, TNF-α, and IL-6 levels were assessed with ELISA. The cell proliferative ability was measured by a CCK-8 assay and cell apoptosis was determined with TUNEL staining. The HE and Masson staining was performed to observe the renal morphology. The RIP assay was conducted to detect the interaction between METTL3/YTHDF2 and PINK1. RESULTS: The m6A content and METTL3 levels were prominently elevated in diabetic kidney disease. METTL3 silencing promoted the cell growth and the expression of LC3 II, PINK1, and Parkin, while inhibiting the cell apoptosis and the expression of LC3 I and p62 in the high glucose (HG) stimulated HK-2 cells. METTL3 silencing also decreased the ROS, TNF-α, and IL-6 levels in diabetic kidney disease. PINK1 silencing neutralized the function of sh-METTL3 in the HG stimulated HK-2 cells. The HE and Masson staining showed that METTL3 silencing alleviated the kidney injury induced by DKD. METTL3 silencing decreased the m6A levels of PINK1, while increased the mRNA levels of PINK1 which depended on YTHDF2. CONCLUSIONS: METTL3 silencing could inhibit the progression of diabetic nephropathy in vivo and in vitro by regulating the m6A modification of PINK1, which depends on YTHDF2. Our research lays the theoretical foundation for the precise treatment of diabetic kidney disease and the development of targeted drugs in the future.

GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21.

As a major neuron type in the brain, the excitatory neuron (EN) regulates the lifespan in C. elegans. How the EN acquires senescence, however, is unknown. Here, we show that growth differentiation factor 11 (GDF11) is predominantly expressed in the EN in the adult mouse, marmoset and human brain. In mice, selective knock-out of GDF11 in the post-mitotic EN shapes the brain ageing-related transcriptional profile, induces EN senescence and hyperexcitability, prunes their dendrites, impedes their synaptic input, impairs object recognition memory and shortens the lifespan, establishing a functional link between GDF11, brain ageing and cognition. In vitro GDF11 deletion causes cellular senescence in Neuro-2a cells. Mechanistically, GDF11 deletion induces neuronal senescence via Smad2-induced transcription of the pro-senescence factor p21. This work indicates that endogenous GDF11 acts as a brake on EN senescence and brain ageing.

Ellagic acid inhibits cell proliferation, migration, and invasion of anaplastic thyroid cancer cells via the Wnt/ß-catenin and PI3K/Akt pathways.

Anaplastic thyroid cancer (ATC) is a rare but lethal human malignant cancer with no known cure. Ellagic acid (EA), a natural plant extract, has shown antitumor activity against multiple cancers; however, its effects on the malignant phenotypes of ATC cells remain unknown. This study aimed to evaluate the effects of EA on proliferation, migration, and invasion of ATC cells and further explore the associated signaling mechanisms. The normal human thyroid cell line Nthy-ori3-1 and ATC cell line BHT-101 were used. Cytotoxicity assay was performed using the Cell Counting kit-8 (CCK-8) assay. Cell proliferation, migration, and invasion assays were performed using the CCK-8 and colony formation, wound healing, and Transwell invasion assays, respectively. Western blotting was used to detect the levels of related proteins. ß-catenin nuclear protein levels were measured to evaluate the Wnt/ß-catenin pathway. The phosphorylation level of the Akt protein was measured and calculated to evaluate the PI3K/Akt pathway. LiCl and IGF-1 were used as pathway agonists to determine the involvement of the corresponding pathway. The results showed that EA inhibited the proliferation, migration, and invasion of ATC cells. Furthermore, both the Wnt/ß-catenin and PI3K/Akt pathways were suppressed by EA treatment, and activation of these two pathways reversed the EA-induced inhibition of the pathological phenotypes of ATC cells. These findings demonstrate that EA inhibits proliferation, migration, and invasion of ATC cells by suppressing the Wnt/ß-catenin and PI3K/Akt pathways, suggesting that EA is a potential drug candidate for treating ATC and provides a theoretical basis for further in vivo experiments and clinical applications.

Inhibition of RIPK1 by ZJU-37 promotes oligodendrocyte progenitor proliferation and remyelination via NF-κB pathway.

Receptor interacting serine/threonine protein kinase 1 (RIPK1) activation and necroptosis have been genetically and mechanistically linked with human multiple sclerosis and neurodegenerative diseases for which demyelination is a common key pathology. Demyelination can be healed through remyelination which is mediated by new oligodendrocytes derived from the adult oligodendrocyte progenitor cells (OPCs). Unfortunately, the efficiency of remyelination declines with progressive aging partially due to the depletion of OPCs following chronic or repeated demyelination. However, to our knowledge, so far there is no drug which enhances proliferation of OPCs, and it is unknown whether inhibiting RIPK1 activity directly affect OPCs, the central player of remyelination. Using TNFα induced RIPK1-dependent necroptosis in Jurkat FADD-/- cells as a cell death assay, we screened from 2112 FDA-approved drugs and the drug candidates of new RIPK1 inhibitors selected by ourselves, and identified ZJU-37, a small molecule modified by introducing an amide bond to Nec-1s, is a new RIPK1 kinase inhibitor with higher potency than Nec-1s which has the best reported potency. We unveil in addition to protecting myelin from demyelination and axons from degeneration, ZJU-37 exhibits a new role on promoting proliferation of OPCs and enhancing remyelination by inhibiting RIPK1 kinase activity with higher potency than Nec-1s. Mechanistically, ZJU-37 promotes proliferation of OPCs by enhancing the transcription of platelet derived growth factor receptor alpha via NF-κB pathway. This work identifies ZJU-37 as a new drug candidate which enhances remyelination by promoting proliferation of OPCs, paving the way for a potential drug to enhance myelin repair.

Impaired metabolism of oligodendrocyte progenitor cells and axons in demyelinated lesion and in the aged CNS.

The key pathology of multiple sclerosis (MS) comprises demyelination, axonal damage, and neuronal loss, and when MS develops into the progressive phase it is essentially untreatable. Identifying new targets in both axons and oligodendrocyte progenitor cells (OPCs) and rejuvenating the aged OPCs holds promise for this unmet medical need. We summarize here the recent evidence showing that mitochondria in both axons and OPCs are impaired, and lipid metabolism of OPCs within demyelinated lesion and in the aged CNS is disturbed. Given that emerging evidence shows that rewiring cellular metabolism regulates stem cell aging, to protect axons from degeneration and promote differentiation of OPCs, we propose that restoring the impaired metabolism of both OPCs and axons in the aged CNS in a synergistic way could be a promising strategy to enhance remyelination in the aged CNS, leading to novel drug-based approaches to treat the progressive phase of MS.

Restoring nuclear entry of Sirtuin 2 in oligodendrocyte progenitor cells promotes remyelination during ageing.

The age-dependent decline in remyelination potential of the central nervous system during ageing is associated with a declined differentiation capacity of oligodendrocyte progenitor cells (OPCs). The molecular players that can enhance OPC differentiation or rejuvenate OPCs are unclear. Here we show that, in mouse OPCs, nuclear entry of SIRT2 is impaired and NAD+ levels are reduced during ageing. When we supplement ß-nicotinamide mononucleotide (ß-NMN), an NAD+ precursor, nuclear entry of SIRT2 in OPCs, OPC differentiation, and remyelination were rescued in aged animals. We show that the effects on myelination are mediated via the NAD+-SIRT2-H3K18Ac-ID4 axis, and SIRT2 is required for rejuvenating OPCs. Our results show that SIRT2 and NAD+ levels rescue the aged OPC differentiation potential to levels comparable to young age, providing potential targets to enhance remyelination during ageing.

Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain.

Microglia dynamically monitor the microenvironment of the central nervous system (CNS) by constantly extending and retracting their processes in physiological conditions, and microglia/macrophages rapidly migrate into lesion sites in response to injuries or diseases in the CNS. Consequently, their migration ability is fundamentally important for their proper functioning. However, the mechanisms underlying their migration have not been fully understood. We wonder whether the voltage-gated proton channel HVCN1 in microglia/macrophages in the brain plays a role in their migration. We show in this study that in physiological conditions, microglia and bone marrow derived macrophage (BMDM) express HVCN1 with the highest level among glial cells, and upregulation of HVCN1 in microglia/macrophages is presented in multiple injuries and diseases of the CNS, reflecting the overactivation of HVCN1. In parallel, myelin debris accumulation occurs in both the focal lesion and the site where neurodegeneration takes place. Importantly, both genetic deletion of the HVCN1 gene in cells in vitro and neutralization of HVCN1 with antibody in the brain in vivo promotes migration of microglia/macrophages. Furthermore, neutralization of HVCN1 with antibody in the brain in vivo promotes myelin debris clearance by microglia/macrophages. This study uncovers a new role of HVCN1 in microglia/macrophages, coupling the proton channel HVCN1 to the migration of microglia/macrophages for the first time.

Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity.

Maintenance of telomere length is essential to delay replicative cellular senescence. It is controversial on whether growth differentiation factor 11 (GDF11) can reverse cellular senescence, and this work aims to establish the causality between GDF11 and the telomere maintenance unequivocally. Using CRISPR/Cas9 technique and a long-term in vitro culture model of cellular senescence, we show here that in vitro genetic deletion of GDF11 causes shortening of telomere length, downregulation of telomeric reverse transcriptase (TERT) and telomeric RNA component (TERC), the key enzyme and the RNA component for extension of the telomere, and reduction of telomerase activity. In contrast, both recombinant and overexpressed GDF11 restore the transcription of TERT in GDF11KO cells to the wild-type level. Furthermore, loss of GDF11-induced telomere shortening is likely caused by enhancing the nuclear entry of SMAD2 which inhibits the transcription of TERT and TERC. Our results provide the first proof-of-cause-and-effect evidence that endogenous GDF11 plays a causal role for proliferative cells to maintain telomere length, paving the way for potential rejuvenation of the proliferative cells, tissues, and organs.

Role of Intrinsic Factors of Polyimides in Glass Transition Temperature: An Atomistic Investigation.

Polyimides (PIs) are in high demand in the field of active matrix organic light-emitting diode displays because of their excellent heat resistance, chemical stability, and mechanical properties. However, the most critical key to their application is to further enhance their glass transition temperature (Tg), which directly affects the processing temperature of thin-film transistors on the PI films. Therefore, it is of great importance to study the factors that have an influence on the Tg of PIs. To accomplish this goal, PIs derived from pyromellitic acid dianhydride and three sets of isomeric imidazole-based diamines were investigated. The investigation, by computational methods, was to clarify the effect of intrinsic factors associated with the molecular structure of the PIs on their Tg and to construct a structure-Tg relationship for these PIs. For each model system, all-atom molecular dynamics simulations were used to identify and distinguish the effects of chain rigidity, fractional free volume (FFV), cohesive energy density, hydrogen-bonding interactions, and charge-transfer complex interactions on Tg. The results showed that the physical property, chain rigidity, has a direct impact on Tg regardless of the polymer backbone structure. A linear correlation between the increase of FFV and the decrease of Tg was not established due to the existence of hydrogen-bonding interactions, but the tendency was maintained. Furthermore, the formation of hydrogen bonds was found to have an indirect relationship with Tg. That is, the increase of intrachain hydrogen bonds would lead to a decrease in chain rigidity and consequently reduce the Tg value.

Ultrasensitive and selective assay of glutathione species in arsenic trioxide-treated leukemia HL-60 cell line by molecularly imprinted polymer decorated electrochemical sensors.

Herein a pair of molecularly imprinted polymer (MIP) modified electrochemical sensors were reported to detect glutathione (GSH) and glutathione disulfide (GSSG) in arsenic trioxide-treated HL-60 cells. MIP film was in situ synthesized onto electrode surface via electro-polymerization in a facile way. The characteristics of the obtained sensors were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Both GSH-MIP and GSSG-MIP sensors exhibit the relatively wide linear detection range and low detection limit of 1.33 × 10(-10) M (S/N=3). It is found that N-acetylcysteine and DL-homocysteine, the precursors of GSH, show little influence on the detection of glutathione species, nor did the reactants of arsenite and GSH. Such strategies were successfully applied to discriminate GSH and GSSG in cell samples with acceptable recoveries of 92.0-109.1%, and the results are comparable with classic o-phthalaldehyde fluorospectrophotometry. Moreover, the presented sensors allow for easy disclosure of the reversion of malignant phenotype in leukemia cells via glutathione species analysis.

Effect of Fanbaicao (Herba Potentillae Discoloris) oil on the expression of p21 and CDK4 in HepG2 cells.

OBJECTIVE: To research the anti-cancer mechanism of the Traditional Chinese Medicine Fanbaicao (Herba Potentillae Discoloris) oil in the human hepatoma cell line HepG2. METHODS: Gas chromatography was used to analyze the components of Fanbaicao (Herba Potentillae Discoloris). We tested the inhibitory effect of Fanbaicao (Herba Potentillae Discoloris) oil on the human hepatoma cell line HepG2 in vitro using 3-(4, 5-Dimet hylt hiazol-2-yl)-2,5-dip henyltetrazolium bromide assays. Fluorescence activating cell sorter analysis was used to examine the levels of apoptosis, and western blot and immunofluorescence were used to detect the expression of p21, p-p21 and CDK4 proteins. RESULTS: Fanbaicao (Herba Potentillae Discoloris) oil contains 45 ingredients, and L-ascorbic acid 2, 6-bispalmitate was the main component and accounted for 44.96% of total drive-off peak area. Other components included (Z)-14-met hyl-8-exadecenal- acetal (8.56%), phytol (7.74%) and lauric acid (6.31% ). Fanbaicao (Herba Potentillae Discoloris) oil treatment reduced the proliferation of HepG2 cells and the half growth inhibition concentration (IC50) was 2.03 mg/mL. Furthermore, we also observed significantly increased HepG2 cell apoptosis in a dose-dependent manner (P < 0.05). Fanbaicao (Herba Potentillae Discoloris) oil significantly increased the expression of p21 and p-p21 and significantly decreased the expression of CDK4 in HepG2 cells compared with controls (P < 0.01). CONCLUSION: Our results showed that Fanbaicao (Herba Potentillae Discoloris) oil has anti-cancer activities in HepG2 cells, which is probably related to the upregulation of p21 and p-p21 and downregulation of CDK4 expression.

Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII α in epileptic hippocampal neurons.

PURPOSE: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated. METHOD: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg(2+) free medium for 3 hours; 3) Model group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg(2+) free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca(2+) turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca(2+) turnover was observed under a laser scanning confocal microscope. RESULTS: The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca(2+) fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes. CONCLUSION: GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.

Intervention effects of ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of neurotrophin-4 and N-cadherin.

Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg(2+) free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg(2+) free medium for 3 hours), iii) GLS group I (incubated with Mg(2+) free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg(2+) free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.

[Intervention effect of ganoderma lucidum spores on the changes of XOD, MPO and SDH in the testis tissue of NIDDM rats].

OBJECTIVE: To investigate the changes of xanthine oxidase (XOD), myeloperoxidase (MPO) and mitochondrial succinate dehydrogenase (SDH) in the testis and the protective effect of ganoderma lucidum spores on the testicular tissue of rats with non-insu- lin-dependent diabetes mellitus (NIDDM). METHODS: Fifty male Wistar rats were divided randomly into a model, a ganoderma and a normal control group, the first two groups injected with 2% STZ (25 mg/kg) through the peritoneum, and the last one with half-and-half sodium citrate/citrate buffer solution. Two weeks after normal diet, glucose tolerance tests were performed and the rats with abnormal glucose tolerance in the model and ganoderma groups received high-fat and high-carbohydrate food, the latter given ganoderma lycium spores (250 mg/kg x d) in addition, both for 10 weeks and all rats fed alone. Glucose tolerance tests were repeated 1 day before the end of the experiment and the testes of the rats were harvested for the determination of XOD, MPO and SDH. RESULTS: SDH was significantly lower (P < 0.05) while XOD and MPO significantly higher in the model group than in the ganoderma and control groups (P < 0.05). The model rats exhibited abnormal convoluted seminiferous tubules, indistinct parietal layers, decreased or abolished gonepoiesis, luminal peripheral fibrous tissue (interstitial substance) accrementition, basal lamina thickening, and vessel wall fibrous tissue accrementition and sclerosis. CONCLUSION: Ganoderma lucidum spores can protect the testis of diabetic rats by reducing free radical-induced damage to the testicular tissue and enhancing the activity of SDH.

[Effects of ganoderma lucidum spores on mitochondrial calcium ion and cytochrome C in epididymal cells of type 2 diabetes rats].

OBJECTIVE: To observe the effects of ganoderma lucidum spores (GLS) on mitochondrial calcium ion and cytochrome C in the epididymal cells of type 2 diabetes rats. METHODS: Fifty adolescent rats were randomly divided into a model group (n=20), a GLS group (n=20) and a control group (n=10). The animals of the former two groups were injected with 2% STZ via vena caudalis for one time to induce type 2 diabetes. Then the model group was given high-fat-sugar diet, the GLS group high-fat-sugar diet + GLS (250 mg/kg x d), and the control group normal diet + CA-citrate sodium buffer. The bilateral epididymides were obtained 10 weeks later and the contents of mitochondrial calcium and cytochrome C detected. RESULTS: Type 2 diabetes models were successfully constructed. The content of mitochondrial calcium in the epididymal cells was significantly higher in the model group ([3.279 +/- 0.502] mg/L) than in the control group ([2.606 +/- 0.048] mg/L, P < 0.01), with no significant difference between the GLS group ([2.693 +/- 0. 196] mg/L) and the control (P > 0.05). In the model group, the content of mitochondrial cytochrome C ([3.213 +/- 1.511] micromol/L) was significantly lower (P < 0.05) while that of cytoplasm cytochrome C ([2.484 +/- 0.661] micromol/L) significantly higher (P < 0.05) than in the control ([5.688 +/- 1.679] micromol/L and [1.574 +/- 0.329] micromol/L, respectively). In the GLS group, the content of mitochondrial cytochrome C ([5.258 +/- 1.560] micromol/L) was higher, with no significant difference (P > 0.05), and that of cytoplasm cytochrome C ([1.727 +/- 0.396] micromol/L) significantly lower than in the model group (P < 0.05), but the difference between the GLS and the control group was not significant (P > 0.05). CONCLUSION: With disequilibrium of calcium homeostasis and damage to mitochondria, there might be excessive apoptosis in the epididymal cells of type 2 diabetes rats. Ganoderma lucidum spores could protect epididymal cells and counteract their apoptosis in diabetic condition.

[Effects of ganoderma lucidum spores on cytochrome C and mitochondrial calcium in the testis of NIDDM rats].

OBJECTIVE: To observe the effects of Ganoderma lucidum spores on Cytochrome C (Cyt-C) and mitochondrial calcium in the testis of NIDDM rats. METHODS: Fifty male Wistar rats were divided randomly into three groups: model, ganoderma and normal control, the first two groups injected with 2% STZ through vena caudalis, and the last one with half-and-half sodium citrate/citrate buffer solution. Two weeks after normal diet, glucose tolerance tests were performed and the rats with abnormal glucose tolerance from the model and ganoderma groups received high-fat and high-carbohydrate food, the ganoderma group given Ganoderma lucidum spores (250mg/[ kg x d] ) in addition, both for 10 weeks. Glucose tolerance tests were repeated 1 day before the end of the experiment and the rats were castrated and relevant indexes measured. RESULTS: The NIDDM model was successfully constructed. In the model group, the levels of mitochondrial Cyt-C and mitochondrial calcium were significantly lower (P <0. 05) while that of the plasma Cyt-C was significantly higher than in the ganoderma and the control groups. CONCLUSION: Cyt-C and calcium ion are involved in the damage of the testis. Ganoderma lucidum spores can protect the testis of NIDDM rats.

[Effects of experimental varicocele on mitochondria calcium and cytochrome C of the epididymis in adolescent rats].

OBJECTIVE: To investigate the effects of experimental varicocele on mitochondria calcium and cytochrome C of the epididymal cells in adolescent rats. METHODS: Forty male adult Wistar rats were divided into two groups randomly: varicocele group (VG) and sham operation group (SOG) by partial ligation or exposure of the left renal vein. Bilateral epididymides were removed after ten RESULTS: The content of mitochondria weeks. Mitochondria calcium and cytochrome C levels of the epididymal cells were detected. calcium decreased (P < 0.001 ) while that of cytochrome C increased (P < 0.05) markedly in the experimental group compared with SOG. CONCLUSION: Calcium dyshomeostasis and mitochondrial damage of the epididymal cells caused by varicocele may play an important role in leading to subfertility.