Longxuetongluo Capsule alleviate ischemia/reperfusion induced cardiomyocyte apoptosis through modulating oxidative stress and mitochondrial dysfunction.

BACKGROUND: Chinese dragon's blood, the red resin of Dracaena cochinchinensis (Lour.) S. C. Chen., is widely used to treat cardiovascular and cerebrovascular diseases in China. Longxuetongluo Capsule (LTC) is a total phenolic compound extracted from Chinese dragon's blood, currently used in treating ischemic stroke. Myocardial injury can be aggravated after reperfusion of ischemic myocardium, which is called myocardial ischemia-reperfusion injury (MIRI), and the mechanism of MIRI is complex. However, the exact effect and mechanism of LTC on MIRI are still unclear. We explore the effect of LTC on alleviating MIRI based on mitochondrial dysfunction and oxidative stress. AIM OF THE STUDY: To explore the cardioprotective mechanism of LTC against MIRI. MATERIALS AND METHODS: A rat MIRI model was constructed through ligation of the left anterior descending coronary artery, and LTC was given continuously for 28 days before surgery. The H9c2 cardiomyocyte injury model was induced by oxygen-glucose deprivation/reperfusion (OGD/R), and LTC was given 24 h before OGD. Myocardial ischemia areas were detected with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiac histopathological changes were detected with hematoxylin-eosin (HE) staining. And biochemical indexes were detected with serum biochemical kit. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining and flow cytometry were used to detect apoptosis. Fluorescent probes were used to observe reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), Ca2+and other indexes. MitoTracker staining and immunofluorescence were used to observe the morphology of mitochondria and translocation of dynamin-related protein 1 (Drp1). Finally, immunohistochemistry and Western blotting were used to examine the expression of proteins related to apoptosis, mitochondrial fission and fusion and oxidative stress. RESULTS: LTC could ameliorate cardiac pathological changes, decrease myocardial infarct area and the content or level of relevant serum cardiac enzymes, indicating that LTC could alleviate MIRI. Meanwhile, LTC could inhibit cardiomyocyte apoptosis via regulating apoptosis-related protein expression, and it could restore mitochondrial morphology, maintain ΔΨm, inhibit mitochondrial ROS generation and Ca2+ accumulation, increase the expression of mitochondrial fusion protein 2 (Mfn2), decrease the level of phosphorylation dynamin-related protein 1 (p-Drp1), and regulate ATP synthesis, thereby significantly ameliorating mitochondrial dysfunction. Moreover, LTC significantly reduced the expression of NADPH oxidase 2 (NOX2), NADPH oxidase 4 (NOX4) and neutrophil cytosolic factor 2 (NOXA2/p67phox), and reduced ROS production. CONCLUSION: The study demonstrated that LTC could inhibit MIRI induced cardiomyocyte apoptosis by inhibiting ROS generation and mitochondrial dysfunction, and these fundings suggested that LTC can be used to alleviate MIRI, which provides a potential therapeutic approach for future treatment of MIRI.

ERK1/2 Regulates Epileptic Seizures by Modulating the DRP1-Mediated Mitochondrial Dynamic.

After seizures, the hyperactivation of extracellular signal-regulated kinases (ERK1/2) causes mitochondrial dysfunction. Through the guidance of dynamin-related protein 1 (DRP1), ERK1/2 plays a role in the pathogenesis of several illnesses. Herein, we speculate that ERK1/2 affects mitochondrial division and participates in the pathogenesis of epilepsy by regulating the activity of DRP1. LiCl-Pilocarpine was injected intraperitoneally to establish a rat model of status epilepticus (SE) for this study. Before SE induction, PD98059 and Mdivi-1 were injected intraperitoneally. The number of seizures and the latency period before the onset of the first seizure were then monitored. The analysis of Western blot was also used to measure the phosphorylated and total ERK1/2 and DRP1 protein expression levels in the rat hippocampus. In addition, immunohistochemistry revealed the distribution of ERK1/2 and DRP1 in neurons of hippocampal CA1 and CA3. Both PD98059 and Mdivi-1 reduced the susceptibility of rats to epileptic seizures, according to behavioral findings. By inhibiting ERK1/2 phosphorylation, the Western blot revealed that PD98059 indirectly reduced the phosphorylation of DRP1 at Ser616 (p-DRP1-Ser616). Eventually, the ERK1/2 and DRP1 were distributed in the cytoplasm of neurons by immunohistochemistry. Inhibition of ERK1/2 signaling pathways downregulates p-DRP1-Ser616 expression, which could inhibit DRP1-mediated excessive mitochondrial fission and then regulate the pathogenesis of epilepsy.

Selenium Treatment Ameliorates the Adverse Effects Caused by Dynamin Gene Knockdown in Bombyx mori.

Our previous research reported the influence of 50 µM selenium (Se) on the cytosolization (endocytosis) pathway, which in turn stimulates the growth and development of Bombyx mori. Lately, dynamin is recognized as one of the key proteins in endocytosis. To explore the underlying mechanisms of Se impact, the dynamin gene was knocked down by injecting siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3). This was followed by an analysis of the target gene and levels of silk protein genes, as well as growth and developmental indices, Se-enrichment capacity, degree of oxidative damage, and antioxidant capacity of B. mori. Our findings showed a considerable decrease in the relative expression of the dynamin gene in all tissues 24 h after the interference and a dramatic decrease in the silkworm body after 48 h. RNAi dynamin gene decreased the silkworm body weight, cocoon shell weight, and the ratio of cocoon. In the meantime, malondialdehyde level increased and glutathione level and superoxide dismutase/catalase activities decreased. 50 µM Se markedly ameliorated these growth and physiological deficits as well as decreases in dynamin gene expression. On the other hand, there were no significant effects on fertility (including produced eggs and laid eggs) between the interference and Se treatments. Additionally, the Se content in the B. mori increased after the dynamin gene interference. The dynamin gene was highly expressed in the silk gland and declined significantly after interference. Among the three siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3), the dynamin-2 displayed the highest interference effects to target gene expression. Our results demonstrated that 50 µM Se was effective to prevent any adverse effects caused by dynamin knockdown in silkworms. This provides practical implications for B. mori breeding industry.

Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics.

BACKGROUND: Human mesenchymal stem cells have attracted interest in regenerative medicine and are being tested in many clinical trials. In vitro expansion is necessary to provide clinical-grade quantities of mesenchymal stem cells; however, it has been reported to cause replicative senescence and undefined dysfunction in mesenchymal stem cells. Quality control assessments of in vitro expansion have rarely been addressed in ongoing trials. Young small extracellular vesicles from the remnant pulp of human exfoliated deciduous teeth stem cells have demonstrated therapeutic potential for diverse diseases. However, it is still unclear whether young small extracellular vesicles can reverse senescence-related declines. RESULTS: We demonstrated that mitochondrial structural disruption precedes cellular dysfunction during bone marrow-derived mesenchymal stem cell replication, indicating mitochondrial parameters as quality assessment indicators of mesenchymal stem cells. Dynamin-related protein 1-mediated mitochondrial dynamism is an upstream regulator of replicative senescence-induced dysfunction in bone marrow-derived mesenchymal stem cells. We observed that the application of young small extracellular vesicles could rescue the pluripotency dissolution, immunoregulatory capacities, and therapeutic effects of replicative senescent bone marrow-derived mesenchymal stem cells. Mechanistically, young small extracellular vesicles could promote Dynamin-related protein 1 translocation from the cytoplasm to the mitochondria and remodel mitochondrial disruption during replication history. CONCLUSIONS: Our findings show that Dynamin-related protein 1-mediated mitochondrial disruption is associated with the replication history of bone marrow-derived mesenchymal stem cells. Young small extracellular vesicles from human exfoliated deciduous teeth stem cells alleviate replicative senescence by promoting Dynamin-related protein 1 translocation onto the mitochondria, providing evidence for a potential rejuvenation strategy.

Acutely blocking excessive mitochondrial fission prevents chronic neurodegeneration after traumatic brain injury.

Progression of acute traumatic brain injury (TBI) into chronic neurodegeneration is a major health problem with no protective treatments. Here, we report that acutely elevated mitochondrial fission after TBI in mice triggers chronic neurodegeneration persisting 17 months later, equivalent to many human decades. We show that increased mitochondrial fission after mouse TBI is related to increased brain levels of mitochondrial fission 1 protein (Fis1) and that brain Fis1 is also elevated in human TBI. Pharmacologically preventing Fis1 from binding its mitochondrial partner, dynamin-related protein 1 (Drp1), for 2 weeks after TBI normalizes the balance of mitochondrial fission/fusion and prevents chronically impaired mitochondrial bioenergetics, oxidative damage, microglial activation and lipid droplet formation, blood-brain barrier deterioration, neurodegeneration, and cognitive impairment. Delaying treatment until 8 months after TBI offers no protection. Thus, time-sensitive inhibition of acutely elevated mitochondrial fission may represent a strategy to protect human TBI patients from chronic neurodegeneration.

Bcl-2 Orthologues, Buffy and Debcl, Can Suppress Drp1-Dependent Age-Related Phenotypes in Drosophila.

The relationship of Amyotrophic Lateral Sclerosis, Parkinson's disease, and other age-related neurodegenerative diseases with mitochondrial dysfunction has led to our study of the mitochondrial fission gene Drp1 in Drosophila melanogaster and aspects of aging. Previously, the Drp1 protein has been demonstrated to interact with the Drosophila Bcl-2 mitochondrial proteins, and Drp1 mutations can lead to mitochondrial dysfunction and neuronal loss. In this study, the Dopa decarboxylase-Gal4 (Ddc-Gal4) transgene was exploited to direct the expression of Drp1 and Drp1-RNAi transgenes in select neurons. Here, the knockdown of Drp1 seems to compromise locomotor function throughout life but does not alter longevity. The co-expression of Buffy suppresses the poor climbing induced by the knockdown of the Drp1 function. The consequences of Drp1 overexpression, which specifically reduced median lifespan and diminished climbing abilities over time, can be suppressed through the directed co-overexpression of pro-survival Bcl-2 gene Buffy or by the co-knockdown of the pro-cell death Bcl-2 homologue Debcl. Alteration of the expression of Drp1 acts to phenocopy neurodegenerative disease phenotypes in Drosophila, while overexpression of Buffy can counteract or rescue these phenotypes to improve overall health. The diminished healthy aging due to either the overexpression of Drp1 or the RNA interference of Drp1 has produced novel Drosophila models for investigating mechanisms underlying neurodegenerative disease.

Mitochondrial fragmentation in early differentiation of human MB135 myoblasts: Role of mitochondrial ROS production in the absence of depolarization.

AIMS: Study of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis. MAIN METHODS: Mitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria. KEY FINDINGS: In immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation. SIGNIFICANCE: Mitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.

Regulation mechanism of GPS2 on PGC-1α/Drp1-mediated mitochondrial dynamics in inflammation of acute lung injury.

Acute lung injury (ALI) has been a hot topic in the field of critical care research in recent years. Mitochondrial dynamics consists of mitochondrial fusion and mitochondrial fission. Dynamin-related protein 1 (Drp1), a key molecule that regulates mitochondrial fission, is important in the oxidative stress and inflammatory response to ALI. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a core protein that mediates mitochondrial biogenesis. G-protein pathway suppressor 2 (GPS2) acts as a transcriptional cofactor with regulatory effects on nuclear-encoded mitochondrial genes. This study aimed to investigate the mechanism of PGC-1α/Drp1-mediated mitochondrial dynamics involved in ALI and to demonstrate the protective mechanism of GPS2 in regulating mitochondrial structure and function and inflammation in ALI. The ALI model was constructed using LPS-induced wild-type mice and human pulmonary microvascular endothelial cells (HPMVECs). It was found that lung injury, oxidative stress and inflammation were exacerbated in the mice ALI model and that mitochondrial structure and function were disrupted in HPMVECs. In vitro studies revealed that LPS led to the upregulated expression of Drp1 and the downregulated expression of PGC-1α and GPS2. Mitochondrial division was reduced and respiratory function was restored in Drp1 knockdown cells, which inhibited oxidative stress and inflammatory response. In addition, the overexpression of PGC-1α and GPS2 significantly inhibited the expression of Drp1, mitochondrial function was restored, and inhibited reactive oxygen species (ROS) production and inflammatory factor release. Moreover, the overexpression of GPS2 promoted the upregulated expression of PGC-1α. This mechanism was also validated in vivo, in which the low expression of GPS2 in mice resulted in the upregulated expression of Drp1 and the downregulated expression of PGC-1α, and further exacerbated LPS-induced ALI. In the present study, we also found that LPS-induced the downregulated expression of GPS2 may be associated with its increased degradation by the proteasome. Therefore, these findings revealed that GPS2 inhibited oxidative stress and inflammation by modulating PGC-1α/Drp1-mediated mitochondrial dynamics to alleviate LPS-induced ALI, which may provide a new approach to the therapeutic orientation for LPS-induced ALI.

Cr(VI) induced hepatocyte apoptosis through the CTH/H2S/Drp1 signaling pathway.

Hexavalent chromium [Cr(VI)] is a highly hazardous heavy metal with multiple toxic effects. Occupational studies indicate that its accumulation in humans can lead to liver damage. However, the exact mechanism underlying Cr(VI)-induced hepatotoxicity remains unknown. In this study, we explored the role of CTH/H2S/Drp1 pathway in Cr(VI)-induced oxidative stress, mitochondrial dysfunction, apoptosis, and liver injury. Our data showed that Cr(VI) triggered apoptosis, accompanied by H2S reduction, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction in both AML12 cells and mouse livers. Moreover, Cr(VI) reduced cystathionine γ-lyase (CTH) and dynamin related protein 1 (Drp1) S-sulfhydration levels, and elevated Drp1 phosphorylation levels at Serine 616, which promoted Drp1 mitochondrial translocation and Drp1-voltage-dependent anion channel 1 (VDAC1) interactions, ultimately leading to mitochondria-dependent apoptosis. Elevated hydrogen sulfide (H2S) levels eliminated Drp1 phosphorylation at Serine 616 by increasing Drp1 S-sulfhydration, thereby preventing Cr(VI)-induced Drp1-VDAC1 interaction and hepatotoxicity. These findings indicated that Cr(VI) induced mitochondrial apoptosis and hepatotoxicity by inhibiting CTH/H2S/Drp1 pathway and that targeting either CTH/H2S pathway or Drp1 S-sulfhydration could serve as a potential therapy for Cr(VI)-induced liver injury.

Novel Drp1 GTPase Inhibitor, Drpitor1a: Efficacy in Pulmonary Hypertension.

BACKGROUND: Drp1 (dynamin-related protein 1), a large GTPase, mediates the increased mitochondrial fission, which contributes to hyperproliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension (PAH). We developed a potent Drp1 GTPase inhibitor, Drpitor1a, but its specificity, pharmacokinetics, and efficacy in PAH are unknown. METHODS: Drpitor1a's ability to inhibit recombinant and endogenous Drp1 GTPase was assessed. Drpitor1a's effects on fission were studied in control and PAH human pulmonary artery smooth muscle cells (hPASMC) and blood outgrowth endothelial cells (BOEC). Cell proliferation and apoptosis were studied in hPASMC. Pharmacokinetics and tissue concentrations were measured following intravenous and oral drug administration. Drpitor1a's efficacy in regressing monocrotaline-PAH was assessed in rats. In a pilot study, Drpitor1a reduced PA remodeling only in females. Subsequently, we compared Drpitor1a to vehicles in control and monocrotaline-PAH females. RESULTS: Drp1 GTPase activity was increased in PAH hPASMC. Drpitor1a inhibited the GTPase activity of recombinant and endogenous Drp1 and reversed the increased fission, seen in PAH hPASMC and PAH BOEC. Drpitor1a inhibited proliferation and induced apoptosis in PAH hPASMC without affecting electron transport chain activity, respiration, fission/fusion mediator expression, or mitochondrial Drp1 translocation. Drpitor1a did not inhibit proliferation or alter mitochondrial dynamics in normal hPASMC. Drpitor1a regressed monocrotaline-PAH without systemic vascular effects or toxicity. CONCLUSIONS: Drpitor1a is a specific Drp1 GTPase inhibitor that reduces mitochondrial fission in PAH hPASMC and PAH BOEC. Drpitor1a reduces proliferation and induces apoptosis in PAH hPASMC and regresses monocrotaline-PAH. Drp1 is a therapeutic target in PAH, and Drpitor1a is a potential therapy with an interesting therapeutic sexual dimorphism.

Role of Excessive Mitochondrial Fission in Seawater Immersion Aggravated Hemorrhagic Shock-Induced Cardiac Dysfunction and the Protective Effect of Mitochondrial Division Inhibitor-1.

Aims: Seawater immersion significantly aggravated organ dysfunction following hemorrhagic shock, leading to higher mortality rate. However, the effective treatment is still unavailable in clinic. Mitochondria were involved in the onset and development of multiple organ function disorders; whether mitochondria participate in the cardiac dysfunction following seawater immersion combined with hemorrhagic shock remains poorly understood. Hence, we investigated the role and possible mechanism of mitochondria in seawater immersion combined with hemorrhage shock-induced cardiac dysfunction. Results: Mitochondrial fission protein dynamin-related protein 1 (Drp1) was activated and translocated from the cytoplasm to mitochondria in the heart following seawater immersion combined with hemorrhagic shock, leading to excessive mitochondrial fission. Excessive mitochondrial fission disrupted mitochondrial function and structure and activated mitophagy and apoptosis. At the same time, excessive mitochondrial fission resulted in disturbance of myocardial structure and hemodynamic disorders and ultimately provoked multiple organ dysfunction and high mortality. Further studies showed that the mitochondrial division inhibitor mitochondrial division inhibitor-1 can significantly reverse Drp1 mitochondrial translocation and inhibit mitochondrial fragmentation, reactive oxygen species (ROS) accumulation, mitophagy, and apoptosis and then protect circulation and vital organ functions, prolonging animal survival. Innovation: Our findings indicate that Drp1-mediated mitochondrial fission could be a novel therapeutic targets for the treatment of seawater immersion combined with hemorrhagic shock. Conclusion: Drp1 mitochondrial translocation played an important role in the cardiac dysfunction after seawater immersion combined with hemorrhage shock. Drp1-mediated excessive mitochondrial fission leads to cardiac dysfunction due to the mitochondrial structure and bioenergetics impairment.

PINK1 insufficiency can be exploited as a specific target for drug combinations inducing mitochondrial pathology-mediated cell death in gastric adenocarcinoma.

There exist very limited non-hazardous therapeutic strategies except for surgical resection and lymphadenectomy against gastric cancer (GC) despite being the third leading cause of cancer deaths worldwide. This study proposes an innovative treatment approach against GC using a drug combination strategy that manipulates mitochondrial dynamics in conjunction with the induction of mitochondrial pathology-mediated cell death. Comparative analysis was done with gastric adenocarcinoma and normal cells by qPCR, western blot, microscopic immunocytochemistry, and live cell imaging. In this study, impairment of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission by Mdivi-1 created an imbalance in mitochondrial structural dynamics in indomethacin-treated AGS cells in which mitophagy-regulator protein PINK1 is downregulated. These drug combinations with the individual sub-lethal doses ultimately led to the activation of cell death machinery upregulating pro-apoptotic proteins like Bax, Puma, and Noxa. Interestingly, this combinatorial therapy did not affect normal gastric epithelial cells significantly and also no significant upregulation of death markers was observed. Moreover, the drug combination strategy also retarded cell migration and reduced stemness in GC cells. In summary, this study offers a pioneering specific therapeutic strategy for GC treatment, sparing normal cells providing opportunities for minimal drug-mediated toxicity utilizing mitochondria as a viable and specific target for anti-cancer therapy in gastric cancer.

Mechanistic insights into metabolic function of dynamin-related protein 1.

Dynamin-related protein 1 (DRP1) plays crucial roles in mitochondrial and peroxisome fission. However, the mechanisms underlying the functional regulation of DRP1 in adipose tissue during obesity remain unclear. To elucidate the metabolic and pathological significance of diminished DRP1 in obese adipose tissue, we utilized adipose tissue-specific DRP1 KO mice challenged with a high-fat diet. We observed significant metabolic dysregulations in the KO mice. Mechanistically, DRP1 exerts multifaceted functions in mitochondrial dynamics and endoplasmic reticulum (ER)-lipid droplet crosstalk in normal mice. Loss of function of DRP1 resulted in abnormally giant mitochondrial shapes, distorted mitochondrial membrane structure, and disrupted cristae architecture. Meanwhile, DRP1 deficiency induced the retention of nascent lipid droplets in ER, leading to perturbed overall lipid dynamics in the KO mice. Collectively, dysregulation of the dynamics of mitochondria, ER, and lipid droplets contributes to whole-body metabolic disorders, as evidenced by perturbations in energy metabolites. Our findings demonstrate that DRP1 plays diverse and critical roles in regulating energy metabolism within adipose tissue during the progression of obesity.

Structural basis for GTPase activity and conformational changes of the bacterial dynamin-like protein SynDLP.

SynDLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length SynDLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of SynDLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular SynDLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional SynDLP assembly states during GTP turnover.

Polyphenol (-)-Epigallocatechin Gallate (EGCG) mitigated kidney injury by regulating metabolic homeostasis and mitochondrial dynamics involvement with Drp1-mediated mitochondrial fission in mice.

The study aimed to examine effects of (-)-epigallocatechin-3-gallate (EGCG) on energy metabolism and mitochondrial dynamics in mouse model of renal injury caused by doxorubicin (DOX). Here, mice were divided into Control group, EGCG-only treated group, DOX group, and three doses of EGCG plus DOX groups. Our results showed that EGCG behaved beneficial effects against kidney injury via attenuation of pathological changes in kidney tissue, which was confirmed by reducing serum creatinine (SCr), blood urea nitrogen (BUN), and apoptosis. Subsequently, changes in reactive oxygen species generation, malondialdehyde content, and activities of antioxidant enzymes were considerably ameliorated in EGCG + DOX groups when compared to DOX group. Furthermore, EGCG-evoked renal protection was associated with increases of mitochondrial membrane potential and decreases of mitochondrial fission protein Dynamin-related protein 1 (Drp1). Moreover, changing glycolysis into mitochondrial oxidative phosphorylation was observed, evidenced by controlling activities of malate dehydrogenase (MDH) and hexokinase (HK) in EGCG + DOX groups when compared to DOX group, indicating that reprogramming energy metabolism was linked to EGCG-induced renal protection in mice. Therefore, EGCG was demonstrated to have a protective effect against kidney injury by reducing oxidative damage, metabolic disorders, and mitochondrial dysfunction, suggesting that EGCG has potential as a feasible strategy to prevent kidney injury.

[Effect of Csn-B Combined with Imatinib on Apoptosis of Chronic Myeloid Leukemia Cells through Nur77/Pim-1/Drp1 Pathway].

OBJECTIVE: To investigate the anti- chronic myelogenous leukemia (CML) activity of Nur77-specific agonist Csn-B combined with imatinib by promoting Nur77 expression, and explore the potential role of its signaling pathway. METHODS: Firstly, CCK-8 and Transwell assay were used to detect the inhibitory effects of Csn-B, imatinib, and their combination on the proliferation and migration of K562 cells. Furthermore, the apoptosis rate of K562 cells treated with Csn-B, imatinib, and their combination was detected by flow cytometry. The expression levels of Nur77, Pim-1, Drp1, p-Drp1 S616, Bcl-2 and Bax in K562 cells were detected by Western blot. Finally, the expression levels of reactive oxygen species (ROS) in K562 cells treated with Csn-B, imatinib and their combination were detected by immunofluorescence assay. RESULTS: The level of Nur77 in CML patients decreased significantly compared with normal population in dataset of GSE43754 (P < 0.001). Csn-B combined with imatinib could significantly inhibit the proliferation and migration of K562 cells (both P < 0.001), and induce apoptosis (P < 0.001). Csn-B promoted Nur77 expression in K562 cells, and synergistically enhanced imatinib sensitivity when combined with imatinib. Csn-B combined with imatinib could significantly enhanced ROS levels in K562 cells and mitochondria compared with single-drug treatment (both P < 0.001). CONCLUSION: Csn-B combined with imatinib can enhance ROS expression and induce apoptosis of K562 cells through Nur77/Pim-1/Drp1 pathway.

Inhibiting the NF-κB/DRP1 Axis Affords Neuroprotection after Spinal Cord Injury via Inhibiting Polarization of Pro-Inflammatory Microglia.

BACKGROUND: Spinal cord injury (SCI) is considered a central nervous system (CNS) disorder. Nuclear factor kappa B (NF-κB) regulates inflammatory responses in the CNS and is implicated in SCI pathogenesis. The mechanism(s) through which NF-κB contributes to the neuroinflammation observed during SCI however remains unclear. METHODS: SCI rat models were created using the weight drop method and separated into Sham, SCI and SCI+NF-κB inhibitor groups (n = 6 rats per-group). We used Hematoxylin-Eosin Staining (H&E) and Nissl staining for detecting histological changes in the spinal cord. Basso-Beattie-Bresnahan (BBB) behavioral scores were utilized for assessing functional locomotion recovery. Mouse BV2 microglia were exposed to lipopolysaccharide (LPS) to mimic SCI-induced microglial inflammation in vitro. RESULTS: Inhibition of NF-κB using JSH-23 alleviated inflammation and neuronal injury in SCI rats' spinal cords, leading to improved locomotion recovery (p < 0.05). NF-κB inhibition reduced expression levels of CD86, interleukin-6 (IL-6), IL-1ß, and inducible Nitric Oxide Synthase (iNOS), and improved expression levels of CD206, IL-4, and tissue growth factor-beta (TGF-ß) in both LPS-treated microglia and SCI rats' spinal cords (p < 0.05). Inhibition of NF-κB also effectively suppressed mitochondrial fission, evidenced by the reduced phosphorylation of dynamin-related protein 1 (DRP1) at Ser616 (p < 0.001). CONCLUSION: We show that inhibition of the NF-κB/DRP1 axis prevents mitochondrial fission and suppresses pro-inflammatory microglia polarization, promoting neurological recovery in SCI. Targeting the NF-κB/DRP1 axis therefore represents a novel approach for SCI.

RIPK3 causes mitochondrial dysfunction and albuminuria in diabetic podocytopathy through PGAM5-Drp1 signaling.

BACKGROUND: Receptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD. METHODS: We analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3. RESULTS: RIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05-4.98]) and incident chronic kidney disease (HR 4.08 [1.10-15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1. CONCLUSIONS: The study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.

SIRT6 prevent chronic cerebral hypoperfusion induced cognitive impairment by remodeling mitochondrial dynamics in a STAT5-PGAM5-Drp1 dependent manner.

Vascular dementia (VaD) is a prevalent form of dementia resulting from chronic cerebral hypoperfusion (CCH). However, the pathogenic mechanisms of VaD and corresponding therapeutic strategies are not well understood. Sirtuin 6 (SIRT6) has been implicated in various biological processes, including cellular metabolism, DNA repair, redox homeostasis, and aging. Nevertheless, its functional relevance in VaD remains unexplored. In this study, we utilized a bilateral common carotid artery stenosis (BCAS) mouse model of VaD to investigate the role of SIRT6. We detected a significant decrease in neuronal SIRT6 protein expression following CCH. Intriguingly, neuron-specific ablation of Sirt6 in mice exacerbated neuronal damage and cognitive deficits after CCH. Conversely, treatment with MDL-800, an agonist of SIRT6, effectively mitigated neuronal loss and facilitated neurological recovery. Mechanistically, SIRT6 inhibited excessive mitochondrial fission by suppressing the CCH-induced STAT5-PGAM5-Drp1 signaling cascade. Additionally, the gene expression of monocyte SIRT6 in patients with asymptomatic carotid stenosis showed a correlation with cognitive outcomes, suggesting translational implications in human subjects. Our findings provide the first evidence that SIRT6 prevents cognitive impairment induced by CCH, and mechanistically, this protection is achieved through the remodeling of mitochondrial dynamics in a STAT5-PGAM5-Drp1-dependent manner.

Drp1 depletion protects against ferroptotic cell death by preserving mitochondrial integrity and redox homeostasis.

Mitochondria are highly dynamic organelles which undergo constant fusion and fission as part of the mitochondrial quality control. In genetic diseases and age-related neurodegenerative disorders, altered mitochondrial fission-fusion dynamics have been linked to impaired mitochondrial quality control, disrupted organelle integrity and function, thereby promoting neural dysfunction and death. The key enzyme regulating mitochondrial fission is the GTPase Dynamin-related Protein 1 (Drp1), which is also considered as a key player in mitochondrial pathways of regulated cell death. In particular, increasing evidence suggests a role for impaired mitochondrial dynamics and integrity in ferroptosis, which is an iron-dependent oxidative cell death pathway with relevance in neurodegeneration. In this study, we demonstrate that CRISPR/Cas9-mediated genetic depletion of Drp1 exerted protective effects against oxidative cell death by ferroptosis through preserved mitochondrial integrity and maintained redox homeostasis. Knockout of Drp1 resulted in mitochondrial elongation, attenuated ferroptosis-mediated impairment of mitochondrial membrane potential, and stabilized iron trafficking and intracellular iron storage. In addition, Drp1 deficiency exerted metabolic effects, with reduced basal and maximal mitochondrial respiration and a metabolic shift towards glycolysis. These metabolic effects further alleviated the mitochondrial contribution to detrimental ROS production thereby significantly enhancing neural cell resilience against ferroptosis. Taken together, this study highlights the key role of Drp1 in mitochondrial pathways of ferroptosis and expose the regulator of mitochondrial dynamics as a potential therapeutic target in neurological diseases involving oxidative dysregulation.