Expression Of PINK1 Research Articles

FGD5-AS1/miR-5590-3p/PINK1 induces Lenvatinib resistance in hepatocellular carcinoma

BackgroundLenvatinib is a common systemic treatment for advanced hepatocellular carcinoma (HCC), the resistance to which presents a great challenge. However, the mechanism of lenvatinib resistance in HCC remains unclear. Therefore, elucidating the underlying and key regulatory molecular mechanisms of lenvatinib resistance is urgently needed. MethodsBioinformatic enrichment analysis was used to investigate the gene associated with lenvatinib resistance. RT-PCR, Western blot, immunohistochemistry, and luciferase assays were used to explore the mechanisms of lenvatinib resistance. The effects of the FGD5-AS1/miR-5590-3p/PINK1 axis on lenvatinib resistance were evaluated by colony formation assay, cell viability, apoptosis, mitochondrial homeostasis, and morphology analyses. ResultsHigher expression of PINK1 was observed in lenvatinib-resistant cells and tissues. PINK1 could be activated by increased FGD5-AS1 expression, thereby maintaining the mitochondrial structure and function and promoting the antioxidative stress response. FGD5-AS1/miR-5590-3p showed competitive regulation of PINK1, which affected lenvatinib sensitivity through regulation of mitochondrial structure and antioxidative stress. ConclusionsPINK1 was identified as a key gene leading to lenvatinib resistance by maintaining the mitochondrial structure and function. The FGD5-AS1/miR-5590-3p/PINK1 axis may be a promising strategy to overcome lenvatinib resistance in treatment-negative patients.

Identification of Rab7 as an autophagy marker: potential therapeutic approaches and the effect of Qi Teng Xiao Zhuo granule in chronic glomerulonephritis

Context Qi Teng Xiao Zhuo granule (QTXZG) is a traditional Chinese medicine (TCM) used for therapeutic effects on chronic glomerulonephritis (CGN). However, the underlying mechanism remains unclear. Objective To investigate the molecular mechanism of QTXZG on CGN by proteomics. Materials and methods The CGN model was induced in Sprague–Dawley rats by injecting adriamycin (3.5 mg/kg, Day 1; 3.0 mg/kg, Day 14) twice through the tail vein. Urine samples were collected on the 21st day; and the rats divided randomly into control, adriamycin, QTXZG administration groups. Rats in the QTXZG group received QTXZG (10.8 g/kg); control and adriamycin groups were given physiological saline once per day for 30 days. Proteomics was applied to identify the candidate proteins combined with autophagy database and verified by immunofluorescence (IF) and western blots (WB). Results 278 differentially expressed proteins (DEPs) were identified based on proteomics and Rab7 was screened as an autophagy protein biomarker. In vitro cell experiments, we found that QTXZG (20%, IC50 = 23.47%) could decrease the expression of NLRP3, Caspase-1, IL-18, IL-1β, while increasing the expression of Pink1, Parkin, Rab7, Podocalyxin. The cell apoptosis rate increased from 6.68 ± 0.07 to 11.03 ± 0.36%. Overexpression of Rab7 resulted in an increase in autophagy relevant protein expression. Discussion and conclusion TCM CGN-regulating herbs (QTXZG) can exert therapeutic effects by affecting the Rab7/Pink1/Parkin pathway to promote mitochondrial autophagy. New breakthroughs in targeted Rab7 may eventually enable such applications.

Resveratrol Inhibits Zinc Deficiency-Induced Mitophagy and Exerts Cardiac Cytoprotective Effects.

Resveratrol (Res) possesses various beneficial effects, including cardioprotective, anti-inflammatory, anti-aging, and antioxidant properties. However, the precise mechanism underlying these effects remains unclear. Here we investigated the protective effects of resveratrol on cardiomyocytes, focusing on the role of Zn2+ and mitophagy. Using the MTT/lactate dehydrogenase assay, we found that addition of a zinc chelator TPEN for 4h induced mitophagy and resulted in a significant reduction in cell viability, increased cytotoxicity, and apoptosis in H9c2 cells. Notably, resveratrol effectively mitigated these detrimental effects caused by TPEN. Similarly, Res inhibited the TPEN-induced expression of mitophagy-associated proteins, namely P62, LC3, NIX, TOM20, PINK1, and Parkin. The inhibitory action of resveratrol on mitophagy was abrogated by the mitophagy inhibitor 3-MA. Additionally, we discovered that silencing of the Mfn2 gene could reverse the inhibitory effects of resveratrol on mitophagy via the AMPK-Mfn2 axis, thereby preventing the opening of the mitochondrial permeability transition pore (mPTP). Collectively, our data suggest that Res can safeguard mitochondria protection by impeding mitophagy and averting mPTP opening through the AMPK-Mfn2 axis in myocardial cells.

Effect of ligustrazine on hypoxic-ischemic encephalopathy in neonatal rats by regulating autophagy through the PINK1/Parkin pathway

To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism. Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62). Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05). Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.

Protective Effect of Astragaloside IV against Cadmium-Induced Damage on Mouse Renal Podocytes (MPC5).

In this study, we investigated the protective effect of Astragaloside IV (Ast) on mouse podocytes and its possible mechanism of action by constructing a cadmium-induced mouse renal podocytes model. We investigated the effects of cadmium (Cd) toxicity on cell number, morphology, the mitochondrial status of subcellular organelles, protein and gene levels, and the protective effects of Ast by constructing a model of Cd-induced damage to mouse renal podocytes (MPC5) and giving Ast protection at the same time. The results showed that exposure of MPC5 cells to CdCl2 culture medium containing 6.25 μM concentration acted with low cell mortality, but the mortality of MPC5 cells increased with the prolongation of cadmium exposure time. Given Ast, the death rate in the low dose group (12.5 μM) was significantly reduced, while the death rate in the medium dose group (25 μM) was extremely significantly reduced. In comparison to the control group, the Cd-exposed group exhibited a significant increase of 166.7% in malondialdehyde (MDA) content and a significant decrease of 17.1% in SOD activity. The mitochondrial membrane potential was also reduced to varying degrees. However, in the Ast-protected group compared to the Cd-exposed group, the MDA content significantly decreased by 20.8%, the SOD activity decreased by 7.14%, and the mitochondrial membrane potential showed a significant increase. Fluorescence staining of mitochondrial membrane potential indicated that Cd exposure caused mitochondrial apoptosis. In the 12-h cadmium-exposed group, the protein expression of Nephrin in mice significantly decreased by 33.4%. However, the expression of the Desmin protein significantly increased by 67.8%, and the expression of the autophagy protein LC3-II significantly increased by 55.5%. Meanwhile, the expression of PINK1, a mitochondrial autophagy pathway protein, was significantly increased in the 12 h and 24 h cadmium exposure groups. The mRNA level of PINK1 was significantly increased, and that of Parkin was decreased in the 48 h cadmium exposure group. Compared to the Cd-exposed group, the Ast group showed more significant improvements in the expression of podocyte structure, functional proteins, and mitochondrial autophagy pathway proteins. The immunological assay of mitochondrial autophagic pathway proteins further indicated that Cd-induced damage to MPC5 cells might be associated with the dysregulation of mitochondrial autophagy.

PINK1 and oxidative stress in lean and obese patients with type 2 diabetes mellitus

AimTo compare mRNA [messenger RNA] expression of PINK1 in whole blood and the levels of biomarkers of Oxidative Stress (mitochondrial DNA [mtDNA] content & Total Antioxidant status [TAS]) in newly diagnosed lean and obese patients with T2DM. MethodsNewly diagnosed patients of T2DM were enrolled in this study. The patients were divided into two groups of 30 patients each, lean (BMI < 18.5 kg/m2) and obese (BMI > 25 kg/m2). mRNA expression of PINK1 & mtDNA content was measured by real time PCR. Serum TAS was measured using a commercially available kit. ResultsThere was a 1.78-fold decrease in mRNA expression of PINK1 in obese group compared to the lean group. Mean mtDNA content was 300.82 ± 169.66 in the obese group and 332.78 ± 147.07 in the lean group (p = 0.06). Mean levels of TAS was 5.39 ± 2.28 μM Trolox Equivalents in the obese group and 3.85 ± 3.33 μM Trolox Equivalents in the lean group (p = 0.001). ConclusionThe T2DM patient with obesity had greater OS than the lean patients. Thus, there is a compensatory increase in antioxidants in obese patients with T2DM. Our findings also suggest that decreased levels of PINK1 in obese group are unable to protect the mitochondria against OS leading to decreased mtDNA content. Does it also result in beta cell dysfunction or contribute to insulin resistance in obese patients with T2DM needs to be explored.

#4478 HIF-1α REGULATED LNCRNA-ATP6V0E2-AS1 MEDIATES HYPOXIA-INDUCED MITOPHAGY IN RENAL TUBULAR CELLS

Abstract Background and Aims Mitophagy activation is crucial for hypoxia signaling in acute kidney injury (AKI). Selective removal of damaged mitochondria is mediated through a concerted function of coding and non-coding RNA expressions. Insights on HIF-1α signaling in lncRNA-regulated mitophagy mechanism are not known. The study aims to investigate the role of hypoxia-inducible factor 1α (HIF-1α) regulated long non-coding RNA (lncRNA) ATP6V0E2-AS1 in acute kidney injury (AKI) by examining its effect on mitophagy. Method Mitophagy proteins (PINK1, p-PARKIN, LC3) were determined through western blot analysis. Immunofluorescence studies of PINK1 mitochondrial translocation and LC3/TOMM20 localization were determined using confocal analysis. HIF-1α interaction with lncRNA-ATP6V0E2-AS1 through promoter binding activity was evaluated through chromatin immunoprecipitation analysis. RNA interaction between lncRNA-ATP6V0E2-AS1 and PINK1 was analyzed by dual-luciferase reporter assay. Results Hypoxia upregulated PINK1, p-PARKIN, LC3 expressions, and mitophagosome in HK-2 cells. Overexpression and knockdown studies of hypoxia-regulated lncRNA-ATP6V0E2-AS1 significantly regulated PINK1/p-PARKIN expressions, subsequent PINK1 mitochondrial co-localization, and mitophagosome formation. shHIF-1α knockdown in HK-2 cells reveals that HIF-1α mediates PINK1/p-PARKIN mitophagy regulation under hypoxic conditions. In detail, HIF-1α binds to the promoter of lncRNA-ATP6V0E2-AS1 and down-regulates its expression under hypoxia. Deficiency of both HIF-1α and lncRNA-ATP6V0E2-AS1 reverted shHIF-1α mediated suppression of PINK1 expression and mitophagosome formation. Further, ATP6V0E2-AS1 post-transcriptionally regulates PINK1 expression by RNA-RNA interaction. Conclusion Altogether, our study shows novel findings on the HIF-1α mediated lncRNA-ATP6V0E2-AS1 in hypoxia-induced mitophagy regulation in the renal tubular cells. Thus, downregulated ATP6V0E2-AS1 expression with subsequent mitophagy activation is critical for hypoxia-induced mitophagy regulation through HIF-1α/ATP6V0E2-AS1/PINK1 axis.

#4990 OMEGA-3 FATTY ACID ACTIVATES MITOCHONDRIAL BIOGENESIS AND PINK1 DEPENDENT MITOPHAGY IN KIDNEY AND HEART OF ADENINE INDUCED UREMIC RATS

Abstract Background and Aims Mitochondrial homeostasis is controlled by biogenesis, dynamics, and mitophagy. Mitochondrial dysfunction plays a central role in cardiovascular and renal disease and omega-3 fatty acids (FA) are beneficial for cardiovascular disease. We aimed to investigate whether omega-3 FA regulates the expression of mediators of mitochondrial biogenesis, dynamics, and mitophagy in kidney and heart of adenine-induced uremic rats. Method Sprague-Dawley rats were divided into three groups: normal control, adenine control, and omega-3 FA. Uremia was induced by feeding diets containing 0.75% adenine and 2.5% protein for the first 3 weeks. During next 4 weeks, they were received 2.5% protein with or without omega-3 FA (300 mg/kg/day). The renal and cardiac expression of PGC-1α, SIRT1/3, Nrf2, DRP-1, OPA1, Mfn1/2, PINK1, BNIP3 and NIX were examined by western blot analysis. The qPCR was used to determine mitochondrial DNA (mtDNA). Results Compared to normal, serum creatinine and heart weight/body weight in adenine control was increased and improved in omega-3 FA group. Compared with normal, PGC-1α, SIRT1/3 and Nrf2 were down-regulated in kidney and heart of adenine control. PGC-1α expression of kidney and heart was recovered in omega-3 FA group. DRP-1 of kidney was up-regulated but DRP-1 of heart was down-regulated in adenine control. DRP-1 of heart was recovered in omega-3 FA group. PINK1, BNIP3 and NIX were down-regulated in heart of adenine control and recovered in omega-3 FA group. PINK1 was down-regulated but BNIP3 and NIX were up-regulated in kidney of adenine control and those were mitigated in omega-3 FA group. MtDNA was decreased in kidney and heart of adenine control group but mtDNA of heart was recovered in omega-3 FA group. Conclusion DRP-1 related with mitochondrial fission may oppositely work in uremic kidney and heart. Omega-3 FA is beneficial for mitochondrial homeostasis by activating mitochondrial biogenesis and PINK1 dependent mitophagy in kidney and heart of uremic rats.

Zinc oxide nanoparticles induce toxicity in diffuse large B-cell lymphoma cell line U2932 via activating PINK1/Parkin-mediated mitophagy.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma. Zinc oxide (ZnO) nanoparticles have excellent anti-tumor properties in the biomedical field. The present study aimed to explore the underlying mechanism by which ZnO nanoparticles induce toxicity in DLBCL cells (U2932) via the PINK1/Parkin-mediated mitophagy pathway. After U2932 cells were exposed to various concentrations of ZnO nanoparticles, the cell survival rate, reactive oxygen species (ROS) generation, cell cycle arrest, and changes in the expression of PINK1, Parkin, P62, and LC3 were monitored. Moreover, we investigated monodansylcadaverine (MDC) fluorescence intensity and autophagosome and further validated the results using the autophagy inhibitor 3-methyladenine (3-MA). The results showed that ZnO nanoparticles could effectively inhibit the proliferation of U2932 cells and induce cell cycle arrest at the G0/G1 phases. Moreover, ZnO nanoparticles significantly increased ROS production, MDC fluorescence intensity, autophagosome formation, and the expression of PINK1, Parkin, and LC3, and decreased the expression of P62 in U2932 cells. In contrast, the autophagy level was reduced after the intervention of the 3-MA. Overall, ZnO nanoparticles can trigger PINK1/Parkin-mediated mitophagy signaling in U2932 cells, which may be a potential therapeutic approach for DLBCL.

Targeted Central Nervous System Irradiation with Proton Microbeam Induces Mitochondrial Changes in Caenorhabditis elegans.

Fifty percent of all patients with cancer worldwide require radiotherapy. In the case of brain tumors, despite the improvement in the precision of radiation delivery with proton therapy, studies have shown structural and functional changes in the brains of treated patients with protons. The molecular pathways involved in generating these effects are not completely understood. In this context, we analyzed the impact of proton exposure in the central nervous system area of Caenorhabditis elegans with a focus on mitochondrial function, which is potentially implicated in the occurrence of radiation-induced damage. To achieve this objective, the nematode C. elegans were micro-irradiated with 220 Gy of protons (4 MeV) in the nerve ring (head region) using the proton microbeam, MIRCOM. Our results show that protons induce mitochondrial dysfunction, characterized by an immediate dose-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with oxidative stress 24 h after irradiation, which is itself characterized by the induction of the antioxidant proteins in the targeted region, observed using SOD-1::GFP and SOD-3::GFP strains. Moreover, we demonstrated a two-fold increase in the mtDNA copy number in the targeted region 24 h after irradiation. In addition, using the GFP::LGG-1 strain, an induction of autophagy in the irradiated region was observed 6 h following the irradiation, which is associated with the up-regulation of the gene expression of pink-1 (PTEN-induced kinase) and pdr-1 (C. elegans parkin homolog). Furthermore, our data showed that micro-irradiation of the nerve ring region did not impact the whole-body oxygen consumption 24 h following the irradiation. These results indicate a global mitochondrial dysfunction in the irradiated region following proton exposure. This provides a better understanding of the molecular pathways involved in radiation-induced side effects and may help in finding new therapies.

Protective effect of Tongdu Tiaoshen acupuncture combined with Xiaoxuming decoction on dopaminergic neurons in Parkinson's disease model.

To explore the possible mechanism of Tongdu Tiaoshen acupuncture combined with Xiaoxuming decoction (, XXMD) in the treatment of Parkinson's disease (PD). C57BL/6 mice were randomly divided into eight groups ( 12), including blank group, model group, medication group, acupuncture group, high-dose XXMD group (XXMD-H), low-dose XXMD group (XXMD-L), acupuncture combined with high-dose XXMD group (A+H), and acupuncture combined with low-dose XXMD group (A+L). After treatment for 6 weeks, dopamine (DA) neurons and the pathological changes of tyrosine hydroxylase (TH) positive cells were observed. The enzyme-linked immunosorbent assay (ELISA) was used to measure the content of DA and the level of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). The mRNA level of PINK1 and Parkin and the protein expression of Nix, PINK1 and Parkin in the substantia nigra were also detected. Combination treatment effectively ameliorated the symptoms of PD. Compared with model group, combined treatment significantly up-regulated the protein expression of Nix, Parkin and PINK1 and the mRNA levels of PINK1 and Parkin in the substantia nigra (<0.0001, <0.001, <0.01 or <0.05). Furthermore, the levels of pro-inflammation cytokines were obviously decreased after combination therapy, while IL-10 content was increased remarkably (<0.01). Compared with each treatment alone, combination therapy improved the pathological damage of DA neurons of PD mice more effectively. The possible mechanism may be attributed to the up-regulated level of mitochondrial autophagy and improved mitochondrial function. These results provide fresh insight into the mechanism of co-treatment with Tongdu Tiaoshen acupuncture and XXMD for PD.

ASTRAGALOSIDE IV IMPROVED OXIDATIVE STRESS INDUCED INJURY THROUGH PINK1/PARKIN-MEDIATED MITOPHAGY IN H9C2 CELLS

Objective: To explore the mechanisms of Astragaloside IV on improving the oxidative stress induced injury in H9c2 cells. Design and method: H9c2 cells were divided into normal control group (cultured without intervention), H2O2 model group (cultured with 200umol/L H2O2 for 2 hours), As-IV with H2O2 group (pretreated with 100umol/L As-IV for 1 hour, then incubated with H2O2 for 2 hours) and Mdivi-1 with H2O2 group (pretreated with 5umol/L Mdivi-1 for 1 hour, then incubated with H2O2 for 2 hours). The activity of lactate dehydrogenase (LDH) in cell culture medium was measured to evaluate the degree of oxidative damage of cells, and the protein levels of Bax, Bcl-2, Drp1, LC3, PINK1 and Parkin in each group were detected by Western blot. The statistical analysis of the experimental results was carried out by SPSS13.0 software. Results: 1. Apoptosis of cells in each group: Compared with the normal control group, the expression of Bax in the H2O2 group increased (P&lt;0.05), and the expression of Bcl-2 decreased [P&lt;0.05]; compared with H2O2 group, the expression of Bax in As-IV group decreased (P&lt;0.05), Bcl-2 The expression increased (P&lt;0.05). 2. Oxidative stress damage of cells in each group: Compared with the normal control group, the LDH activity of cells in the H2O2 group increased, and compared with the H2O2 group Compared with that, the LDH activity of As-IV group decreased. 3. Mitochondrial dynamic expression: Compared with the normal control group, the expression of Drp1 in the H2O2 group increased; compared with the H2O2 group, the expression of Drp1 in the As-IV group and the Mdivi-1 group decreased. 4. The expression of mitophagy-related proteins in each group: Compared with the normal control group, the ratio of autophagy-related proteins LC3-II/LC3-I in the H2O2 group increased, and the expression of PINK1 increased (P&lt;0.05), the expression of Parkin was significantly increased (P&lt;0.05); compared with the H2O2 group, LC3-II/LC3-I in As-IV group and Mdivi-1 group decreased, the expression of PINK1 and Parkin proteins in As-IV group and Mdivi-1 group decreased . Conclusions: As-IV inhibited mitophagy through the PINK1/Parkin pathway, and alleviated oxidative stress induced injury in H9c2 cells.

THE EMERGING ROLE OF IDH2-MEDIATED MITOPHAGY AND MTUPR IN ATHEROSCLEROSIS

Objective: Mitochondrial dysfunction is a risk factor for vascular disease by overexpressing ROS caused by some stimuli in mitochondria which causes oxidative stress and enhances vascular inflammatory response. Isocitrate dehydrogenase 2 (IDH2) is NADP+ dependent mitochondrial enzyme and an antioxidant enzyme that produces NADPH in the antioxidant system. Mitophagy and mitochondrial Unfolding Protein Response (mtUPR) are internal defense mechanism in mitochondria. In this study, we investigated whether IDH2 knockdown causes mitochondrial dysfunction then Mitophagy and mtUPR in vitro in HUVECs and in vivo in IDH2 knock out mice. Design and method: We examined expression of mitophagy and mtUPR-related gene in IDH2-deficient human umbilical vein endothelial cells (HUVECs) and its role in mitophagy and mtUPR in the endothelial cell and IDH2 KO animal tissues. Mitophagy and mtUPR were measured using the western blotting and qPCR. Results: We showed that knockdown of IDH2 expression induced depolarization of mitochondrial membrane potential (MMP). Mitochondrial dynamics is mitochondrial fusion and fission. Knockdown of IDH2 increased Drp1 (fission protein) and mfn1 (fusion protein) compared with Tom20 (control). IDH2 deficiency increase Mitophagy related protein PINK-1 and Parkin expression and mRNA level (PINK-1, Parkin, BNIP3, NIX, FUNDC-1). Moreover, knockdown of IDH2 induced mtUPR mRNA level (USP30, Clpp) in vitro. In addition, IDH2 deficiency increases mtUPR mRNA level and decreases PINK-1 and Parkin protein expression in vivo. Conclusions: Our data show that IDH2 deficiency induces mitochondrial dysfunction and then Mitophagy and mtUPR expression in endothelial cells. These findings provide novel strategy for the development of therapeutic agents for restoring mitochondrial and endothelial function.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy.

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 µM) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Silibinin alleviates ferroptosis of rat islet β cell INS-1 induced by the treatment with palmitic acid and high glucose through enhancing PINK1/parkin-mediated mitophagy

Type 2 diabetes (T2DM) is induced by the abundance of glucose and lipids, which causes glucolipotoxicity to the pancreatic β-cells. Silibinin is a natural flavonoid possessing the regulatory activity on insulin production and therapeutic activity in diabetic mice; however, its effect on glucolipotoxicity is not fully explained. This in vitro study investigates the effects of silibinin on palmitic acid (PA) and high glucose (HG)-induced cell loss and ferroptosis of rat insulinoma INS-1 cells. In the cells treated with PA and HG, expressions of glucose transporter 4 (Glut4) and carnitine acyltransferase I (CPT1) for β-oxidation of fatty acids are reduced. Mitochondria are the metabolic organelles for glucose and fatty acids. The mitochondrial membrane potential (MMP) and ATP production were decreased, while the ROS level was elevated in the cells treated with PA and HG, indicating an induction of mitochondrial disorder. Cell loss was partially rescued by ferroptosis inhibition, suggesting an involvement of ferroptosis in the cells treated with PA and HG. More importantly, the increases in total iron, lipid ROS, MDA and COX-2, and the decrease in ferroptosis inhibitory molecules GSH, GPX4 and FSP1 appeared in the cells treated with PA and HG, confirming the occurrence of ferroptosis. Moreover, PINK1/parkin-mediated mitophagy, a vital process for selective elimination of damaged mitochondria, was blocked. Interestingly, silibinin rescued the mitochondria, restricted the ferroptosis and restored the mitophagy. By using the pharmacological stimulator and inhibitor of mitophagy, and si-RNA transfection to silence PINK1 expression, silibinin's protective effect against ferroptosis caused by PA and HG treatment was found to depend on mitophagy. Collectively, our current study reveals the new mechanisms for the protection of silibinin against the injury of INS-1 cells treated with PA and HG, elucidates the participation of ferroptosis in glucolipotoxicity, highlighting the involvement of mitophagy in defense against ferroptotic cell death.

Bacterial extracellular vesicles induced oxidative stress and mitophagy through mTOR pathways in colon cancer cells, HT-29: Implications for bioactivity

Bacterial-extracellular-vesicles (BEVs) derived from Escherichia coli, strain-A5922, were used as a therapeutic tool to treat colon cancer cells, HT-29. BEVs induced oxidative stress, and observed mitochondrial autophagy, known as mitophagy, were crucial in initiation of treatment. The mitophagy, induced by the BEVs in HT-29 cells, produced adenocarcinomic cytotoxicity, and stopped the cells growth. The trigger for mitophagy, and an increase in productions of reactive oxygen species led to cellular oxidative stress, that eventually led to cells death. A reduction in the mitochondrial membrane potential, and an increase in the PINK1 expressions confirmed the oxidative stress involvements. The BEVs triggered cytotoxicity, and mitophagy in the HT-29 carcinoid cells, channelized through the Akt/mTOR pathways connecting the cellular oxidative stress, effectively played its part to cause cells death. These findings substantiated the BEVs' potential as a plausible tool for treating, and possibly preventing the colorectal cancer.

The role of CK2 in the regulation of mitochondrial autophagy induced by rotenone

CK2 regulates receptor-mediated mitophagy that removes damaged mitochondria. The PINK1/Parkin pathways also involve mitochondrial clearance through mitophagy. However, it is not clear whether CK2 regulates PINK1/Parkin-dependent mitophagy in response to stress. Rotenone treatment showed a decrease of FUNDC1 expression in the mitochondrial fraction of SH-SY5Y and HeLa cells, but an increase of PINK1/Parkin expression only in SH-SY5Y cells. Interestingly, CK2 inhibition increased mitochondrial LC3II expression in rotenone-treated HeLa cells, whereas it decreased in SH-SY5Y cells, indicating that CK2 mediates rotenone-induced mitophagy in dopaminergic neurons. Furthermore, FUNDC1 expression increased in rotenone-treated SH-SY5Y cells by CK2 inhibition, whereas it decreased in HeLa cells. CK2 inhibition also blocked the increase of Drp1, PINK1 and Parkin translocation into mitochondria and decrease of PGAM5 expression in rotenone-treated SH-SY5Y cells. As expected, rotenone treatment in PGAM5-knockdown cells reduced the expression of PINK1 and Parkin and decrease of LC3II expression. Interestingly, we observed that knockdown of CK2α or PGAM5 induced a further increase in caspase-3 expression. These results suggest that PINK1/Parkin-dependent mitophagy was dominant over FUNDC1 receptor-mediated mitophagy. Collectively, our findings suggest that CK2 can positively induce PINK1/Parkin-dependent mitophagy, and that mitophagy regulates cytoprotective effects by CK2 signaling in dopaminergic neurons. Data availability statementAll data generated or analyzed during this study are available upon request.

WITHDRAWN: Revealing the neuroprotection and neurodegeneration efficacy of marine sponge extract in Parkinson's disease using gene expression and molecular docking

Parkinson's disease is one of the most prevalent neurological conditions (PD). The pathogenesis of PD was thought to involve mitochondrial dysfunction and oxidative stress. In this study, PD was induced in zebrafish models, and the effectiveness of MS01 sponge extract in neuroprotective effects was assessed. In zebrafish models, rotenone and MS01-mixed pellets were fed, tested for sensitivity and motor function, and euthanized. Histopathology and confocal images of the zebrafish brain were then analyzed for the expression of the PINK1 and Parkin genes. Gene expression study showed MS01 was effective against neurodegeneration at a dose-dependent concentration. The compound xenin identified by LC-MS/MS analysis was selected for in silico studies. Using molecular docking and modeling techniques, the gene expression of PINK1 and Parkin were also investigated. The analysis showed binding of the xenin peptide to the PINK1-Ubiquitin complex may enhance the stability of the complex and increase the production of Parkin, which reduces Parkinson's disease symptoms (PD). This demonstrates the neuroprotective effects of the xenin peptide.

Hydrogen regulates mitochondrial quality to protect glial cells and alleviates sepsis-associated encephalopathy by Nrf2/YY1 complex promoting HO-1 expression.

Sepsis-associated encephalopathy (SAE) is a complication of the central nervous system in patients with sepsis. Currently, no effective treatment for sepsis is available. Hydrogen plays a protective role in different diseases; however, the detailed mechanism of hydrogen-treated disease remains unclear. The purpose of this study was to investigate the effect of hydrogen on SAE in vitro and in vivo and the mechanism of hydrogen in mitochondrial dynamics and its function in astrocytes and microglia stimulated by lipopolysaccharides (LPSs). Animal models of SAE were generated by cecal ligation and puncture, and the SAE model was established by in vitro LPS stimulation. MTT, lactate dehydrogenase (LDH), reactive oxygen species (ROS), heme oxygenase-1 (HO-1) activity, mitochondrial membrane potential (MMP), and cell apoptosis assays were used to determine the effect of hydrogen on astrocytes and microglia stimulated by LPSs. The relationships between nuclear factor erythroid 2-related factor 2 (Nrf2), YY1, and HO-1 were examined by chromatin immunoprecipitation and co-immunoprecipitation. Mitochondrial homeostasis-related proteins in LPS-stimulated glial cells and brain tissues of SAE mice were detected by western blotting. The effects of hydrogen treatment in the SAE mouse model were investigated using Morris water maze and Y-maze analyses. After performing experiments with different concentrations of LPSs in vitro, we selected 1000ng/ml for subsequent experiments. Hydrogen attenuated the increase in ROS, LDH, and apoptosis and promoted decreases in cell activity and MMP, further promoting an increase in HO-1 expression induced by LPSs in astrocytes and microglia. Moreover, hydrogen further promoted the expression of Nrf2, HO-1, PGC-1α, TFAM, PARKIN, and PINK1, inhibited LPS-induced OPA1 and MFN2 expression in astrocytes and microglia, and downregulated the expression of DRP1 after LPS induction. Intriguingly, hydrogen treatment enhanced the binding between Nrf2 and YY1. However, silencing Nrf2 or YY1 abolished the protective effects of hydrogen on cell activity, LDH, ROS, and MMP; apoptosis; and regulation of Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, and PINK1 in microglia. Finally, hydrogen treatment improved the results of behavioral detection, apoptosis, Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, PINK1, and cytokines in SAE in vivo. Hydrogen improved cell injury and mitochondrial quality, which were associated with HO-1 expression promoted by the Nrf2/YY1 complex in vitro. Thus, hydrogen treatment may represent a novel therapeutic method for treating SAE.

Swietenine Alleviates Vascular Remodelling by Enhancing Mitophagy of Pulmonary Arterial Smooth Muscle Cells in Experimental Pulmonary Hypertension.

Vascular remodelling during pulmonary hypertension (PH) is characterized by the phenotypic transformation of pulmonary arterial smooth muscle cells (PASMCs). Swietenine (Swi), extracted from the seeds of traditional medicine Swietenia mahagoni, has been used to treat cardiac remodelling, but the effect of Swi on PH is unknown. This study aims to evaluate the effect of Swi on hypoxia-induced phenotypic transformation of PASMCs in experimental PH. In our research, C57BL/6 mice were treated with SU5416 and exposed to hypoxia for 4 weeks to establish HySu-PH model. Mice in the Swi treatment group were subjected to HySu with daily administration of Swi. Hemodynamic parameters, echocardiography, and degree of vascular muscularization were measured to evaluate the PH model. Proliferation of PASMC was assessed by Ki67 and EdU assay. Cell migration was detected by wound-healing assay. Mitophagy levels were evaluated by mito-tracker and lyso-tracker, autophagic flux, and protein expression of Pink1 and Lc3Ⅱ. The molecular docking was used to validate the interaction of Swi with Nrf2. Immunofluorescence and immunohistochemical staining was applied to determine the subcellular localization of Nrf2. The results showed that Swi attenuated hypoxia-induced increase of right ventricle systolic pressure, Fulton index, and vascular remodelling and decreased PASMC proliferation, migration, and enhanced mitophagy. Furthermore, the interaction of Swi with Nrf2 promoted the translocation of Nrf2 into the nucleus, resulting in the induction of Pink1. This study demonstrates that Swi prevents vascular remodelling in experimental PH through inhibition of phenotypic transformation and hyperproliferation of PASMCs caused by reversing hypoxia-induced inhibition of mitophagy.