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BACKGROUND: Exercises can reduce blood lipids and slow down the development of atherosclerosis. Atherosclerosis begins with mitochondrial dysfunction, and prohibitin 2 is involved in mitophagy by endurance training. OBJECTIVE: To explore the role of endurance training in the intervention of prohibitin 2 protein in the mitophagy autophagy pathway in atherosclerosis. METHODS: A total of 40 Wistar rats were randomly divided into control group, exercise group, atherosclerosis group and atherosclerosis combined with exercise group, with 10 rats in each group. A rat model of atherosclerosis was constructed by combining a high-fat diet (9 weeks) with vitamin D injections (weeks 1, 3, and 6) in the latter two groups, while the two exercise groups were subjected to progressing intensity endurance training for 9 weeks. After the intervention, lipid and pathological detections were conducted to observe the modeling and interventional effects. Mitochondrial membrane potential and mitophagy proteins were detected by microplate reader and western blot. Immunofluorescence staining was used to observe the co-localization of mitophagy proteins in aortic tissue. RESULTS AND CONCLUSION: Lipid and pathological sections showed that compared with the atherosclerosis group, the serum low-density lipoprotein cholesterol and total cholesterol levels and aortic lipid deposition area were significantly reduced in the atherosclerosis combined with exercise group (P < 0.001). The results of mitochondrial membrane potential showed that the significant decrease in mitochondrial membrane potential of the aorta in the atherosclerosis combined with exercise group was reversed (P < 0.01). The results of western blot assay showed that compared with the control group, the mitochondrial protein expression of prohibitin 2, LC3II/I, PINK1 and Parkin was significantly increased (P < 0.05), and the protein expression of PARL and PGAM5 decreased (P < 0.05). Compared with the atherosclerosis group, the protein expression of PINK1 and Parkin in the mitchondria of rats in the atherosclerosis combined with exercise group was significantly decreased (P < 0.05), and the protein expressions of prohibitin 2, LC3II/I, PARL and PGAM5 were significantly increased (P < 0.05). Immunofluorescence results showed that compared with the control group, the co-localization of LC3 and PINK1 with TOMM20 was significantly increased in the atherosclerosis group (P < 0.05), while compared with the atherosclerosis group, the co-localization of LC3 and PINK1 with TOMM20 was significantly increased in the atherosclerosis combined with exercise group (P < 0.05). Co-localization of LC3 and PARL with prohibitin 2 was significantly increased in the atherosclerosis group compared with the control group (P < 0.01), co-localization of LC3 with prohibitin 2 was significantly increased in the atherosclerosis combined with exercise group compared with the atherosclerosis group (P<0.01), and co-localization of PARL protein with prohibitin 2 was significantly decreased in the atherosclerosis combined with exercise group compared with the atherosclerosis group (P < 0.01). To conclude, endurance training can induce the expression of prohibitin 2 in the inner mitochondrial membrane and promote the binding of prohibitin 2 to the mitophagy junction protein to complete mitophagy, restore mitochondrial function, and slow down the occurrence of atherosclerosis. [ABSTRACT FROM AUTHOR]

ABSTRACT: Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the immortalized bovine mammary epithelial cell line (MAC-T cells). First, mammary gland tissue and blood samples were collected from healthy cows (n = 15, BHB 3.0 mM). Compared with healthy cows, cows with clinical ketosis exhibited lower DMI, milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in cows with clinical ketosis. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (MAP1LC3-II, also called LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in cows with clinical ketosis. The ATP content of mammary gland tissue in cows with clinical ketosis was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). The MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, and MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. The MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA-induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.

Abstract Diabetic encephalopathy (DE) is a severe complication of diabetes, but its pathogenesis remains unclear. This study aimed to investigate the roles and underlying mechanisms of high glucose (HG)- and advanced glycosylation end product (AGE)-induced oxidative stress (OS) in the cognitive decline in DE. The DE mouse model was established using a high-fat diet and streptozotocin, and its cognitive functions were evaluated using the Morris Water Maze, novel object recognition, and Y-maze test. The results revealed increased reactive oxygen species (ROS) generation, mitophagy inhibition, and decreased prohibitin 2 (PHB2) expression in the hippocampal neurons of DE mice and HG- or AGE-treated HT-22 cells. However, overexpression of PHB2 reduced ROS generation, reversed mitophagy inhibition, and improved mitochondrial function in the HG- or AGE-treated HT-22 cells and ameliorated cognitive decline, improved mitochondrial structural damage, and reversed mitophagy inhibition of hippocampal neurons in DE mice. Further analysis revealed that the Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was involved in the HG- or AGE-mediated downregulation of PHB2 in HT-22 cells. These results demonstrate that HG- or AGE-induced OS inhibits the mitophagy of hippocampal neurons via the Keap1–Nrf2–PHB2 pathway, thereby contributing to the cognitive decline in DE.

Diabetic encephalopathy (DE) is a severe complication of diabetes, but its pathogenesis remains unclear. This study aimed to investigate the roles and underlying mechanisms of high glucose (HG)- and advanced glycosylation end product (AGE)-induced oxidative stress (OS) in the cognitive decline in DE. The DE mouse model was established using a high-fat diet and streptozotocin, and its cognitive functions were evaluated using the Morris Water Maze, novel object recognition, and Y-maze test. The results revealed increased reactive oxygen species (ROS) generation, mitophagy inhibition, and decreased prohibitin 2 (PHB2) expression in the hippocampal neurons of DE mice and HG- or AGE-treated HT-22 cells. However, overexpression of PHB2 reduced ROS generation, reversed mitophagy inhibition, and improved mitochondrial function in the HG- or AGE-treated HT-22 cells and ameliorated cognitive decline, improved mitochondrial structural damage, and reversed mitophagy inhibition of hippocampal neurons in DE mice. Further analysis revealed that the Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was involved in the HG- or AGE-mediated downregulation of PHB2 in HT-22 cells. These results demonstrate that HG- or AGE-induced OS inhibits the mitophagy of hippocampal neurons via the Keap1–Nrf2–PHB2 pathway, thereby contributing to the cognitive decline in DE. [ABSTRACT FROM AUTHOR]

High expression of truncated O‐glycans Tn antigen predicts adverse clinical outcome in patients with clear cell renal cell carcinoma (ccRCC). To understand the biosynthetic underpinnings of Tn antigen changes in ccRCC, we focused on N‐acetylgalactosaminyltransferases (GALNTs, also known as GalNAcTs) known to be involved in Tn antigen synthesis. Data from GSE15641 profile and local cohort showed that GALNT6 was significantly upregulated in ccRCC tissues. The current study aimed to determine the role of GALNT6 in ccRCC, and whether GALNT6‐mediated O‐glycosylation aggravates malignant behaviors. Gain‐ and loss‐of‐function experiments showed that overexpression of GALNT6 accelerated ccRCC cell proliferation, migration, and invasion, as well as promoted ccRCC‐derived xenograft tumor growth and lung metastasis. In line with this, silencing of GALNT6 yielded the opposite results. Mechanically, high expression of GALNT6 led to the accumulation of Tn antigen in ccRCC cells. By undertaking immunoprecipitation coupled with liquid chromatography/mass spectrometry, vicia villosa agglutinin blot, and site‐directed mutagenesis assays, we found that O‐glycosylation of prohibitin 2 (PHB2) at Ser161 was required for the GALNT6‐induced ccRCC cell proliferation, migration, and invasion. Additionally, we identified lens epithelium‐derived growth factor (LEDGF) as a key regulator of GALNT6 transcriptional induction in ccRCC growth and an upstream contributor to ccRCC aggressive behavior. Collectively, our findings indicate that GALNT6‐mediated abnormal O‐glycosylation promotes ccRCC progression, which provides a potential therapeutic target in ccRCC development. [ABSTRACT FROM AUTHOR]

Objective To explore the effects of prohibitin 2(PHB2) on sensitivity of non-small cell lung cancer cell line A549 to erlotinib (Erl) and its potential mechanisms. Methods RACK1-specific small interfering RNA was transfected in A549 cells for knocking-down of PHB2. The effects of PHB2 inhibition on cell proliferation and apoptosis induced by Erl were observed. The colocalization of microtuble-associated protein light chain 3 alpha (LC3) and mitochondria was visualized by MitoTracker staining and green fluorescent protein-microtuble-associated protein light chain 3 alpha (GFP-LC3) transfection. Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine (EdU) staining. Cell colony formation was evaluated by colony forming assay. Apoptotic index of A549 cells was evaluated by TUNEL. Western blot was used to measure the expressions of PHB2 and LC3Ⅱ. Mitochondrial transmembrane potential, cytochrome c and respiratory chain complex Ⅰ/Ⅱ/Ⅴ activity were analyzed by the commercially available kits. Results Compared with the siPHB2 and siCtrl+Erl group, the EdU-positive A549 cells and the number of cell colonies decreased significantly (P＜0.05), while the TUNEL-positive A549 cells increased significantly(P＜0.05) in the siPHB2+Erl group. In addition, compared with the siPHB2 and siCtrl+Erl group, mitochondrial transmembrane potential and respiratory chain complex Ⅰ/Ⅱ/Ⅴ activity decreased significantly (all P＜0.05) and the levels of cytochrome c increased in mitochondrial fractions (P＜0.05) and decreased in cytosolic fractions(P＜0.05) in the siPHB2+Erl group. Conclusions PHB2 inhibition significantly improves sensitivity of A549 cells to Erl,which may be explained by inhibition of PHB2-mediated mitochondrial autophagy.

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Objective: To explore the effects of prohibitin 2 on right ventricular remodeling in hypoxia-induced pulmonary hypertension（HPH） mice and potential mechanisms. Methods: Eight-week-old male mice were randomly allocated to the following 4 groups:control group, HPH group, HPH+vector group and HPH+PHB2 group. HPH group, HPH+vector group and HPH+PHB2 group were placed in low-pressure and low-oxygen artificial chamber, and control group were placed in atmospheric oxygen environment for 4weeks. Adeno-associated virus serotype 9（AAV9） carrying smooth muscle promoter-driven SM22ap encoding PHB2 and empty vectors were injected via tail vein 3 weeks prior to model construction. Hemodynamics, the level of right ventricle remodeling, inflammation, oxidative stress and vasoactive substances, and the expression levels of PHB2 and p-STAT3 were determined in mice. Results: Compared with control group, RVSP, RVAW, RVHI and CVP were increased significantly（P＜0. 05） and RVID was decreased in HPH group and HPH+vector group（P＜0. 05), with upregulated plasma ET-1 and BNP（P＜0. 05） and downregulated NO, t-NOS and i NOS（P＜0. 05), as well as increased IL-1β, IL-6 and TNF-α and decreased SOD and GSH-Px in right ventricular tissue（P＜0. 05), and the levels of PHB2 expression in the lung tissue were decreased（P＜0. 05） and p-STAT3 expression was increased（P＜0. 05） in HPH group and HPH+vector group. Compared with HPH group and HPH+vector group, RVSP, RVAW, RVHI and CVP were decreased significantly（P＜0. 05） and RVID was increased in HPH+PHB2 group（P＜0. 05), with downregulated plasma ET-1 and BNP and upregulated NO, t-NOS and i NOS（P＜0. 05), as well as decreased IL-1β, IL-6 and TNF-α and increased SOD and GSH-Px in right ventricular tissue（P＜0. 05), and the levels of PHB2 expression in the lung tissue were increased（P＜0. 05） and p-STAT3 expression was decreased（P＜0. 05） in HPH+PHB2 group. Conclusion: PHB2 alleviates right ventricular remodeling in HPH mice, which may be related to the inhibition of STAT3 phosphorylation by PHB2 in pulmonary artery smooth muscle. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (SA) is a major cause of sepsis, leading to acute lung injury (ALI) characterized by inflammation and oxidative stress. However, the role of the Nrf2/PHB2 pathway in SA‐induced ALI (SA‐ALI) remains unclear. In this study, serum samples were collected from SA‐sepsis patients, and a SA‐ALI mouse model was established by grouping WT and Nrf2−/− mice after 6 h of intraperitoneal injection. A cell model simulating SA‐ALI was developed using lipoteichoic acid (LTA) treatment. The results showed reduced serum Nrf2 levels in SA‐sepsis patients, negatively correlated with the severity of ALI. In SA‐ALI mice, downregulation of Nrf2 impaired mitochondrial function and exacerbated inflammation‐induced ALI. Moreover, PHB2 translocation from mitochondria to the cytoplasm was observed in SA‐ALI. The p‐Nrf2/total‐Nrf2 ratio increased in A549 cells with LTA concentration and treatment duration. Nrf2 overexpression in LTA‐treated A549 cells elevated PHB2 content on the inner mitochondrial membrane, preserving genomic integrity, reducing oxidative stress, and inhibiting excessive mitochondrial division. Bioinformatic analysis and dual‐luciferase reporter assay confirmed direct binding of Nrf2 to the PHB2 promoter, resulting in increased PHB2 expression. In conclusion, Nrf2 plays a role in alleviating SA‐ALI by directly regulating PHB2 transcription and maintaining mitochondrial function in lung cells. [ABSTRACT FROM AUTHOR]

Abstract Staphylococcus aureus (SA) is a major cause of sepsis, leading to acute lung injury (ALI) characterized by inflammation and oxidative stress. However, the role of the Nrf2/PHB2 pathway in SA‐induced ALI (SA‐ALI) remains unclear. In this study, serum samples were collected from SA‐sepsis patients, and a SA‐ALI mouse model was established by grouping WT and Nrf2−/− mice after 6 h of intraperitoneal injection. A cell model simulating SA‐ALI was developed using lipoteichoic acid (LTA) treatment. The results showed reduced serum Nrf2 levels in SA‐sepsis patients, negatively correlated with the severity of ALI. In SA‐ALI mice, downregulation of Nrf2 impaired mitochondrial function and exacerbated inflammation‐induced ALI. Moreover, PHB2 translocation from mitochondria to the cytoplasm was observed in SA‐ALI. The p‐Nrf2/total‐Nrf2 ratio increased in A549 cells with LTA concentration and treatment duration. Nrf2 overexpression in LTA‐treated A549 cells elevated PHB2 content on the inner mitochondrial membrane, preserving genomic integrity, reducing oxidative stress, and inhibiting excessive mitochondrial division. Bioinformatic analysis and dual‐luciferase reporter assay confirmed direct binding of Nrf2 to the PHB2 promoter, resulting in increased PHB2 expression. In conclusion, Nrf2 plays a role in alleviating SA‐ALI by directly regulating PHB2 transcription and maintaining mitochondrial function in lung cells.

Autophagy of ovarian granulosa cells is one of the reasons which results in follicular atresia. PHB2 regulates many fundamental biological processes and is pivotal in the mitophagy of cells; nevertheless, the autophagy in the porcine ovary and how PHB2 regulates the follicular cells are unknown. Here we report a protein complex that induces autophagy in porcine granulosa cells (PGCs) through the direct interaction of ERβ and PHB2. In this study, we aimed to elucidate the autophagy and the role of PHB2 in porcine ovaries using porcine primary ovarian granulosa cells (PGCs). The results showed that PHB2 induces PGCs autophagy because of the change in related genes and protein expression levels. In addition, the results of Co-IP and the distribution of the combination of PHB2 and ERβ showed that this complex is also indicated as an essential role of PHB2 in PGCs autophagy. Based on our results, it can be concluded that PHB2 combined with ERβ induces PGCs autophagy by targeting the mTOR pathway. This study pinpoints a novel regulatory mechanism of autophagy and demonstrates the existence of a protein complex that may underlie its roles in autophagy in PGCs. • PHB2 induced autophagy in porcine granulosa cells. • PHB2 bound to ERβ to form a novel dimer in porcine granulosa cells. • The dimer stimulated mTOR phosphorylation to induce autophagy in porcine granulosa cells. [ABSTRACT FROM AUTHOR]

Objective To observe the effects of dihydromyricetin on mitophagy in vascular endothelial cells, and to clarify the underlying role of Prohibitin 2 in the process. Methods After human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of dihydromyricetin (0, 0.1, 1, 10, 100 μmol/L) for 12 h, intensity of MitoTracker® Deep Red (MTDR, a widely used mitochondria-selective probe) was determined for mitochondrial staining by fluorescence microscopy and flow cytometry, and the expression of Prohibitin 2 at mRNA and protein levels as determined by qRT-PCR and Western blotting, respectively. Furthermore, after treatment with of without control/Prohibitin 2 siRNA, HUVECs were incubated with dihydromyricetin for 12 h or carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) for 6 h in the presence of the lysosomal blocker chloroquine, and then subjected to flow cytometry analysis to determine mitophagy. Results As compared with the non-dihydromyricetin group, dihydromyricetin (≥ 0.1μmol/L) treatment markedly decreased MTDR fluorescence levels (P < 0.05), and meanwhile, the treatment significantly increased the mRNA and protein levels of Prohibitin 2 (P < 0.05). After pretreated with chloroquine, dihydromyricetin (≥10 μmol/L) significantly enhanced mitophagy in HUVECs, which was consistent with the effect of CCCP (P < 0.05). After Prohibitin 2 siRNA transfection, mitophagy induced by dihydromyricetin was significantly suppressed when compared with the control siRNA treatment group (P < 0.05). Conclusion Dihydromyricetin enhances mitophagy in vascular endothelial cells by regulating Prohibitin 2.

Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40%–50% of all AML cases. Despite advances in understanding the molecular pathophysiology of CNAML, its clinical outcome remains unsatisfactory and unpredictable. To investigate its clinical implication in CN-AML, we measured the expression of prohibitin 2 (PHB2) using immunohistochemical staining (IHCS) of paraffin-embedded bone marrow sections from 134 CN-AML patients. IHCS results were semi-quantitatively scored. Clinical outcome was analyzed in comparison with other prognostic markers, including NPM1 polymorphism and FLT3 internal tandem duplication, and WT1 and BAALC mRNA expression. Except for BAALC mRNA expression, the known molecular markers showed no prognostic effect in the CN-AML patients. PHB2 protein overexpression was significantly associated with adverse prognosis in CN-AML patients. The PHB2 protein expression status may serve as an independent prognostic indicator in CN-AML. [ABSTRACT FROM AUTHOR]

Age‐related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly, but its mechanism remains unclear. Scaffold protein prohibitin 2 (PHB2) has been widely involved in aging and neurodegeneration. However, the role of PHB2 in ARHL is undeciphered to date. To investigate the expression pattern and the role of PHB2 in ARHL, we used C57BL/6 mice and HEI‐OC1 cell line as models. In our study, we have found PHB2 exists in the cochlea and is expressed in hair cells, spiral ganglion neurons, and HEI‐OC1 cells. In mice with ARHL, mitophagy is reduced and correspondingly the expression level of PHB2 is decreased. Moreover, after H2O2 treatment the mitophagy is activated and the PHB2 expression is increased. These findings indicate that PHB2 may exert an important role in ARHL through mitophagy. Findings from this study will be helpful for elucidating the mechanism underlying the ARHL and for providing a new target for ARHL treatment. [ABSTRACT FROM AUTHOR]

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of Cg PHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. Cg PHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of Cg PHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of Cg PHB2 protein was also enhanced at 12–24 h compared to control group. Furthermore, the green signals of Cg PHB2 were colocalized respectively with the red signals of mitochondria and Cg LC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of Cg PHB2 and mtphagy Dye was significantly increased. The direct interaction between Cg PHB2 and Cg LC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that Cg PHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters. • Cg PHB2 translocated to mitochondria after V. splendidus stimulation. • Cg PHB2 functioned as a mitophagy receptor through its LIR motif. • Cg PHB2 was involved in mitophagy after V. splendidus stimulation. [ABSTRACT FROM AUTHOR]

Abstract Background Leishmaniasis, a disease caused by parasites of the genus Leishmania, infects roughly 12 million people worldwide, with about two million new cases per year. Prohibitins (PHBs) are highly conserved proteins belonging to the stomatin-prohibitin flotillin-HflC/K (SPFH) protein superfamily. In this study, we examine the potential functions of two proteins of Leishmania major, PHB1 and PHB2, as well as how they might help protect the protozoan against oxidative stress. Results By immunolocalization in the parasite cells, PHB1 appeared in the mitochondria and plasma membrane, whereas PHB2 was grouped in the nucleus. When Leishmania cells were under oxidative stress, PHB1 migrates towards the plasma membrane and the paraxial rod, while PHB2 remained in the nucleus and near the kinetoplast. PHB1 presented higher mRNA levels than PHB2 in the amastigotes and the infective metacyclic forms. The mRNA expression of both prohibitins was affected by the presence of the Fe3+ ion. PHBs inhibited the Fenton reaction, where reactive oxygen species could nick DNA, implying that they play a crucial role in controlling oxidative stress. Conclusions Here, we propose that PHBs may help to protect membranes and DNA against superoxide ions, thus enhancing the survival capacity of the protozoan by controlling the ROS within the phagosome of the macrophages where the parasite multiplies.

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) interacts with and inhibits the tumor suppressor function of prohibitin-2 (PHB2), and recent in vivo studies have demonstrated that the BIG3-PHB2 interaction is a promising target for breast cancer therapy. However, little biophysical characterization on BIG3 and its interaction with PHB2 has been reported. Here we compared the calculated 8-class secondary structure of the N-terminal domains of BIG family proteins and identified a loop region unique to BIG3. Our biophysical characterization demonstrated that this loop region significantly affects the colloidal and thermodynamic stability of BIG3 and the thermodynamic and kinetic profile of its interaction with PHB2. These results establish a model for the BIG3-PHB2 interaction and an entry for drug discovery for breast cancer. • The interaction between BIG3 and PHB2 was characterized by using ITC and SPR. • Non-conserved loop region of BIG3 affects the colloidal and thermodynamic stability of BIG3. • A mutein of the non-conserved loop BIG3 (1-373Δloop) showed higher binding affinity. • The N-terminal domain of BIG3 is regulated via the molecular states of the unique loop region. [ABSTRACT FROM AUTHOR]

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, when not effectively treated. The aim of this study was to discover new targets for the diagnosis and treatment of MN. A reliable mouse model of MN was used by the administration of cationic bovine serum albumin (cBSA). Mice with MN exhibited proteinuria, histopathological changes, and accumulation of immune complexes in the glomerular basement membrane. Label-free proteomics analysis was performed to identify changes in protein expression, and the overexpressed proteins were evaluated. There were 273 proteins that showed significantly different expression in mice with MN, as compared to the controls. String analysis showed that functions related to cellular catabolic processes were downregulated in MN. Among the differentially expressed proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2) were upregulated in the kidneys of mice with MN, as demonstrated by immunohistochemistry (IHC), and this upregulation was observed in both the tubular cells and glomeruli. Both shRNA-mediated knockdown of PHB1 or PHB2 inhibited tumor suppressor p53 expression and significantly promoted podocyte proliferation. In addition, both PHB1 and PHB2 were responsible for cBSA-induced cytotoxicity. Microarray analysis further revealed that the upregulation of PHB1 and PHB2 may be due to a blockage of proteasome activity. These data demonstrate that the upregulation of PHB2 is involved in cBSA-mediated podocyte cytotoxicity, which may lead to MN development. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is involved in the mechanism of early brain injury (EBI) following subarachnoid hemorrhage (SAH). Blood-brain barrier disruption is a devastating outcome in the early stage of SAH. In this study, we aimed to investigate the role of a mitochondria-related drug Mitoquinone (MitoQ) in blood-brain barrier disruption after SAH in rats. A total of 181 male Sprague–Dawley SAH rats with the endovascular perforation model were utilized. Intraperitoneal MitoQ was given 1 h (h) post-SAH. Cerebroventricular ML385, an inhibitor of NF-E2-related factor 2 (Nrf2) and Small interfering ribonucleic acid (siRNA) for Prohibitin 2 (PHB2) were injected respectively 24 h and 48 h before SAH. Neurological function evaluation was performed before sacrifice. SAH grade was measured during the sacrifice of each animal. Brain water content was performed at 24 h. Co-immunoprecipitation was used to demonstrate the relationship of proteins Nrf2 and PHB2. Mitochondrial and cytoplasmic fractions were gathered using mitochondria isolation kits. Pathway related proteins were investigated with Western blot and immunofluorescence staining. Transmission electron microscopy was performed for mitochondrial morphology. Expression of Nrf2 levels peaked at the 3 h time point following SAH and then decreased to normal levels at 24 h, while PHB2 and Optic Atrophy 1 (OPA1) decreased at 24 h and 72 h after SAH compared with the Sham group. MitoQ treatment attenuated neurological deficits and brain edema, thereby resulting in a decreased expression of Albumin, while an increase of Nrf2, PHB2, OPA1 and Claudin-5 proteins compared with SAH + vehicle group. With co-immunoprecipitation, Nrf2 and PHB2 were further demonstrated to show their interaction. And MitoQ administration lead to more binding of the two proteins. ML385 abolished the effects of MitoQ on neurobehavior and protein levels post-SAH. Similarly, PHB2 siRNA reversed the neuroprotection of MitoQ administration with the decreased expression of PHB2 and OPA1 after SAH. Further, MitoQ treatment improved mitochondrial morphology after SAH with an increase of PHB2 and OPA1 in mitochondrial extraction. MitoQ attenuates blood-brain barrier disruption via Nrf2/PHB2/OPA1 pathway after SAH in rats. MitoQ may serve as a potential therapeutic strategy for SAH patients. [ABSTRACT FROM AUTHOR]

Tumor cell-targeting molecules play a vital role in cancer diagnosis, targeted therapy, and biomarker discovery. Aptamers are emerging as novel targeting molecules with unique advantages in cancer research. In this work, we have developed several DNA aptamers through cell-based systematic evolution of ligands by exponential enrichment (Cell-SELEX). The selected SYL-6 aptamer can bind to a variety of cancer cells with high signal. Tumor tissue imaging demonstrated that SYL-6-Cy5 fluorescent probe was able to recognize multiple clinical tumor tissues but not the normal tissues, which indicates great potential of SYL-6 for clinical tumor diagnosis. Meanwhile, we identified prohibitin 2 (PHB2) as the molecular target of SYL-6 using mass spectrometry, pull-down and RNA interference assays. Moreover, SYL-6 can be used as a delivery vehicle to carry with doxorubicin (Dox) chemotherapeutic agents for antitumor targeted chemotherapy. The constructed SYL-6-Dox can not only selectively kill tumor cells in vitro, but also inhibit tumor growth with reduced side effects in vivo. This work may provide a general tumor cell-targeting molecule and a potential biomarker for cancer diagnosis and targeted therapy. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD). HFMD caused by EV-A71 seriously endangers children’s health. Although autophagy is an important antiviral defense mechanism, some viruses have evolved strategies to utilize autophagy to promote self-replication. EV-A71 can utilize autophagy vesicles as replication scaffolds, indicating that EV-A71 infection is closely related to its autophagy induction mechanism. VP1, a structural protein of EV-A71, has been reported to induce autophagy, but the underlying mechanism is still unclear. In this study, we found that the C-terminus (aa 251–297) of VP1 induces autophagy. Mass spectrometry analysis suggested that prohibitin 2 (PHB2) interacts with the C-terminus of the EV-A71 VP1 protein, and this was further verified by coimmunoprecipitation assays. After PHB2 knockdown, EV-A71 replication, viral particle release, and viral protein synthesis were reduced, and autophagy was inhibited. The results suggest that PHB2 interaction with VP1 is essential for induction of autophagy and the infectivity of EV-A71. Furthermore, we confirmed that EV-A71 induced complete autophagy that required autolysosomal acidification, thus affecting EV-A71 infection. In summary, this study revealed that the host protein PHB2 is involved in an autophagy mechanism during EV-A71 infection.

Accumulating evidence demonstrates that mitochondrial dysfunction and inflammasome activation play a critical role in the pathogenesis of renal tubular injury through the production of reactive oxygen species and cytokines. Prohibitin 2 (PHB2) is a newly identified intracellular receptor of mitophagy (a type of autophagy) that mediates selective removal of damaged mitochondria, and it could possibly play a renoprotective role in kidney disease. In this study, we confirmed that autophagy is activated in tubular epithelial cells treated with angiotensin II and that inhibition of autophagy results in tubular cell injury. Strikingly, PHB2 knockdown reduced the level of mitophagy and augmented cell death, while overexpression of PHB2 provided protection against pyrin domain-containing protein 3 (NLRP3)-induced inflammatory pathways through amelioration of mitochondrial dysfunction. Our research is the first to experimentally demonstrate the role of PHB2 in renal proximal tubular cells and thereby to provide a better understanding of how autophagy modulates inflammation in renal tubules. These data highlight PHB2 as a therapeutic target in the future treatment of CKD. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis, a disease caused by parasites of the genus Leishmania, infects roughly 12 million people worldwide, with about two million new cases per year. Prohibitins (PHBs) are highly conserved proteins belonging to the stomatin-prohibitin flotillin-HflC/K (SPFH) protein superfamily. In this study, we examine the potential functions of two proteins of Leishmania major, PHB1 and PHB2, as well as how they might help protect the protozoan against oxidative stress. Results: By immunolocalization in the parasite cells, PHB1 appeared in the mitochondria and plasma membrane, whereas PHB2 was grouped in the nucleus. When Leishmania cells were under oxidative stress, PHB1 migrates towards the plasma membrane and the paraxial rod, while PHB2 remained in the nucleus and near the kinetoplast. PHB1 presented higher mRNA levels than PHB2 in the amastigotes and the infective metacyclic forms. The mRNA expression of both prohibitins was affected by the presence of the Fe3+ ion. PHBs inhibited the Fenton reaction, where reactive oxygen species could nick DNA, implying that they play a crucial role in controlling oxidative stress. Conclusions: Here, we propose that PHBs may help to protect membranes and DNA against superoxide ions, thus enhancing the survival capacity of the protozoan by controlling the ROS within the phagosome of the macrophages where the parasite multiplies. [ABSTRACT FROM AUTHOR]

We sought to investigate the underlying mechanism of action of the long noncoding RNA (lncRNA) LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target proteins were investigated by RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Subcellular fractionation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the subcellular localization of LOC283070. Western blotting was performed to detect the expression of prohibitin 2 (PHB2). Luciferase activity assays were performed to evaluate the effects of LOC283070 and PHB2 on the androgen receptor (AR) signaling pathway. A methyl thiazolyl tetrazolium (MTT) assay and a growth curve assay were used to test cell viability. Flow cytometry was performed to analyze cell cycles. A transwell assay was employed to test cell migration. We identified PHB2 as an interaction partner of LOC283070 in the pull-down and RIP experiments. Furthermore, we confirmed that the enrichment of LOC283070 with PHB2 in androgen-independent LNCaP (LNCaP-AI) cells was much greater than that in LNCaP cells. Moreover, the expression of PHB2 was not significantly different between the two cell lines, and the expression of LOC283070 in the nuclei of the LNCaP-AI cells was significantly greater than that in the LNCaP cells. In vitro data revealed that PHB2 overexpression significantly inhibited AR activity and cell proliferation and migration and induced accumulation of prostate cancer cells in G0/G1 phase. Moreover, the overexpression of LOC283070 fully abrogated the effects of PHB2 overexpression. In conclusion, we found that LOC283070 can bind to PHB2 located in the nucleus and inhibit its effect, and this is one of the mechanisms by which LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells. [ABSTRACT FROM AUTHOR]

Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are proteins that are nearly ubiquitously expressed. They are localized in mitochondria, cytosol and cell nuclei. In the healthy CNS, they occur in neurons and non-neuronal cells (oligodendrocytes, astrocytes, microglia, and endothelial cells) and fulfill pivotal functions in brain development and aging, the regulation of brain metabolism, maintenance of structural integrity, synapse formation, aminoacidergic neurotransmission and, probably, regulation of brain action of certain hypothalamic-pituitary hormones.With regard to the diseased brain there is increasing evidence that prohibitins are prominently involved in numerous major diseases of the CNS, which are summarized and discussed in the present review (brain tumors, neurotropic viruses, Alzheimer disease, Down syndrome, Fronto-temporal and vascular dementia, dementia with Lewy bodies, Parkinson disease, Huntington disease, Multiple sclerosis, Amyotrophic lateral sclerosis, stroke, alcohol use disorder, schizophrenia and autism). Unfortunately, there is no PHB-targeted therapy available for any of these diseases. [ABSTRACT FROM AUTHOR]

Background: Phenotypic transition of vascular smooth muscle cells (VSMCs) accounts for the pathogenesis of a variety of vascular diseases during the early stage. Recent studies indicate the metabolic reprogramming may be involved in VSMC phenotypic transition. However, the definite molecules that link energy metabolism to distinct VSMC phenotype remain elusive., Methods: A carotid artery injury model was used to study postinjury neointima formation as well as VSMC phenotypic transition in vivo. RNA-seq analysis, cell migration assay, collagen gel contraction assay, wire myography assay, immunoblotting, protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay were performed to clarify the phenotype and elucidate the molecular mechanisms., Results: We collected cell energy-regulating genes by using Gene Ontology annotation and applied RNA-Seq analysis of transforming growth factor-β or platelet-derived growth factor BB stimulated VSMCs. Six candidate genes were overlapped from energy metabolism-related genes and genes reciprocally upregulated by transforming growth factor-β and downregulated by platelet-derived growth factor BB. Among them, prohibitin 2 has been reported to regulate mitochondrial oxidative phosphorylation. Indeed, prohibitin 2-deficient VSMCs lost the contractile phenotype as evidenced by reduced contractile proteins. Consistently, Phb2 SMCKO mice were more susceptible to postinjury VSMC proliferation and neointima formation compared with Phb2 flox/flox mice. Further protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay revealed that prohibitin 2, through its C-terminus, directly interacts with hnRNPA1, a key modulator of pyruvate kinase M1/2 (PKM) mRNA splicing that promotes PKM2 expression and glycolysis. Prohibitin 2 deficiency facilitated PKM1/2 mRNA splicing and reversion from PKM1 to PKM2, and enhanced glycolysis in VSMCs. Blocking prohibitin 2-hnRNPA1 interaction resulted in increased PKM2 expression, enhanced glycolysis, repressed contractile marker genes expression in VSMCs, as well as aggravated postinjury neointima formation in vivo., Conclusions: Prohibitin 2 maintains VSMC contractile phenotype by interacting with hnRNPA1 to counteract hnRNPA1-mediated PKM alternative splicing and glucose metabolic reprogramming.

Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40%-50% of all AML cases. Despite advances in understanding the molecular pathophysiology of CN-AML, its clinical outcome remains unsatisfactory and unpredictable. To investigate its clinical implication in CN-AML, we measured the expression of prohibitin 2 (PHB2) using immunohistochemical staining (IHCS) of paraffin-embedded bone marrow sections from 134 CN-AML patients. IHCS results were semi-quantitatively scored. Clinical outcome was analyzed in comparison with other prognostic markers, including NPM1 polymorphism and FLT3 internal tandem duplication, and WT1 and BAALC mRNA expression. Except for BAALC mRNA expression, the known molecular markers showed no prognostic effect in the CN-AML patients. PHB2 protein overexpression was significantly associated with adverse prognosis in CN-AML patients. The PHB2 protein expression status may serve as an independent prognostic indicator in CN-AML.

Abstract: Prohibitin 2 (PHB2) is an evolutionarily conserved and ubiquitously expressed multifunctional protein which is present in various cellular compartments including the nucleus. However, mechanisms underlying various functions of PHB2 are not fully explored yet. Previously we showed that PHB2 interacts with Akt and inhibits muscle differentiation by repressing the transcriptional activity of both MyoD and MEF2. Here we show that Calcium/Calmodulin-dependent kinase IV (CaMK IV) specifically binds to the C terminus of PHB2 and phosphorylates PHB2 at serine 91. Ectopic expression of CaMK IV and PHB2 in C2C12 cells results effectively in decreased PHB2-mediated repression of MEF2-dependent gene expression. Conversely, PHB2 mutant (S91A) resistant to CaMK IV phosphorylation has less effective in relieving the inhibition of MEF2 transcription by PHB2. Our findings suggest that CaMK IV interacts with and regulates PHB2 through phosphorylation, which could be one of the mechanisms underlying the CaMK-mediated activation of MEF2. [Copyright &y& Elsevier]

Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERα are coexpressed in mammary epithelial cells. REA heterozygous (REA+/-) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA+/- mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA+/-/ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such as progesterone receptor and cyclin D1 in the mammary gland. Our analysis also revealed that REA is an important repressor of ER transcriptional activity in the mammary gland under natural, as well as ovariectomized and estrogen-replaced, hormonal conditions. Our results indicate that REA is a physiological modulator of ER function in the mammary gland and that its correct gene dosage is required for maintenance of normal ER activity and normal mammary gland development. Consequently, a reduction or loss of REA function may cause overactivation of ER and increase breast cancer risk in humans. [ABSTRACT FROM AUTHOR]

In a yeast two-hybrid screen using the full-length Akt as bait, we found that prohibitin 2 (PHB2) specifically interacts with Akt. The C terminus of Akt (amino acids 413-480) and a central region of PHB2 (amino acids 120232) are responsible for their mutual interaction. PHB2 acts as a transcriptional repressor in cells. PHB2 interacts with both MyoD and MEF2, and represses both MyoD and MEF2-dependent gene transcription. Furthermore, binding of PHB2 to both MyoD and MEF2 significantly decreases upon myogenic differentiation. When stably expressed in C2C12 myogenic cells, PHB2 inhibits myogenin induction and phenotypic muscle differentiation. PHB2 was found to specifically recruit histone deacetylase 1, which is probably responsible for its repressive activity. Co-expression of Akt can partially reduce PHB2 binding to MyoD and relieve the repressive effect of PHB2 on myogenic reporters, which could be one of the mechanisms underlying Akt-mediated MyoD activation and accelerated muscle differentiation. [ABSTRACT FROM AUTHOR]

Platelet mitophagy is a major pathway involved in the clearance of injured mitochondria during hemostasis and thrombosis. Prohibitin 2 (PHB2) has recently emerged as an inner mitochondrial membrane receptor involved in mitophagy. However, the mechanisms underlying PHB2-mediated platelet mitophagy and activation are not completely understood. PHB2 is a highly conserved inner mitochondrial membrane protein that regulates mitochondrial assembly and function due to its unique localization on the mitochondrial membrane. The present study aimed to investigate the role and mechanism underlying PHB2 in platelet mitophagy and activation. Phorbol-12-myristate-13-acetate (PMA) was used to induce MEG-01 cells maturation and differentiate into platelets following PHB2 knockdown. Cell Counting Kit-8 assays were performed to examine platelet viability. Flow cytometry was performed to assess platelet mitochondrial membrane potential. RT-qPCR and western blotting were conducted to measure mRNA and protein expression levels, respectively. Subsequently, platelets were exposed to CCCP and the role of PHB2 was assessed. The results of the present study identified a crucial role for PHB2 in platelet mitophagy and activation, suggesting that PHB2-mediated regulation of mitophagy may serve as a novel strategy for downregulating the expression of platelet activation genes. Although further research into mitophagy is required, the present study suggested that PHB2 may serve as a novel therapeutic target for thrombosis-related diseases due to its unique localization on the mitochondrial membrane. [ABSTRACT FROM AUTHOR]

Simple Summary: In the present study, we found the novel pleiotropic regulation of the oncogene product thymidylate synthase (TS) by a chemical biology approach to identify rabdosianone I-binding proteins. Rabdosianone I, which is extracted from a traditional Asian herb Isodon japonicus Hara for longevity, suppressed TS expression at mRNA and protein levels. We immobilized rabdosianone I onto nano-magnetic beads and identified two mitochondrial proteins, adenine nucleotide translocase 2 (ANT2) and prohibitin 2 (PHB2), as the direct targets of rabdosianone I in cancer cells. Mechanistically, the knockdown of ANT2 or PHB2 promoted proteasomal degradation of the TS protein. In addition, PHB2 reduced TS mRNA levels. Thus, we provide previously unknown mechanisms of TS regulation by ANT2 and PHB2 and propose the possibility of rabdosianone I as a promising lead compound for the discovery of a novel TS suppressor. Natural products have numerous bioactivities and are expected to be a resource for potent drugs. However, their direct targets in cells often remain unclear. We found that rabdosianone I, which is a bitter diterpene from an oriental herb for longevity, Isodon japonicus Hara, markedly inhibited the growth of human colorectal cancer cells by downregulating the expression of thymidylate synthase (TS). Next, using rabdosianone I-immobilized nano-magnetic beads, we identified two mitochondrial inner membrane proteins, adenine nucleotide translocase 2 (ANT2) and prohibitin 2 (PHB2), as direct targets of rabdosianone I. Consistent with the action of rabdosianone I, the depletion of ANT2 or PHB2 reduced TS expression in a different manner. The knockdown of ANT2 or PHB2 promoted proteasomal degradation of TS protein, whereas that of not ANT2 but PHB2 reduced TS mRNA levels. Thus, our study reveals the ANT2- and PHB2-mediated pleiotropic regulation of TS expression and demonstrates the possibility of rabdosianone I as a lead compound of TS suppressor. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD). HFMD caused by EV-A71 seriously endangers children's health. Although autophagy is an important antiviral defense mechanism, some viruses have evolved strategies to utilize autophagy to promote self-replication. EV-A71 can utilize autophagy vesicles as replication scaffolds, indicating that EV-A71 infection is closely related to its autophagy induction mechanism. VP1, a structural protein of EV-A71, has been reported to induce autophagy, but the underlying mechanism is still unclear. In this study, we found that the C-terminus (aa 251–297) of VP1 induces autophagy. Mass spectrometry analysis suggested that prohibitin 2 (PHB2) interacts with the C-terminus of the EV-A71 VP1 protein, and this was further verified by coimmunoprecipitation assays. After PHB2 knockdown, EV-A71 replication, viral particle release, and viral protein synthesis were reduced, and autophagy was inhibited. The results suggest that PHB2 interaction with VP1 is essential for induction of autophagy and the infectivity of EV-A71. Furthermore, we confirmed that EV-A71 induced complete autophagy that required autolysosomal acidification, thus affecting EV-A71 infection. In summary, this study revealed that the host protein PHB2 is involved in an autophagy mechanism during EV-A71 infection. [ABSTRACT FROM AUTHOR]

Background and Purpose- Mitoquinone has been reported as a mitochondria-targeting antioxidant to promote mitophagy in various chronic diseases. Here, our aim was to study the role of mitoquinone in mitophagy activation and oxidative stress-induced neuronal death reduction after subarachnoid hemorrhage (SAH) in rats. Methods- Endovascular perforation was used for SAH model of male Sprague-Dawley rats. Exogenous mitoquinone was injected intraperitoneally 1 hour after SAH. ML385, an inhibitor of Nrf2 (nuclear factor-E2-related factor 2), was given intracerebroventricularly 24 hours before SAH. Small interfering RNA for PHB2 (prohibitin 2) was injected intracerebroventricularly 48 hours before SAH. Nuclear, mitochondrial, and cytoplasmic fractions were gathered using nucleus and mitochondria isolation kits. SAH grade evaluation, short- and long- term neurological function tests, oxidative stress, and apoptosis measurements were performed. Pathway related proteins were investigated with Western blot and immunofluorescence staining. Results- Expression of Keap1 (Kelch-like epichlorohydrin-associated protein 1, 2.84× at 24 hours), Nrf2 (2.78× at 3 hours), and LC3II (light chain 3-II; 1.94× at 24 hours) increased, whereas PHB2 (0.46× at 24 hours) decreased after SAH compared with sham group. Mitoquinone treatment attenuated oxidative stress and neuronal death, both short-term and long-term. Administration of mitoquinone resulted in a decrease in expression of Keap1 (0.33×), Romo1 (reactive oxygen species modulator 1; 0.24×), Bax (B-cell lymphoma-2 associated X protein; 0.31×), Cleaved Caspase-3 (0.29×) and an increase in Nrf2 (2.13×), Bcl-xl (B-cell lymphoma-extra large; 1.67×), PINK1 (phosphatase and tensin-induced kinase 1; 1.67×), Parkin (1.49×), PHB2 (1.60×), and LC3II (1.67×) proteins compared with SAH+vehicle group. ML385 abolished the treatment effects of mitoquinone on behavior and protein levels. PHB2 small interfering RNA reversed the outcomes of mitoquinone administration through reduction in protein expressions downstream of PHB2. Conclusions- Mitoquinone inhibited oxidative stress-related neuronal death by activating mitophagy via Keap1/Nrf2/PHB2 pathway after SAH. Mitoquinone may serve as a potential treatment to relieve brain injury after SAH.