Chemical mitophagy modulators: Drug development strategies and novel regulatory mechanisms.

Maintaining mitochondrial homeostasis is a potential therapeutic strategy for various diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic disorders, and cancer. Selective degradation of mitochondria by autophagy (mitophagy) is a fundamental mitochondrial quality control mechanism conserved from yeast to humans. Indeed, small-molecule modulators of mitophagy are valuable pharmaceutical tools that can be used to dissect complex biological processes and turn them into potential drugs. In the past few years, pharmacological regulation of mitophagy has shown promising therapeutic efficacy in various disease models. However, with the increasing number of chemical mitophagy modulator studies, frequent methodological flaws can be observed, leading some studies to draw unreliable or misleading conclusions. This review attempts (a) to summarize the molecular mechanisms of mitophagy; (b) to propose a Mitophagy Modulator Characterization System (MMCS); (c) to perform a comprehensive analysis of methods used to characterize mitophagy modulators, covering publications over the past 20 years; (d) to provide novel targets for pharmacological intervention of mitophagy. We believe this review will provide a panorama of current research on chemical mitophagy modulators and promote the development of safe and robust mitophagy modulators with therapeutic potential by introducing high methodological standards.

Nono deficiency impedes the proliferation and adhesion of H9c2 cardiomyocytes through Pi3k/Akt signaling pathway.

Congenital heart disease (CHD) is the most common type of birth defect and the main noninfectious cause of death during the neonatal stage. The non-POU domain containing, octamer-binding gene, NONO, performs a variety of roles involved in DNA repair, RNA synthesis, transcriptional and post-transcriptional regulation. Currently, hemizygous loss-of-function mutation of NONO have been described as the genetic origin of CHD. However, essential effects of NONO during cardiac development have not been fully elucidated. In this study, we aim to understand role of Nono in cardiomyocytes during development by utilizing the CRISPR/Cas9 gene editing system to deplete Nono in the rat cardiomyocytes H9c2. Functional comparison of H9c2 control and knockout cells showed that Nono deficiency suppressed cell proliferation and adhesion. Furthermore, Nono depletion significantly affected the mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, resulting in H9c2 overall metabolic deficits. Mechanistically we demonstrated that the Nono knockout impeded the cardiomyocyte function by attenuating phosphatidyl inositol 3 kinase-serine/threonine kinase (Pi3k/Akt) signaling via the assay for transposase-accessible chromatin using sequencing in combination with RNA sequencing. From these results we propose a novel molecular mechanism of Nono to influence cardiomyocytes differentiation and proliferation during the development of embryonic heart. We conclude that NONO may represent an emerging possible biomarkers and targets for the diagnosis and treatment of human cardiac development defects.

Copy Number Analyses Identified a Novel Gene: APOBEC3A Related to Lipid Metabolism in the Pathogenesis of Preeclampsia.

Background: Preeclampsia is a heterogeneous and complex disease with its pathogenesis mechanism not fully elucidated. A certain subset of patients with preeclampsia exhibit disturbances in lipid metabolism before clinical symptoms. Moreover, there is a tendency for preeclampsia to run in families. Whether genetic factors play a role in abnormal lipid metabolism during the incidence of preeclampsia has not been well investigated. Methods: Preeclampsia patients (n = 110) and healthy age- and gravidity-matched pregnant women (n = 110) were enrolled in this study. Peripheral blood specimens were used for genomic analysis (n = 10/group) or laboratory validation (n = 100/group). We retrospectively obtained the baseline clinical characteristics of 68 preeclampsia patients and 107 controls in early pregnancy (12-14 gestational weeks). Correlation analyses between differential genes and baseline lipid profiles were performed to identify candidate genes. In vitro and in vivo gain-of-function models were constructed with lentivirus and adeno-associated virus systems, respectively, to investigate the role of candidate genes in regulating lipid metabolism and the development of preeclampsia. Results: We observed that preeclampsia patients exhibited significantly elevated plasma TC (P = 0.037) and TG (P < 0.001) levels and increased body mass index (P = 0.006) before the disease onset. Within the region of 27 differential copy number variations, six genes potentially connected with lipid metabolism were identified. The aberrant copies of APOBEC3A, APOBEC3A_B, BTNL3, and LMF1 between preeclampsia patients and controls were verified by quantitative polymerase chain reaction. Especially, APOBEC3A showed a significant positive correlation with TC (P < 0.001) and LDL (P = 0.048) in early pregnancy. Then, our in vitro data revealed that overexpression of APOBEC3A disrupted lipid metabolism in HepG2 cells and affected both cholesterol and fatty acid metabolisms. Finally, in vivo study in a hepatic-specific overexpressed APOBEC3A mouse model revealed abnormal parameters related to lipid metabolism. Pregnant mice of the same model at the end of pregnancy showed changes related to preeclampsia-like symptoms, such as increases in sFlt-1 levels and sFlt-1/PLGF ratios in the placenta and decreases in fetal weight. Conclusion: Our findings established a new link between genetics and lipid metabolism in the pathogenesis of preeclampsia and could contribute to a better understanding of the molecular mechanisms of preeclampsia.

Artificial intelligence for distinguishment of hammering sound in total hip arthroplasty.

Recent studies have focused on hammering sound analysis during insertion of the cementless stem to decrease complications in total hip arthroplasty. However, the nature of the hammering sound is complex to analyse and varies widely owing to numerous possible variables. Therefore, we performed a preliminary feasibility study that aimed to clarify the accuracy of a prediction model using a machine learning algorithm to identify the final rasping hammering sound recorded during surgery. The hammering sound data of 29 primary THA without complication were assessed. The following definitions were adopted. Undersized rasping: all undersized stem rasping before the rasping of the final stem size, Final size rasping: rasping of the final stem size, Positive example: hammering sound during final size rasping, Negative example A: hammering sound during minimum size stem rasping, Negative example B: hammering sound during all undersized rasping. Three datasets for binary classification were set. Finally, binary classification was analysed in six models for the three datasets. The median values of the ROC-AUC in models A-F among each dataset were dataset a: 0.79, 0.76, 0.83, 0.90, 0.91, and 0.90, dataset B: 0.61, 0.53, 0.67, 0.69, 0.71, and 0.72, dataset C: 0.60, 0.48, 0.57, 0.63, 0.67, and 0.63, respectively. Our study demonstrated that artificial intelligence using machine learning was able to distinguish the final rasping hammering sound from the previous hammering sound with a relatively high degree of accuracy. Future studies are warranted to establish a prediction model using hammering sound analysis with machine learning to prevent complications in THA.

Acoustic characteristics of broaching procedure for post-operative stem subsidence in cementless total hip arthroplasty.

PURPOSE: Avoiding stem subsidence is crucial for achieving better outcome for cementless total hip arthroplasty (THA). The aim of this study was to develop a prediction model for the incidence of post-operative stem subsidence using full quantitative acoustic parameters in hammering sound during the broaching procedure and to assess the accuracy of this prediction model. METHODS: The acoustic parameters of the hammering sounds during a broaching procedure for 55 hips in 49 patients who underwent THAs with cementless taper-wedged stem were analysed. The stem subsidence was assessed at one month post-operatively, and the relationship between the acoustic parameters and the value of stem subsidence was investigated. RESULTS: The average stem subsidence was 2.15 ± 2.91 mm. The subsidence 3 mm or more was observed in eleven hips (20%), and 5 mm or more was observed in seven hips (12.7%). Basic patient's characteristics, preoperative femoral morphology and immediate post-operative canal fill ratio and stem alignment were not significantly related to the volume of stem subsidence. Nine acoustic parameters were significantly correlated with the value of subsidence. The prediction model for post-operative subsidence using only acoustic parameters during broaching procedure was established, and this model showed a positive prediction value of 100% and a negative prediction value of 90.6% for post-operative stem subsidence at 5 mm or more. CONCLUSION: Post-operative stem subsidence can be predicted by using acoustic parameters of the hammering sound during the broaching procedure. Our results suggest that we are at the start of a new era in which novel and innovative smart technologies can be used to assist in orthopaedic surgery.

PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica.

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

Lycorine, a natural alkaloid, promotes the degradation of alpha-synuclein via PKA-mediated UPS activation in transgenic Parkinson's disease models.

BACKGROUND: Parkinson's disease (PD) is one of the most common neurodegenerative motor disorders, and is characterized by the presence of Lewy bodies containing misfolded α-synuclein (α-syn) and by selective degeneration of midbrain dopamine neurons. Studies have shown that upregulation of ubiquitin-proteasome system (UPS) activity promotes the clearance of aggregation-prone proteins such as α-syn and Tau, so as to alleviate the neuropathology of neurodegenerative diseases. PURPOSE: To identify and investigate lycorine as a UPS enhancer able to decrease α-syn in transgenic PD models. METHODS: Dot blot was used to screen α-syn-lowering compounds in an inducible α-syn overexpression cell model. Inducible wild-type (WT) and mutant α-syn-overexpressing PC12 cells, WT α-syn-overexpressing N2a cells and primary cultured neurons from A53T transgenic mice were used to evaluate the effects of lycorine on α-syn degradation in vitro. Heterozygous A53T transgenic mice were used to evaluate the effects of lycorine on α-syn degradation in vivo. mCherry-GFP-LC3 reporter was used to detect autophagy-dependent degradation. Ub-R-GFP and Ub-G76V-GFP reporters were used to detect UPS-dependent degradation. Proteasome activity was detected by fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC). RESULTS: Lycorine significantly promoted clearance of over-expressed WT and mutant α-syn in neuronal cell lines and primary cultured neurons. More importantly, 15 days' intraperitoneal administration of lycorine effectively promoted the degradation of α-syn in the brains of A53T transgenic mice. Mechanistically, lycorine accelerated α-syn degradation by activating cAMP-dependent protein kinase (PKA) to promote proteasome activity. CONCLUSION: Lycorine is a novel α-syn-lowering compound that works through PKA-mediated UPS activation. This ability to lower α-syn implies that lycorine has the potential to be developed as a pharmaceutical for the treatment of neurodegenerative diseases, such as PD, associated with UPS impairment and protein aggregations.

Use of Dual Mobility Acetabular Component and Anterior Approach in Patients With Displaced Femoral Neck Fracture.

BACKGROUND: Although the antidislocation effect of total hip arthroplasty (THA) via the direct anterior approach (DAA) with dual mobility cup (DMC) for displaced femoral neck fracture (FNF) has already been reported, the clinical result of DMC-DAA-THA for displaced FNF in terms of mortality, complications, and walking ability are still unclear. METHODS: 106 cases with DMC-DAA-THA for displaced FNF were investigated of dislocation; perioperative complications; 3-, 6-, and12-month mortality rate; and pre/early postoperative walking ability. The walking ability was stratified into the following four categories: (1) use of a wheelchair (no walking), (2) walking alongside a support (including walkers designed for the elderly), (3) walking using one stick, and (4) unaided walking. RESULTS: There was no dislocation withing one-year postoperative. The 3-, 6-, and 12-month mortality rate was 2.8%, 4.7%, and 5.7%. Total complications occurred in 14 cases (14.7%). Although there was no revision surgery, two cases (1.9%) of intraoperative fracture treated without additional fixation, and one case of postoperative fracture was occurred. Among patients with preinjury walking category 2, 3, and 4 (total 94 cases), the number of patients who recovered same walking category at two-week postoperation was 56 cases (59.6%). CONCLUSIONS: Our study demonstrated that DMC-DAA-THA for displaced FNF offered quick recovery of walking ability with no dislocation and low one-year mortality rate. We believe that the combination of early postoperative recovery due to the minimal invasiveness of the DAA and decreased dislocation rate due to increased range of motion by DMC and adequate soft tissue tension by DAA contributed to null dislocation.

Does total hip arthroplasty via the direct anterior approach using dual mobility increase leg length discrepancy compared with single mobility?

BACKGROUND: Total hip arthroplasty (THA) via the direct anterior approach (DAA) using dual mobility cup (DMC) is considered to effectively prevent postoperative dislocation. However, the dislocation and reduction procedure using a trial implant during the surgery is difficult because of high soft tissue tension. Thereby, leg length discrepancy (LLD) is difficult to assess when using DM via the DAA. PURPOSE: To compare the LLD between cases using conventional SM and those using DMC in THA via the DAA with fluoroscopy. PATIENTS AND METHODS: We retrospectively investigated 34 hips treated with DMC (DMC-DAA group) and 31 hips treated with SM (SM-DAA group). The LLD was defined as the difference in the distance from the teardrop to the medial-most point of the lesser trochanter between the operative and nonoperative sides at immediate postoperative X-ray. RESULTS: The mean LLD in the DMC-DAA group and SM-DAA group was 0.68 ± 7.7 mm and 0.80 ± 5.5, respectively, with no significant difference. The absolute value of the LLD in the DMC-DAA group and SM-DAA group was 6.3 ± 4.4 mm and 5.9 ± 5.5, respectively, with no significant difference. CONCLUSION: Despite the difficulty in assessment of the LLD during THA via the DAA using DMC, this technique does not increase the LLD compared with the use of SM. LEVEL OF EVIDENCE: III, matched case-control study.

Does a fluoro-assisted direct anterior approach for total hip arthroplasty pose an excessive risk of radiation exposure to the surgeon?

INTRODUCTION: Intraoperative fluoroscopy can be easily used because patients are placed in the supine position during total hip arthroplasty via direct anterior approach (DAA-THA) to reduce complications. However, the cumulative level of radiation exposure by intraoperative fluoroscopy increases as the annual number of cases increases, increasing the risk of influencing the health of both the patients and medical workers. The objective of the study was to compare the radiation exposure time of DAA-THA with osteosynthesis and to determine if the level of radiation exposure exceeded safety limits. MATERIAL AND METHODS: DAA-THA was performed in 313 patients between January 2016 and July 2018 and 60 patients with proximal femoral fracture were treated with osteosynthesis. The intraoperative fluoroscopy time was retrospectively surveyed and compared between these two groups. A total of eight surgeons operated DAA-THA employing the same procedure using a traction table. A total of nine surgeons operated osteosynthesis and fluoroscopy was appropriately used during reduction and implant insertion. RESULTS: The mean operative time of DAA-THA was 103.3 min and that of osteosynthesis was 83.3 min, showing a significant difference (p < 0.05). The mean intraoperative fluoroscopy time was 0.83 min (SD ± 0.68) in DAA-THA and 8.91 min (SD ± 8.34) in osteosynthesis showing a significant difference (p < 0.05). CONCLUSIONS: The intraoperative exposure level was significantly lower and the fluoroscopy time was significantly shorter in DAA-THA than in osteosynthesis for proximal femoral fracture. It was clarified that the annual cumulative radiation exposure level in DAA-THA does not exceed the tissue dose limit.

Pharmacological enhancement of TFEB-mediated autophagy alleviated neuronal death in oxidative stress-induced Parkinson's disease models.

Autophagy, a conserved cellular degradation and recycling process, can be enhanced by nutrient depletion, oxidative stress or other harmful conditions to maintain cell survival. 6-Hydroxydopamine/ascorbic acid (6-OHDA/AA) is commonly used to induce experimental Parkinson's disease (PD) lesions by causing oxidative damage to dopaminergic neurons. Activation of autophagy has been observed in the 6-OHDA-induced PD models. However, the mechanism and exact role of autophagy activation in 6-OHDA PD model remain inconclusive. In this study, we report that autophagy was triggered via mucolipin 1/calcium/calcineurin/TFEB (transcription factor EB) pathway upon oxidative stress induced by 6-OHDA/AA. Interestingly, overexpression of TFEB alleviated 6-OHDA/AA toxicity. Moreover, autophagy enhancers, Torin1 (an mTOR-dependent TFEB/autophagy enhancer) and curcumin analog C1 (a TFEB-dependent and mTOR-independent autophagy enhancer), significantly rescued 6-OHDA/AA-induced cell death in SH-SY5Y cells, iPSC-derived DA neurons and mice nigral DA neurons. The behavioral abnormality of 6-OHDA/AA-treated mice can also be rescued by Torin 1 or C1 administration. The protective effects of Torin 1 and C1 can be blocked by autophagy inhibitors like chloroquine (CQ) or by knocking down autophagy-related genes TFEB and ATG5. Taken together, this study supports that TFEB-mediated autophagy is a survival mechanism during oxidative stress and pharmacological enhancement of this process is a neuroprotective strategy against oxidative stress-associated PD lesions.

Application value of Caprini risk assessment model and elevated tumor-specific D-dimer level in predicting postoperative venous thromboembolism for patients undergoing surgery of gynecologic malignancies.

AIM: Venous thromboembolism (VTE) is a major cause of morbidity and mortality in gynecologic malignant patients after surgery. We aimed to validate the Caprini risk assessment model (RAM) and elevated tumor-specific D-dimer as predictive marker of postoperative VTE for patients undergoing surgery of gynecologic malignancies. METHODS: Inpatients were divided into five groups (low: score = 0-1; moderate: score = 2; high: score = 3-4; higher: score = 5-7; sup-high: score > 7) and treated according to their risk level after the surgery during the hospitalization according to the Caprini RAM. D-dimer level was detected during the perioperative period. If D-dimer did not fall to normal reference range on the seventh day after operation, the use of low-molecular-weight heparin was prolonged to 28 days after surgery. RESULTS: The majority (853/974, 87.6%) of the patients was in the Caprini score ≥5, with an overall VTE incidence of 1.75%. The VTE group had significantly higher Caprini score, CA125, vascular invasion rate and lymph node metastasis rate. If 1.5 µg/mL was used as the D-dimer cut-off value to predicting VTE, the sensitivity was 87.5%, the specificity was 93.8% and the negative predictive value was 99.2%. The D-dimer level was a marker for prolonging the anticoagulants use during the perioperative period, especially for the sup-high group. CONCLUSION: The Caprini RAM is an effective and reliable VTE risk prediction tool for patients undergoing gynecological malignant tumor surgery. The group (score ≥ 5) can be divided into two subgroups (higher: score = 5-7 and sup-high: score > 7), which may better predict the occurrence of VTE for malignant tumor patients. Great than 1.5 µg/mL D-dimer before operation should be given more attention for the presence of VTE.

Polyprenols mitigate cognitive dysfunction and neuropathology in the APP/PS1 mouse.

Alzheimer's disease (AD) is a very common neurodegenerative disorder in the elderly and brings considerable financial and social problems worldwide. In this study, polyprenols were firstly evaluated the effects on the cognitive deficits and neuropathology in APP/PS1 mice model of AD. At 3 months old, the APP/PS1 mice were divided into model group; polyprenols low, middle, and high dosage group; and positive drug group. Age-matched wild-type mice were chosen in control group. The administration by oral gavage lasted 6 months. Polyprenols treatment significantly improved cognitive impairment of double transgenic mice compared with vehicle control treatment in behavioral tests. In addition, immunohistochemistry and enzyme-linked immunosorbent assay showed that there were significantly reductions in neuritic plaques and the level of hyperphosphorylated tau in brain of polyprenols-treated mice. Furthermore, we found that polyprenols treatment reduced the apoptotic cells in brain sections of 9-month-old APP/PS1 mice. These results reveal that polyprenols exert neuroprotective effects in APP/PS1 mice and could represent an effective treatment for AD.

ANGPTL4 mediates the protective role of PPARγ activators in the pathogenesis of preeclampsia.

Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be a therapeutic target for preeclampsia (PE). Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional secretory protein involved in regulating lipid metabolism and angiogenesis in various tissues. However, the expression of PPARγ and ANGPTL4 and their interaction in PE remain elusive. Here we showed that PPARγ agonist rosiglitazone upregulated the expression and secretion of ANGPTL4 in a dose-dependent manner in HTR8/SVneo cells, human umbilical vein endothelial cells (HUVECs) and placental explants. More importantly, we confirmed that the PPARγ/retinoid X receptor α heterodimer specifically binds to the ANGPTL4 promoter region and enhances its transcriptional activity. In addition, the levels of ANGPTL4 and PPARγ activators in the serum and their expression in placental tissues were significantly reduced in preeclamptic patients compared with normal pregnant subjects. Furthermore, functional studies demonstrated that ANGPTL4 mediates the facilitative effects of the PPARγ agonist on the survival, proliferation, migration and invasion of HTR8/SVneo cells, placental explants outgrowth and angiogenesis in HUVECs. Taken together, our results suggest that ANGPTL4 is a potential target gene for PPARγ and mediates the protective role of PPARγ activators in the pathogenesis of PE.

Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines.

AIMS: To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR. METHODS: Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit. RESULTS: We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated. CONCLUSIONS: Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines.

[Effects of caloric restriction on the oxidative stress injury and the expression of SIRT3 in PC12 cell].

OBJECTIVE: To study the regulation effects of CR (caloric restriction) and SIRT3 in the H2O2-induced oxidative stress injury of PC12 cell. METHODS: The cells were divided into four groups: H2O2, H2O2 + CR, CR and control (high glucose). For control and H2O2 group, cells were cultured in DMEM containing 0.45% glucose; for group in CR condition, cells were treated with the medium containing 0.1% glucose. For groups with H2O2, the H2O2 was diluted in EMEM medium to obtain the final concentration containing 60 µmol/L. Viability of PC12 cells were measured by MTT assay. The medium was refreshed with different concentration of H2O2 (from 10 to 120 µmol/L). The absorbance of the samples was measured at 492 nm using a microtiter plate reader. We detected TUNEL-positive cells using the In Situ Cell Apoptosis Detection kit pretreated with CR and H2O2. Immunofluorescence double staining detected the expression and localization of SIRT3. RT-PCR and Western-blot methods detected the expression of SIRT3, Caspase-3. RESULTS: After pretreating with 60 µmol/L H2O2 for 6 h, the viability of PC12 cells in H2O2 group (74.01 ± 2.21)% retained above 70%, and have statistical significance contrasted with control group (P < 0.05); After pretreating with 120 µmol/L H2O2, the viability of PC12 cells declined significantly (38.22 ± 3.34)%. So 60 µmol/L H2O2 is our experiment concentration. The viability of H2O2 group (74.01 ± 2.21)% was much lower than CR + H2O2 group (97.26 ± 1.92)% (P < 0.05). After pretreating with H2O2, TUNEL staining showed the apoptosis cells of CR + H2O2 group decreased significantly contrasted with H2O2 group. The immunofluorescence double staining results showed that SIRT3 was a mitochondria protein. Western-blot showed the expression of SIRT3 in CR group (6857 ± 157) (P < 0.05) increased and decreased in H2O2 group (3786 ± 160) (P < 0.05) contrasted with control group (5256 ± 143). The expression of SIRT3 in CR + H2O2 group (5056 ± 121) (P < 0.05) increased contrasted with H2O2 group (3786 ± 160). We also detected that Caspase-3 in H2O2 group (8499 ± 426) (P < 0.001) was much higher than control group than (5342 ± 420), but in the CR + H2O2 group (5750 ± 438) the expression of Caspase-3 was much lower than H2O2 group (8499 ± 426) (P < 0.001). RT-PCR also showed that the expression of SIRT3 in CR group (7214 ± 148) increased and decreased in H2O2 group (4807 ± 143) (P < 0.05) contrasted with control group (6204 ± 134). The expression of SIRT3 in CR + H2O2 group (6195 ± 166) increased contrasted with H2O2 group (4807 ± 143) (P < 0.05). CONCLUSION: CR causes anti-oxidative injury and has apoptotic effects in PC12 cell. It up-regulates the expression of SIRT3 and the effects of CR-SIRT3 can prevent PC12 cell from H2O2-induced apoptosis. And SIRT3 may be a novel molecule of regulating target in the delay of neuronal senescence.