Involvement of SIRT1 and SIRT3 in Effects of Hypoxic Hypobaric Preconditioning on Hippocampal Damage Induced by Chronic Cerebral Hypoperfusion in C57/6j and a7nAchRs(-/-) Mice / Participación de SIRT1 y SIRT3 en los Efectos del Precondicionamiento Hipobárico Hipóxico sobre el Daño del Hipocampo Inducido por Hipoperfusión Cerebral Crónica en Ratones C57/6j y a7nAchRs (-/-)

SUMMARY: Hypoxic preconditioning is known to induce neuroprotection, but its effects and pathways in chronic brain pathology still unknown. The aim was to establish an involvement of a7 subunit of nicotinic acetylcholine receptors (a7nAchRs), and sirtuins of 1 (SIRT1) and 3 (SIRT3) types in the effects of hypoxic hypobaric preconditioning on brain damage in mice with chronic cerebral hypoperfusion caused by the left common carotid artery occlusion. The male C57/6j (C57, wild type) and a7nAchRs(-/-) mice were divided to six experimental groups (10 mice per group): sham-operated C57, C57 with chronic cerebral hypoperfusion, C57 with hypoxic hypobaric preconditioning and chronic cerebral hypoperfusion, sham-operated a7nAchRs(-/-) mice, a7nAchRs(-/-) with chronic cerebral hypoperfusion, a7nAchRs(-/-) with hypoxic hypobaric preconditioning and chronic cerebral hypoperfusion. For preconditioning, mice were exposed to hypoxia by "lifting" in barochamber to simulated altitude of 5600 m a.s.l. for 1 h/day on 3 consecutive days before surgical manipulation. Expressions of SIRT1, SIRT3 in brain tissue, and histopathological changes of the hippocampi were examined. It was shown that 8-week chronic hypoperfusion of the brain, caused by unilateral occlusion of the common carotid artery, was accompanied by injury to the neurons of the hippocampi of both hemispheres, which was more pronounced on the side of the occlusion. This damage, as well as the mechanisms of neuroprotection induced by hypoxic preconditioning, were maintained for at least 8 weeks by mechanisms mediated through a7nAChRs. Deficite of a7nAChRs was accompanied with reduction of neuronal damage caused CCH in 8 weeks, as well as preconditioning effects, and lead to compensatory activation of regulatory and protective mechanisms mediated by SIRT1, in normal conditions and in CCH. In wild-type (C57) mice, protective mechanisms in CCH were realized to a greater extent by increased expression of SIRT3 in both hemispheres of the brain.

Se sabe que el precondicionamiento hipóxico induce neuroprotección, pero aún se desconocen sus efectos y vías en la patología cerebral crónica. El objetivo fue establecer la participación de la subunidad a7 de los receptores nicotínicos de acetilcolina (a7nAchR) y las sirtuinas de tipo 1 (SIRT1) y 3 (SIRT3) en los efectos del precondicionamiento hipóxico hipobárico sobre el daño cerebral en ratones con hipoperfusión cerebral crónica causada por la oclusión de la arteria carótida común izquierda. Los ratones macho C57/6j (C57, tipo salvaje) y a7nAchRs(-/-) se dividieron en seis grupos experimentales (10 ratones por grupo): C57 con operación simulada, C57 con hipoperfusión cerebral crónica, C57 con precondicionamiento hipobárico hipóxico y crónica. hipoperfusión cerebral, ratones a7nAchRs(-/-) operados de forma simulada, a7nAchRs(-/-) con hipoperfusión cerebral crónica, a7nAchRs(-/-) con precondicionamiento hipobárico hipóxico e hipoperfusión cerebral crónica. Para el preacondicionamiento, los ratones fueron expuestos a hipoxia "levantándolos" en una cámara de barro a una altitud simulada de 5600 m s.n.m. durante 1 h/día durante 3 días consecutivos antes de la manipulación quirúrgica. Se examinaron las expresiones de SIRT1, SIRT3 en tejido cerebral y los cambios histopatológicos de los hipocampos. Se demostró que la hipoperfusión cerebral crónica de 8 semanas, causada por la oclusión unilateral de la arteria carótida común, se acompañaba de lesión de las neuronas del hipocampo de ambos hemisferios y que era más pronunciada en el lado de la oclusión. Este daño, así como los mecanismos de neuroprotección inducidos por el precondicionamiento hipóxico, se mantuvieron durante al menos 8 semanas mediante mecanismos mediados por a7nAChR. El déficit de a7nAChR se acompañó de una reducción del daño neuronal causado por CCH en 8 semanas, así como de efectos de precondicionamiento, y condujo a una activación compensatoria de mecanismos reguladores y protectores mediados por SIRT1, en condiciones normales y en CCH. En ratones de tipo salvaje (C57), los mecanismos de protección en CCH se realizaron en mayor medida mediante una mayor expresión de SIRT3 en ambos hemisfe- rios del cerebro.

Inhibition of Oxidative Stress of Sciatic Nerve in Diabetic Rats by Tangbikang Granules Regulating AMPK/PGC-1α/SIRT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Tangbikang granules on oxidative stress of sciatic nerve in diabetic rats by regulating adenylate activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α/mitochondrial Sirtuins 3 （AMPK/PGC-1α/SIRT3） signaling pathway. MethodThe spontaneous obesity type 2 diabetes model was established using ZDF rats. After modeling， they were randomly divided into high， medium， and low dose Tangbikang granule groups （2.5， 1.25， 0.625 g·kg-1·d-1） and lipoic acid group （0.026 8 g·kg-1·d-1）， and the normal group was set up. The rats were administered continuously for 12 weeks after modeling. The blood glucose of rats was detected before intervention and at 4， 8， 12 weeks after intervention. At the 12th week， motor nerve conduction velocity （MNCV）， sensory nerve conduction velocity （SNCV）， nerve blood flow velocity， mechanical pain threshold， and thermal pain threshold were detected. The sciatic nerve was taken for hematoxylin-eosin （HE） staining to observe the tissue morphology. The ultrastructure of the sciatic nerve was observed by transmission electron microscope. The expression levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） in sciatic nerve were determined by enzyme-related immunosorbent assay （ELISA）. The mRNA expressions of AMPKα， AMPKβ， PGC-1α， and SIRT3 in sciatic nerve were determined by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the normal group， fasting blood glucose in the model group was increased at each time point （P<0.01）. The mechanical pain threshold was decreased （P<0.05）， and the incubation time of the hot plate was extended （P<0.01）. MNCV， SNCV， and nerve blood flow velocity decreased （P<0.05）. The expression level of SOD was decreased （P<0.01）. The expression levels of MDA， IL-1β， and TNF-α were increased （P<0.01）. The mRNA expression levels of AMPKα， AMPKβ， PGC-1α， and SIRT3 were decreased （P<0.01）. The structure of sciatic nerve fibers in the model group was loose， and the arrangement was disordered. The demyelination change was obvious. Compared with the model group， the fasting blood glucose of rats in the high dose Tangbikang granule group was decreased after the intervention of eight weeks and 12 weeks （P<0.01）. The mechanical pain threshold increased （P<0.05）. The incubation time of the hot plate was shortened （P<0.01）. MNCV， SNCV， and Flux increased （P<0.05）. The expression level of SOD was increased （P<0.01）. The expression levels of MDA， IL-1β， and TNF-α were decreased （P<0.01）. The mRNA expression levels of AMPKα， AMPKβ， PGC-1α， and SIRT3 were increased （P<0.01）. The sciatic nerve fibers in the high-dose Tangbikang granule group were tighter and more neatly arranged， with only a few demyelinating changes. The high， medium， and low dose Tangbikang granule groups showed a significant dose-effect trend. ConclusionTangbikang granules may improve sciatic nerve function in diabetic rats by regulating AMPK/PGC-1α/SIRT3 signaling pathway partly to inhibit oxidative stress.

Construction of hepatocyte-specific Sirt3 gene knockout mouse model / 安徽医科大学学报

Objective @#To construct hepatocyte-specific silence information regulator 3 ( Sirt3 ) gene knockout (Sirt3Δhep ) mice by Cre-loxP technique , and to provide an important animal model for further studying the biological function of the hepatocyte Sirt3 gene in diseases . @*Methods @#LoxP-labeled Sirt3flox/flox mice were mated with Alb-Cre homozygous (Alb-Cre + / + ) mice , and the F1 generation Sirt3flox/ - /Alb-Cre + / - mice were then mated with Sirt3flox/flox mice , and the F2 genotype of Sirt3flox/flox/Alb-Cre + / - mice were the Sirt3Δhep mice constructed in this ex- periment. Sirt3flox/flox/Alb-Cre - / - (Sirt3flox/flox ) mice were the control mice . Mouse tail genome DNA was extracted and PCR was used to identify the genotypes of the offspring mice . Immunofluorescence was used to detect Sirt3 ex- pression in mouse hepatocytes . Primary hepatocytes and tissue proteins of Sirt3Δhep mice were extracted , and the ex- pression of Sirt3 in mouse hepatocytes and other tissues was verified by Western blot. HE staining was used to ob- serve mice ′s liver , heart , spleen , and lung tissue structure . @*Results @#Sirt3Δhep mice were successfully identified .Immunofluorescence and Western blot results demonstrated a significant decrease in the expression of Sirt3 in the hepatocytes of these mice compared to the control group ( P < 0. 01) . At the same time , there was no significant difference in the expression of Sirt3 in the heart , spleen , kidney , and lung tissues of Sirt3Δhep mice compared with the control group (P > 0. 05) . The results of HE staining showed that the histological characteristics of the liver , heart , spleen , lungs , kidneys , and other major organs of Sirt3Δhep mice were not significantly different from those of the control group mice . @*Conclusion @#Hepatocyte-specific Sirt3 gene knockout mice are successfully constructed , which provides an animal model to explore further the role and molecular mechanism of the hepatocyte Sirt3 gene in diseases .

Zoledronic acid affects osteogenic differentiation and bone formation by regulating the expression of SIRT3/P53 / 中华内分泌外科杂志

Objective:To explore the mechanism of zoledronic acid (ZOL) affects osteogenic differentiation and bone formation through the regulation of sirtuin 3 (SIRT3) / P53 expression.Methods:Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate into osteogenic cells, the expression of SIRT3 in the cells was detected, and the targeting regulation relationship between SIRT3 and P53 was analyzed. The intracellular expressions of SIRT3 and P53 were intervened and ZOL was used to treat the cells. MTT method, Western blot method and kit were used to detect cell viability, osteogenesis-related genes Osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining, respectively. Ovariectomy (OVX) was used to construct a rat model and explore the effect of ZOL on the progression of osteoporosis (OP) in vivo.Results:ZOL promoted osteogenic differentiation of BMSCs. The expression of SIRT3 was down-regulated in the serum of OP patients (0.78±0.23) compared with that of healthy subjects (1.00±0.26 vs. 0.78±0.23. t=3.85, P<0.001). During the osteogenic differentiation of BMSCs, the expression level of SIRT3 gradually increased with the prolonged induction of osteogenesis. Compared with the p53 protein expression and BMSCs activity in the control group, SIRT3 knockout could increase the expression level of p53 protein (0.59±0.05 vs. 1.01±0.11. t=6.02, P=0.004) but inhibited the activity of BMSCs (100.00±8.41 vs. 51.26±5.59. t=8.36, P=0.001). After ZOL treatment, the inhibitory effect of SIRT3 on cell viability (49.61±5.11 vs. 87.61±7.31. t=7.38, P=0.002) and osteogenesis was relieved, and the level of P53 was inhibited (1.10±0.10 vs. 0.69±0.04. t=6.59, P=0.003). P53 overexpression partially offseted the effects of ZOL on cell viability (84.61±6.52 vs. 66.54±5.47. t=3.68, P=0.021) and osteogenesis. Compared with the sham surgery group, the OVX group showed inhibition of osteogenesis in rats, and ZOL treatment significantly improved osteogenic inhibition. ZOL treatment increased the expression level of SIRT3 protein in bone tissue of OVX rats, but inhibited the expression level of P53. Conclusion:ZOL promoted osteogenic differentiation and bone formation of BMSCs by promoting the ubiquitination and degradation of P53 by SIRT3.

Effect of Linggui Zhugantang on Mitochondrial Fusion-fission and Sirt3/ AMPK Signaling Pathway in Chronic Heart Failure Rats After Myocardial Infarction / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg-1） and Captopril group （0.002 57 g·kg-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.

Effects of silver needle-thermal conduction therapy on skeletal muscle mitochondria and silent information regulator homolog 3 expression in a rat model of myofascial pain syndrome / 中国组织工程研究

BACKGROUND:Clinical studies have found good analgesic effects of silver needle-thermal conduction therapy in patients with myofascial pain syndrome,but the exact mechanism remains unclear. OBJECTIVE:To observe the effect of silver needle-thermal conduction therapy on silent information regulator homolog 3(SIRT3)changes and mitochondrial ultrastructure in a rat model of myofascial pain syndrome. METHODS:Twenty rats were randomly selected from 26 Sprague-Dawley rats and were subjected to percussion combined with motor fatigue for replicating the rat model of myofascial pain syndrome.Sixteen rats that were successfully modeled were randomly divided into model group and silver needle-thermal conduction therapy group(treatment group),with eight rats in each group.The remaining rats were used as controls(normal group).The treatment group was treated with silver needle-thermal conduction therapy.Mechanical withdrawal threshold and thermal withdrawal latency of rats were measured at 1 day before modeling,1 day after modeling and 14 days after treatment.Electromyographic activities of the right medial femoral muscle were measured at 14 days after treatment.The right medial femoral muscle tissue was taken for hematoxylin-eosin staining to observe the local morphology and for transmission electron microscopy to observe the mitochondrial ultrastructure.Western blot assay was performed to detect SIRT3 expression. RESULTS AND CONCLUSION:Pain threshold:The mechanical withdrawal threshold and thermal withdrawal latency of the model and treatment groups were significantly decreased compared with those in the normal group and before modeling(P＜0.01).After treatment,the mechanical withdrawal threshold and thermal withdrawal latency of rats were significantly higher in the treatment group compared with the model group(P＜0.01).Electromyography:The rats in the model group showed spontaneous electrical activity in the right medial femur,while the rats in the treatment group showed reduced spontaneous electrical activity,longer time frame(P＜0.01)and lower wave amplitude(P＜0.05)compared with the model group.Hematoxylin-eosin staining:In the normal group,rat muscle fibers arranged closely and regularly.In the model group,the muscle fibers of rats were atrophied,degenerated,and disordered in arrangement.In the treatment group,rat muscle structure disorder improved.Mitochondrial microstructure:Under the transmission electron microscope,mitochondrial structure in the normal group was normal;mitochondrial swelling with broken or disappeared cristae appeared in the model group;mitochondrial swelling in the treatment group was obviously relieved or tended to be normal.SIRT3 expression:SIRT3 expression was significantly downregulated in the model group compared with the normal group,but was significantly upregulated in the treatment group compared with the model group(P＜0.05).To conclude,abnormalities in local muscle mitochondria and downregulation of SIRT3 expression suggest the presence of impaired energy metabolism in the rat model of myofascial pain syndrome.Mitochondrial changes recover and are close to normal after the silver needle-thermal conduction therapy,and the expression of SIRT3 is also upregulated close to the normal group,indicating the silver needle-thermal conduction therapy may play a therapeutic role by promoting mitochondrial repair and improving energy metabolism disorder.

Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells via ROS/Sirt3/SOD2 Pathway / 中国肺癌杂志

BACKGROUND@#Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC.@*METHODS@#PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8.@*RESULTS@#CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).@*CONCLUSIONS@#The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.

Research progress on the role of Sirt3 in myocardial infarction / 局解手术学杂志

Myocardial infarction is a serious cardiovascular disease that can disrupt the myocardial energy metabolism.Sirt3,as an important mitochondrial deacetylase,plays a crucial role in the regulation of energy metabolism after myocardial infarction.This review aims to analyze and summarize the research progress of Sirt3 in the regulation of energy metabolism after myocardial infarction,so as to provide new theoretical and practical basis for the treatment of cardiovascular diseases.

Research Progress in Sirt1/Sirt3 Signal Targeted Mitochondrial Quality Control for Cardiovascular Disease Intervention and New Ideas Combined with Traditional Chinese Medicine / 世界科学技术-中医药现代化

Cardiovascular disease has become a major public health problem to be solved due to its high incidence rate,high mortality and heavy economic and social burden.Mitochondrial quality control is the cornerstone of maintaining mitochondrial homeostasis and plays an important role in the occurrence and progression of cardiovascular diseases.Some studies have found that Sirt1/Sirt3 signal is involved in the mechanism of myocardial mitochondrial quality control,and has gradually become an important target for cardiovascular disease prevention and treatment.At the same time,research shows that traditional Chinese medicine can regulate the quality control of myocardial mitochondria based on Sirt1/Sirt3 signal,and improve cardiovascular disease,but research is still insufficient.Therefore,this article reviews the research related to the influence of Sirt1/Sirt3 signal on the mitochondrial quality control of cardiovascular disease and proposes new ideas in combination with traditional Chinese medicine,with a view to providing a basis and direction for the research on the target of cardiovascular disease and the mechanism of traditional Chinese medicine in the prevention and treatment of cardiovascular disease.

The protective effect and mechanism of N-acetylcysteine on acute kidney injury by up-regulating Sirtuin3 protein expression in septic mice / 中华急诊医学杂志

Objective:To explore the protective effect and mechanism of the antioxidant N-acetylcysteine (NAC) regulating silent information regulator 3 (Sirt3) on acute kidney injury (AKI) in septic mice.Methods:Male C57BL/6 mice were randomly ( random number) divided into the sham operation group (sham), cecal ligation and perforation group (CLP), CLP + NAC (50 mg/kg) and CLP + NAC (100 mg/kg) groups, with 10 mice in each group. The mice were sacrificed 24 h after CLP, and blood and kidney tissue samples were collected. HE staining was used to evaluate the pathological damage of the kidney tissue of mice in each group. ELISA was used to detect serum creatinine (Scr), urea nitrogen (BUN), kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated apolipoprotein (NGAL) levels. Immunohistochemistry was used to detect the expression of Sirt3 protein in kidney tissue. RT-qPCR was used to detect the level of Sirt3 mRNA. Mitochondrial damage of renal tubular epithelial cells was observed under transmission electron microscope, and the mitochondrial density was calculated. Meanwhile, the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and malondialdehyde (MDA) in the renal cortex were also detected. Results:Compared with the sham group, in the CLP group, the pathological damage of renal tissue was significantly aggravated ( P<0.001), and the levels of renal function indicators (Scr, BUN, KIM-1 and NGAL) were all increased significantly (all P<0.001). The protein and mRNA expression of Sirt3 were all significantly decreased (all P<0.001), the mitochondrial structure damage of renal tubular epithelial cells was increased, and the mitochondrial density was significantly decreased ( P<0.001). The levels of antioxidant enzymes (SOD, GSH-Px and CAT) in the renal cortex were all significantly decreased (all P<0.001), while the lipid peroxide MDA was significantly increased ( P<0.001). Compared with the CLP group, the renal injury score and renal function indexes (Scr, BUN, KIM-1 and NGAL levels) in the 50 mg/kg NAC pretreatment group were decreased, and the levels of SOD, GSH-Px and CAT in renal tissue were increased, but the differences were not significant. However, pretreatment with 100 mg/kg NAC significantly reduced the pathological damage of kidney tissue caused by CLP ( P<0.001), and significantly decreased the levels of Scr, BUN, KIM-1 and NGAL (all P<0.001). The expression of Sirt3 protein [(50.20±2.79) vs.(20.00±0.75), P<0.001] and mRNA [(0.57±0.07) vs. (0.41±0.07), P<0.001] were all significantly increased. The mitochondrial structure of renal tubular epithelial cells was more stable, and the mitochondrial density was significantly increased [(0.60±0.05) vs. (0.43±0.06), P<0.001]. The levels of SOD [(67.37±3.79) U/mg vs. (21.09±0.89) U/mg, P<0.001], GSH-Px [(265.61±9.61) U/mg vs. (180.00±3.31) U/mg, P<0.001] and CAT [(8.58±0.65) U/mg vs. (5.19±0.58) U/mg, P<0.001] were all significantly increased, while the expression level of MDA was significantly reduced [(40.36 ±1.79) vs. (83.81 ±1.70), P<0.001]. Conclusions:NAC can significantly reduce renal pathological damage, improve renal function, maintain mitochondrial structure stability and reduce oxidative stress levels in septic mice by up-regulating Sirt3 protein expression, and has a significant protective effect on CLP-induced AKI.

Nicotinamide mononucleotide attenuates ischemia-reperfusion injury induced by donor liver from cardiac death through Sirt3 / 器官移植

Objective To evaluate the effect and mechanism of nicotinamide mononucleotide (NMN) on ischemia-reperfusion injury (IRI) induced by donor liver after cardiac death in rat models. Methods Rat models of orthotopic liver transplantation were established by "magnetic ring + double cuff" method. SD rats were randomly divided into the sham operation group (Sham group), orthotopic liver transplantation group (OLT group), NMN treatment + orthotopic liver transplantation group (NMN group), NMN+sirtuin-3 (Sirt3) inhibitor (3-TYP) + orthotopic liver transplantation group (NMN+3-TYP group), respectively. Pathological changes and hepatocyte apoptosis of the rats were observed in each group. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined. Superoxide dismutase (SOD) and malondialdehyde (MDA) contents in liver tissues were detected. The expression levels of Sirt3, microtubule-associated protein 1 light chain 3 (LC3)Ⅱ, PTEN-induced putative kinase 1 (PINK1), Parkin and translocase of the outer mitochondrial membrane 20 (TOMM20) in liver tissues were measured. Postoperative survival of the rats in each group was analyzed. Results Compared with the Sham group, serum ALT and AST levels were higher in the OLT group. Compared with the OLT group, the levels of ALT and AST were decreased in the NMN group. Compared with the NMN group, the levels of ALT and AST were increased in the NMN +3-TYP group (all P < 0.05). The liver tissue structure of rats in the Sham group was basically normal. In the OLT group, pathological changes, such as evident congestion, vacuolar degeneration and hepatocyte necrosis, were observed in the liver tissues. Compared with the Sham group, Suzuki score and apoptosis rate were higher in the OLT group. Suzuki score and apoptosis rate in the NMN group were lower than those in the OLT group. Suzuki score and apoptosis rate in the NMN+3-TYP group were higher compared with those in the NMN group (all P < 0.05). Compared with the Sham group, the SOD content was decreased, whereas the MDA content was increased in the OLT group. Compared with the OLT group, the SOD content was increased, whereas the MDA content was decreased in the NMN group. Compared with the NMN group, the SOD content was decreased, whereas the MDA content was increased in the NMN+3-TYP group (all P < 0.05). Compared with the Sham group, the relative expression levels of Sirt3 and TOMM20 proteins were down-regulated, whereas those of PINK1, Parkin and LC3Ⅱproteins were up-regulated in the OLT group. Compared with the OLT group, the relative expression levels of Sirt3, PINK1, Parkin and LC3Ⅱproteins were up-regulated, whereas that of TOMM20 protein was down-regulated in the NMN group. Compared with the NMN group, the relative expression levels of PINK1, Parkin and LC3Ⅱproteins were down-regulated, whereas that of TOMM20 protein was up-regulated in the NMN+3-TYP group (all P < 0.05). In the Sham group, the 7 d survival rate of rats was 100%, 50% in the OLT group, 75% in the NMN group and 58% in the NMN+3-TYP group, respectively. Conclusions NMN may enhance the antioxidative capacity of the liver, induce PINK1/Parkin-mediated mitochondrial autophagy, and alleviate IRI of the liver by up-regulating Sirt3, thereby playing a protective role in the donor liver after cardiac death.

Role and mechanism of SIRT3 in attenuation of intestinal ischemia-reperfusion injury by dexmedetomidine in mice / 中国临床药理学与治疗学

AIM: To explore the role and mechanism of silent mating type information regulator 2 homolog 3 (SIRT3) in attenuation of intestinal ischemia-reperfusion (I/R) injury by dexmedetomidine in mice. METHODS: Twenty-four healthy male C57BL mice were divided into 4 groups randomly (n=6): sham operation group (Sham group), intestinal ischemia-reperfusion group (I/R group), dexmedetomidine group (Dex group), SIRT3 inhibitor 3-TYP group (3-TYP group). Superior mesenteric artery was clamped for 45 min followed by reperfusion for 2 h to establish intestinal I/R model in I/R group, Dex group, and 3-TYP group. Sham group received sole sham operation. 1 h prior to onset of ischemia, 3-TYP was injected into mice in 3-TYP group intraperitoneally (5 mg/kg, diluted to 0.3 mL), and 0.3 mL normal saline into mice in Dex group intraperitoneally. 30 min prior to onset of ischemia, dexmedetomidine was injected into mice in 3-TYP group and Dex group intraperitoneally (25 μg/kg, diluted to 0.3 mL). 1 h and 30 min prior to onset of ischemia, 0.3 mL normal saline was injected into mice in Sham group and I/R group intraperitoneally, respectively. 2 h of after reperfusion, the mice were sacrificed under anesthesia. Intestinal tissues were took and observed for pathological changes under light microscope after HE staining, and the injury was assessed via the Chiu's score method, and activities of SIRT3 and superoxide dismutase 2 (SOD2) were detected via spectrophotometry, and malondialdehyde (MDA) via spectrophotometry. RESULTS: The pathological injury was exacerbated, and the Chiu's score, the MDA level elevated remarkably, while the activity level of SIRT3 and SOD2 declined remarkably in I/R group, Dex group and 3-TYP group compared to Sham group (P<0.05). The pathological injury was alleviated, and the Chiu's score declined remarkably in Dex group and 3-TYP group compared to I/R group (P<0.05); and the MDA level declined remarkably, while activity level of SIRT3 and SOD2 elevated remarkably in Dex group compared to I/R group (P<0.05); and there was no significant difference both in the activity level of SIRT3 and SOD2 and in the MDA level between 3-TYP group and I/R group. The pathological injury was exacerbated, and the Chiu's score, the MDA level elevated remarkably, while the activity level of SIRT3 and SOD2 declined remarkably in 3-TYP group compared to Dex group (P<0.05). CONCLUSION: SIRT3 and its downstream SOD2 are involved in mediating the effect of attenuation of intestinal ischemia-reperfusion injury through inhibiting oxidative stress response by dexmedetomidine.

Role of Sirt3 in lipopolysaccharide-induced abnormal mitochondrial dynamics in renal tubular epithelial cells / 中华肾脏病杂志

Objective:To observe the expression of sirtuin 3 (Sirt3) and mitochondrial damage-associated proteins in lipopolysaccharide (LPS)-induced acute kidney injury mouse model and renal tubular epithelial cells, and to explore the role of Sirt3 in LPS-induced abnormal mitochondrial dynamics in renal tubular epithelial cells.Methods:Eighteen specific pathogen free (SPF) male C57BL/6 mice were randomly assigned to control group, LPS 24 h group and LPS 48 h group. The control group was intraperitoneally injected with physiological saline (0.1 ml/10 g), and LPS 24 h group and LPS 48 h group were intraperitoneally injected with LPS (10 mg/kg) solution. Renal functional indexes of mice were analyzed by automatic biochemical analyzer. The pathological change of the kidney was observed by HE staining, and the expressions of dynamin-related protein-1 (Drp1), optic atrophy type 1 (Opa1) and Sirt3 were evaluated by Western blotting. Expression and distribution of Sirt3 in kidney was assessed by immunohistochemistry. Human renal tubular epithelial cells (HK-2) were exposed to 10 μg/ml LPS for 24 h, and the expression of Drp1, Opa1 and Sirt3 were detected by Western blotting. Cell apoptosis was assessed by Hoechst-33342 staining. After transfection to HK-2 cells with pcDNA3.1-Sirt3 recombinant plasmid, the expressions of Sirt3, Drp1, Opa1 and cell apoptosis were detected by the same methods as above.Results:(1) The levels of blood urea nitrogen and serum creatinine in LPS group were significantly higher than those in control group (both P<0.05), and the pathological changes of kidney were obvious. (2) Compared with the control group, the expression of mitochondrial fission-associated protein Drp1 in renal tissue of LPS group was significantly higher ( P<0.05), and the expression of mitochondrial fusion associated protein Opa1 was significantly lower ( P<0.05). (3) Compared with the control group, the expression of Sirt3 in LPS group was significantly lower ( P<0.05), and immunohistochemistry results showed that Sirt3 was mainly expressed in glomerular vascular endothelial cells and renal tubular epithelial cells. (4) In vitro, LPS stimulation induced increased Drp1 expression in HK-2 cells ( P<0.05), decreased Opa1 and Sirt3 expression (both P<0.05), and increased apoptosis ( P<0.05). (5) LPS-induced mitochondrial dynamics disturbance and apoptosis were alleviated by pcDNA3.1-Sirt3 recombinant plasmid transfection. Conclusions:LPS can induce down-regulation of Sirt3 expression and disturbance of mitochondrial dynamics, and Sirt3 may play a protective role in LPS-induced acute kidney injury by regulating mitochondrial dynamics.

Ginsenoside Rb1 Protects Human Umbilical Vein Endothelial Cells against High Glucose-Induced Mitochondria-Related Apoptosis through Activating SIRT3 Signalling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate whether ginsenoside Rb1 (Rb1) can protect human umbilical vein endothelial cells (HUVECs) against high glucose-induced apoptosis and examine the underlying mechanism.@*METHODS@#HUVECs were divided into 5 groups: control group (5.5 mmol/L glucose), high glucose (HG, 40 mmol/L) treatment group, Rb1 (50 µ mol/L) treatment group, Rb1 plus HG treatment group, and Rb1 and 3-(@*RESULTS@#Rb1 ameliorated survival in cells in which apoptosis was induced by high glucose (P<0.05 or P<0.01). Upon the addition of Rb1, mitochondrial and intracellular reactive oxygen species generation and malondialdehyde levels were decreased (P<0.01), while the activities of antioxidant enzymes were increased (P<0.05 or P<0.01). Rb1 preserved the mitochondrial membrane potential and reduced the release of Cyt-c from the mitochondria into the cytosol (P<0.01). In addition, Rb1 upregulated mitochondrial biogenesis-associated proteins (P<0.01). Notably, the cytoprotective effects of Rb1 were correlated with SIRT3 signalling pathway activation (P<0.01). The effect of Rb1 against high glucose-induced mitochondria-related apoptosis was restrained by 3-TYP (P<0.05 or P<0.01).@*CONCLUSION@#Rb1 could protect HUVECs from high glucose-induced apoptosis by promoting mitochondrial function and suppressing oxidative stress through the SIRT3 signalling pathway.

The protective effect of N-acetylcysteine on mitochondrial oxidative damage of airway epithelial cells induced by cigarette smoke extract / 西安交通大学学报(医学版)

【Objective】 To investigate the effect of N-acetylcysteine （NAC） on mitochondrial damage of airway epithelial cells induced by cigarette smoke extract （CSE）. 【Methods】 Human airway epithelial cells （BEAS-2B） were cultured and divided into three groups as follows： normal control group， 7.5% （75 mL/L） CSE-treated group and 7.5% CSE plus NAC group. After stimulation for 24 hours， cell viability was determined by MTT， and the levels of mitochondrial reactive oxygen species （ROS） and mitochondrial membrane potential （MMP） were observed under the fluorescence microscope. MMP was also measured by flow cytometry， the protein expressions of Sirt3 and manganese superoxide dismutase （MnSOD） were detected by Western blotting， and MnSOD activity was measured by colorimetry. 【Results】 Pretreatment with NAC significantly improved the viability of airway epithelial cells （P<0.05）. The results of fluorescence microscopy and flow cytometry showed that NAC pretreatment significantly attenuated MMP decline in airway epithelial cells exposed to 7.5% CSE （P<0.05）. Compared with 7.5% CSE-treated group， mitochondrial ROS in airway epithelial cells was significantly decreased in 7.5% CSE plus NAC group （P<0.05）. In addition， pretreatment with NAC significantly inhibited the decrease of Sirt3 and MnSOD protein expression and improved MnSOD activity in airway epithelial cells exposed to 7.5% CSE （P<0.05）. 【Conclusion】 NAC attenuates CSE-induced airway epithelial mitochondrial damage through the regulation of Sirt3-MnSOD signaling pathway， which reveals a new mechanism of NAC treatment for chronic obstructive pulmonary disease.

Rosuvastatin relieves neuronal mitochondrial damage by regulating UCP2-SIRT3 pathway after cerebral ischemia/reperfusion / 中国药理学通报

Aim To explore the biological role and related mechanism of rosuvastatin (RS) in mitochondrial damage of neurons after cerebral ischemia/reperfusion (CIR) through UCP2-SIRT3 signaling pathway. Methods Human neuroblastoma cell (SH-SY5Y cell) cerebral infarction reperfusion model (OGD/R) was established, different concentrations of RS (40 and 2. 5 (mol • L

Rosuvastatin regulates cerebral I/R damage to neurons by UCP2-SIRT3 signal / 中国临床药理学与治疗学

AIM: To study the protective effect and mechanism of rosuvastatin on cerebral ischemia-reperfusion injury. METHODS: (1) Cerebral infarction and OGD/R cell models were established to detect the effects of different concentrations of rosuvastatin on cell proliferation and apoptosis; (2) Different concentrations of rosuvastatin were used to treat OGD/R cell models and to observe rosuvastatin effects on cell morphology and expression and localization of UCP2-SIRT3 in cells; (3) UCP2 silent cell line was constructed to observe cell mitochondrial morphology and expression and localization of TOMM20 and SIRT3 molecules in cells, and to study the channels and mechanisms that play a protective role of rosuvastatin in OGD/R cell model; (4) The mitochondrial membrane potential, mitochondrial gene PGC1, Drp1 and Opa1 expression were detected to study the protective effect of rosuvastatin on mitochondria. RESULTS: (1) Rosuvastatin of different concentrations could significantly reduce OGD/R cell apoptosis and increase cell survival rate; (2) Rosuvastatin exerted cell protection by affecting the expression of UCP2 and SIRT3 in cells, thereby protecting cells from OGD/R injury; (3) Rosuvastatin affected the expression of TOMM20 by regulating UCP2, increased mitochondrial transmembrane transport and energy metabolism, enhanced mitochondrial function, and improved cell state and reduced apoptosis. CONCLUSION: Rosuvastatin inhibits mitochondrial damage of OGD/R cells by regulating UCP2/SIRT pathway, thereby exerting neuron protection.

Maternal hypothyroidism during pregnancy alters the function of the retinol-binding protein 4-mediated mitochondrial permeability conversion pore in the kidneys of offspring rats

OBJECTIVES To determine the role of the RBP4/PiC/SIRT3 signaling pathway in the opening of the mitochondria permeability transition pore (mPTP) in offspring rats with hypothyroidism during pregnancy. METHODS Sixty Sprague-Dawley (SD) rats were employed in this study. Pregnancy was deemed successful when a sperm was found in the uterus. After one week of pregnancy, offspring rats were divided into the following groups: overall hypothyroidism group (OH group), subclinical hypothyroidism group (SCH group), and normal control group (CON group). The establishment of the hypothyroidism model was confirmed when the serum thyroid stimulating hormone (TSH) levels were higher than normal value and TT4 level was within the normal range. The renal mitochondria of offspring rats were extracted on the 14th postnatal day (P14) and 35th postnatal day (P35). RESULTS At P14, no significant differences in the degree of mPTP opening and expression of phosphoric acid carrier vector (PiC) were detected between the rats in the OH group and the SCH group. However, the expression level of silent mating-type information regulation 3 homolog (SIRT3) was markedly reduced. Retinol-binding protein 4 (RBP4) expression increased in the rats from the OH group, relative to that in those from the SCH group. At P35, the degree of mPTP opening and the expression levels of PiC and RBP4 in the OH group were higher than those in the SCH group. However, SIRT3 expression in the OH group was lower than that observed in the SCH group. CONCLUSION RBP4 plays an important role in early renal mitochondrial damage and renal impairment in rats suffering from hypothyroidism during pregnancy. The RBP4/PiC/SIRT3 pathway is thus involved in the opening of the renal mPTP in offspring rats with hyperthyroidism.

Metformin promotes autophagy in muscle cells exposed to palmitic acid through AMPK/SIRT3 pathway / 中华内分泌代谢杂志

Objective@#To investigate the effect of metformin on autophagy in muscle cells exposed to palmitic acid, and to explore its mechanism.@*Methods@#L6 rat myoblasts were incubated with palmitic acid at various concentrations(0.1, 0.2, 0.4, 0.6 mmol/L) and metformin(0.5, 1, 2, 5, 10 mmol/L) for 24 h. CCK8 method was used to detect the survival rate of muscle cells. After muscle cells were treated with palmitic acid and metformin for 24 h, mRNA and protein expressions of microtubule-associated protein11ight chain3(LC3Ⅱ), Beclin 1, p62, and silent mating type information regulation2 homolog-3(SIRT3) were detected by RT-PCR and Western blot, respectively. AMP-activated protein kinase(AMPK) phosphorylation level was detected by Western blot.@*Results@#Palmitic acid dose-dependently decreased the survival rate of muscle cells, which was attenuated by metformin at the concentration of 2 mmol/L. After muscle cells were incubated with 0.4 mmol/L palmitic acid and 2 mmol/L metformin for 24 h, palmitic acid significantly reduced the mRNA and protein expressions of LC3Ⅱ, Beclin1, and SIRT3 as well as phosphorylation level of AMPK(all P<0.05), and increased p62 mRNA and protein expressions(P<0.05). Those effects were all antagonized by metformin(all P<0.05).@*Conclusions@#Metformin treatment may promote the autophagy of muscle cells exposed to palmitic acid through AMPK/STRT3 pathway.

Platelet derived growth factor-BB regulates phenotype transformation of pulmonary artery smooth muscle cells via SIRT3 affecting glycolytic pathway / 中华心血管病杂志

Objective@#To investigate whether platelet-derived growth factor-BB (PDGF-BB) can regulate phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) via SIRT3 affecting glycolytic pathway.@*Methods@#The PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into 3 groups by using 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway: normal control group, PDGF-BB group(30 ng/ml) and PDGF-BB (30 ng/ml)+2-DG (10 mmol/L) group. In lentivirus-mediated overexpression assay, cells were divided into control group, PDGF-BB group(30 ng/ml), PDGF-BB+deacetylase sirtuin-3 (SIRT3) overexpression group and PDGF-BB+empty vector group. The expression levels of phenotype related index such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), calponin, vimentin were detected by qRT-PCR and Western blot. Meanwhile, the expression of α-SMA was detected by cellular immunofluorescence staining. EDU staining was used to detect the proliferation of PASMCs. The expression of SIRT3 was detected by Western blot. The expressions of glucose transporter 1 and aerobic glycolytic enzymes were detected by qRT-PCR and Western blot in lentivirus-mediated overexpression assay.@*Results@#(1) PDGF-BB affects PASMCs phenotypic transformation through glycolytic pathway: compared with normal control group, PDGF-BB significantly decreased the expressions of contractile phenotype markers such as α-SMA, SM-MHC, calponin mRNA and protein (all P<0.05), but it increased the expressions of the synthetic phenotype marker vimentin mRNA and protein (both P<0.05). Cellular immunofluorescence assay showed that PDGF-BB significantly decreased the number of α-SMA positive cells, while 2-DG reversed the process. (2) PDGF-BB promoted cell proliferation through glycolytic pathway: the proliferation of PASMCs was significantly higher in PDGF-BB group than in control group (P<0.05), and which could be significantly reduced by 2-DG (P<0.05). (3) PDGF-BB inhibited the expression of SIRT3 protein in PASMCs: the expression of SIRT3 protein in PDGF-BB group was lower than that in control group (P<0.05). (4) PDGF-BB affected glycolytic pathway through SIRT3:compared with the control group, PDGF-BB significantly increased the expression levels of glucose transporter 1 (Glut1), hexokinase 2 (HK2) and 6-phosphfructo-2-kinase 3 (PFKFB3) mRNA (all P<0.05), which was reserved by over-expression of SIRT3. There were no significant difference in mRNA expression levels between PDGF-BB group and PDGF-BB+empty vector group (P>0.05).Compared with the control group, PDGF-BB significantly increased the expression levels of Glut1, HK2 and PFKFB3 protein(all P<0.05), which was reserved by over-expression of SIRT3. There were no significant differences in protein expression levels between PDGF-BB group and PDGF-BB+empty vector group (all P>0.05).@*Conclusion@#PDGF-BB regulates phenotypic transformation of PASMCs via SIRT3 affecting glycolytic pathway.