To investigate the effect of Huazhi Rougan Granules(HZRG) on autophagy in a steatotic hepatocyte model of free fatty acid(FFA)-induced nonalcoholic fatty liver disease(NAFLD) and explore the possible mechanism. FFA solution prepared by mixing palmitic acid(PA) and oleic acid(OA) at the ratio of 1∶2 was used to induce hepatic steatosis in L02 cells after 24 h treatment, and an in vitro NAFLD cell model was established. After termination of incubation, cell counting kit-8(CCK-8) assay was performed to detect the cell viability; Oil red O staining was employed to detect the intracellular lipid accumulation; enzyme-linked immunosorbnent assay(ELISA) was performed to measure the level of triglyceride(TG); to monitor autophagy in L02 cells, transmission electron microscopy(TEM) was used to observe the autophagosomes; LysoBrite Red was used to detect the pH change in lysosome; transfection with mRFP-GFP-LC3 adenovirus was conducted to observe the autophagic flux; Western blot was performed to determine the expression of autophagy marker LC3B-Ⅰ/LC3B-Ⅱ, autophagy substrate p62 and silent information regulator 1(SIRT1)/adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway. NAFLD cell model was successfully induced by FFA at 0.2 mmol·L~(-1) PA and 0.4 mmol·L~(-1) OA. HZRG reduced the TG level(P<0.05, P<0.01) and the lipid accumulation of FFA-induced L02 cells, while elevated the number of autophagosomes and autophagolysosomes to generate autophagic flux. It also affected the functions of lysosomes by regulating their pH. Additionally, HZRG up-regulated the expression of LC3B-Ⅱ/LC3B-Ⅰ, SIRT1, p-AMPK and phospho-protein kinase A(p-PKA)(P<0.05, P<0.01), while down-regulated the expression of p62(P<0.01). Furthermore, 3-methyladenine(3-MA) or chloroquine(CQ) treatment obviously inhibited the above effects of HZRG. HZRG prevented FFA-induced steatosis in L02 cells, and its mechanism might be related to promoting autophagy and regulating SIRT1/AMPK signaling pathway.
Humans ; Non-alcoholic Fatty Liver Disease/metabolism* ; Sirtuin 1/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Fatty Acids, Nonesterified/metabolism* ; Autophagy ; Liver

Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). In this study, we tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. We found a mild but significant transcriptional increase of Drosophila Sir2 (dSir2) by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. We further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, we showed that Aβ aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila β-secretase (dBACE). Our data suggest that RES rescues the APP-induced behavioral deficits and Aβ burden largely, but not exclusively, via dSir2.
Animals ; Alzheimer Disease/metabolism* ; Amyloid beta-Peptides ; Amyloid beta-Protein Precursor/metabolism* ; Drosophila/physiology* ; Drosophila Proteins/metabolism* ; Resveratrol/pharmacology* ; Sirtuin 1 ; Sleep

OBJECTIVE: To investigate the role and mechanism of silent information regulator 1 (SIRT1) in regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in oxidative stress and inflammatory response to sepsis-induced liver injury. METHODS: A total of 24 male Sprague-Dawley (SD) rats were randomly divided into sham operation (Sham) group, cecal ligation and puncture (CLP) group, SIRT1 agonist SRT1720 pretreatment (CLP+SRT1720) group and SIRT1 inhibitor EX527 pretreatment (CLP+EX527) group, with 6 rats in each group. Two hours before operation, SRT1720 (10 mg/kg) or EX527 (10 mg/kg) were intraperitoneally injected into the CLP+SRT1720 group and CLP+EX527 group, respectively. Blood was collected from the abdominal aorta at 24 hours after modeling and the rats were sacrificed for liver tissue. The serum levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by microplate method. Hematoxylin-eosin (HE) staining was used to observe the pathological injury of rats in each group. The levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH) and superoxide dismutase (SOD) in liver tissue were detected by corresponding kits. The mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RESULTS: Compared with the Sham group, the serum levels of IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were significantly increased; histopathological results showed that liver cords were disordered, hepatocytes were swollen and necrotic, and a large number of inflammatory cells infiltrated; the contents of MDA and 8-OHdG in liver tissue increased, while the contents of GSH and SOD decreased; and the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were significantly decreased. These results suggest that sepsis rats have liver dysfunction, and the levels of SIRT1, Nrf2, HO-1 and antioxidant protein in liver tissues were decreased, while the levels of oxidative stress and inflammation were increased. Compared with the CLP group, the levels of inflammatory factors and oxidative stress were significantly decreased in the CLP+SRT1720 group, the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were significantly increased [IL-6 (ng/L): 34.59±4.21 vs. 61.84±3.78, IL-1β (ng/L): 41.37±2.70 vs. 72.06±3.14, TNF-α (ng/L): 76.43±5.23 vs. 130.85±5.30, ALT (U/L): 30.71±3.63 vs. 64.23±4.59, AST (U/L): 94.57±6.08 vs. 145.15±6.86, MDA (μmol/g): 6.11±0.28 vs. 9.23±0.29, 8-OHdG (ng/L): 117.43±10.38 vs. 242.37±11.71, GSH (μmol/g): 11.93±0.88 vs. 7.66±0.47, SOD (kU/g): 121.58±5.05 vs. 83.57±4.84, SIRT1 mRNA (2^-ΔΔCt): 1.20±0.13 vs. 0.46±0.02, Nrf2 mRNA (2^-ΔΔCt): 1.21±0.12 vs. 0.58±0.03, HO-1 mRNA (2^-ΔΔCt): 1.71±0.06 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.89±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.87±0.08 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.93±0.14 vs. 0.54±0.12, all P < 0.05], these results indicated that SIRT1 agonist SRT1720 pretreatment could improve liver injury in sepsis rats. However, pretreatment with SIRT1 inhibitor EX527 showed the opposite effect [IL-6 (ng/L): 81.05±6.47 vs. 61.84±3.78, IL-1β (ng/L): 93.89±5.83 vs. 72.06±3.14, TNF-α (ng/L): 177.67±5.12 vs. 130.85±5.30, ALT (U/L): 89.33±9.52 vs. 64.23±4.59, AST (U/L): 179.59±6.44 vs. 145.15±6.86, MDA (μmol/g): 11.39±0.51 vs. 9.23±0.29, 8-OHdG (ng/L): 328.83±11.26 vs. 242.37±11.71, GSH (μmol/g): 5.07±0.34 vs. 7.66±0.47, SOD (kU/g): 59.37±4.28 vs. 83.57±4.84, SIRT1 mRNA (2^-ΔΔCt): 0.34±0.03 vs. 0.46±0.02, Nrf2 mRNA (2^-ΔΔCt): 0.46±0.04 vs. 0.58±0.03, HO-1 mRNA (2^-ΔΔCt): 0.21±0.03 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.47±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.32±0.07 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.19±0.09 vs. 0.54±0.12, all P < 0.05]. CONCLUSIONS SIRT1 can inhibit the release of proinflammatory factors and alleviate the oxidative damage of hepatocytes by activating Nrf2/HO-1 signaling pathway, thus playing a protective role against CLP-induced liver injury.
Animals ; Male ; Rats ; Actins/metabolism* ; Chemical and Drug Induced Liver Injury, Chronic ; Heme Oxygenase-1/metabolism* ; Interleukin-6 ; NF-E2-Related Factor 2/metabolism* ; Rats, Sprague-Dawley ; RNA, Messenger ; Sepsis/metabolism* ; Signal Transduction ; Sirtuin 1/metabolism* ; Superoxide Dismutase/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: In this study, the combined effect of two stressors, namely, electromagnetic fields (EMFs) from mobile phones and fructose consumption, on hypothalamic and hepatic master metabolic regulators of the AMPK/SIRT1-UCP2/FOXO1 pathway were elucidated to delineate the underlying molecular mechanisms of insulin resistance. METHODS: Weaned Wistar rats (28 days old) were divided into 4 groups: Normal, Exposure Only (ExpO), Fructose Only (FruO), and Exposure and Fructose (EF). Each group was provided standard laboratory chow ad libitum for 8 weeks . Additionally, the control groups, namely, the Normal and FruO groups, had unrestricted access to drinking water and fructose solution (15%), respectively. Furthermore, the respective treatment groups, namely, the ExpO and EF groups, received EMF exposure (1,760 MHz, 2 h/day x 8 weeks). In early adulthood, mitochondrial function, insulin receptor signaling, and oxidative stress signals in hypothalamic and hepatic tissues were assessed using western blotting and biochemical analysis. RESULT: In the hypothalamic tissue of EF, SIRT1, FOXO 1, p-PI3K, p-AKT, Complex III, UCP2, MnSOD, and catalase expressions and OXPHOS and GSH activities were significantly decreased ( P < 0.05) compared to the Normal, ExpO, and FruO groups. In hepatic tissue of EF, the p-AMPKα, SIRT1, FOXO1, IRS1, p-PI3K, Complex I, II, III, IV, V, UCP2, and MnSOD expressions and the activity of OXPHOS, SOD, catalase, and GSH were significantly reduced compared to the Normal group ( P < 0.05). CONCLUSION The findings suggest that the combination of EMF exposure and fructose consumption during childhood and adolescence in Wistar rats disrupts the closely interlinked and multi-regulated crosstalk of insulin receptor signals, mitochondrial OXPHOS, and the antioxidant defense system in the hypothalamus and liver.
Humans ; Rats ; Animals ; Adult ; Rats, Wistar ; Fructose/metabolism* ; Catalase ; Receptor, Insulin/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Electromagnetic Fields/adverse effects* ; Sirtuin 1/metabolism* ; Cell Phone ; Phosphatidylinositol 3-Kinases/metabolism* ; Forkhead Box Protein O1/metabolism* ; Uncoupling Protein 2

This study aimed to explore the possible effect of Xixin Decoction(XXD) on the learning and memory ability of Alzheimer's disease(AD) model senescence-accelerated mouse-prone 8(SAMP8) and the related mechanism in enhancing neuroprotective effect and reducing neuroinflammation. Forty SAMP8 were randomly divided into a model group(10 mL·kg~(-1)·d~(-1)), a probiotics group(0.39 g·kg~(-1)·d~(-1)), a high-dose group of XXD granules(H-XXD, 5.07 g·kg~(-1)·d~(-1)), a medium-dose group of XXD granules(M-XXD, 2.535 g·kg~(-1)·d~(-1)), and a low-dose group of XXD granules(L-XXD, 1.267 5 g·kg~(-1)·d~(-1)). Eight senescence-accelerated mouse-resistant 1(SAMR1) of the same age and strain were assigned to the control group(10 mL·kg~(-1)·d~(-1)). After ten weeks of intragastric administration, the Morris water maze was used to test the changes in spatial learning and memory ability of mice after treatment. Meanwhile, immunofluorescence staining was used to detect the positive expression of receptor for advanced glycation end products(AGER), Toll-like receptor 1(TLR1), and Toll-like receptor 2(TLR2) in the hippocampal CA1 region of mice. Western blot was employed to test the protein expression levels of silencing information regulator 2 related enzyme 1(SIRT1), AGER, TLR1, and TLR2 in the hippocampus of mice. Enzyme linked immunosorbent assay(ELISA) was applied to assess the levels of Aβ_(1-42) in the hippocampus of mice and the levels of nuclear factor κB p65(NF-κB p65), NOD-like receptor protein 3(NLRP3), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in the serum and hippocampus of mice. Compared with the model group, XXD significantly improved the spatial learning and memory ability of SAMP8, increased the expression of neuroprotective factors in the hippocampus, decreased the levels of neuroinflammatory factors, and inhibited the expression of Aβ_(1-42). In particular, H-XXD significantly increased the expression of SIRT1 in the hippocampus of mice, reduced the expression levels of NF-κB p65, NLRP3, TNF-α, and IL-1β in the serum and hippocampus of mice, and decreased the expression of AGER, TLR1, and TLR2 in the hippocampus of mice(P<0.05 or P<0.01). XXD may improve the spatial learning and memory ability of AD model SAMP8 by enhancing the neuroprotective effect and inhibiting neuroinflammation.
Humans ; Neuroprotective Agents/therapeutic use* ; Sirtuin 1/metabolism* ; Toll-Like Receptor 2/metabolism* ; NF-kappa B/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Neuroinflammatory Diseases ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Toll-Like Receptor 1/metabolism* ; Alzheimer Disease/genetics* ; Hippocampus

OBJECTIVE: To interpret the pharmacology of quercetin in treatment of atherosclerosis (AS). METHODS: Fourteen apolipoprotein E-deficient (ApoE^-/-) mice were divided into 2 groups by a random number table: an AS model (ApoE^-/-) group and a quercetin treatment group (7 in each). Seven age-matched C57 mice were used as controls (n=7). Quercetin [20 mg/(kg·d)] was administered to the quercetin group intragastrically for 8 weeks for pharmacodynamic evaluation. Besides morphological observation, the distribution of CD11b, F4/80, sirtuin 1 (Sirt1) and P21 was assayed by immunohistochemistry and immunofluorescence to evaluate macrophage infiltration and tissue senescence. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MSC/MS) was performed to study the pharmacology of quercetin against AS. Then, simultaneous administration of an apelin receptor antagonist (ML221) with quercetin was conducted to verify the possible targets of quercetin. Key proteins in apelin signaling pathway, such as angiotensin domain type 1 receptor-associated proteins (APJ), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), tissue plasminogen activator (TPA), uncoupling protein 1 (UCP1) and angiotensin II receptor 1 (AT1R), were assayed by Western blot. RESULTS: Quercetin administration decreased lipid deposition in arterial lumen and improved the morphology of ApoE^-/- aortas in vivo. Quercetin decreased the densities of CD11b, F4/80 and P21 in the aorta and increased the level of serum apelin and the densities of APJ and Sirt1 in the aorta in ApoE^-/- mice (all P<0.05). Plasma metabolite profiling identified 118 differential metabolites and showed that quercetin affected mainly glycerophospholipids and fatty acyls. Bioinformatics analysis suggested that the apelin signaling pathway was one of the main pathways. Quercetin treatment increased the protein expressions of APJ, AMPK, PGC-1α, TPA and UCP1, while decreased the AT1R level (all P<0.05). After the apelin pathway was blocked by ML221, the effect of quercetin was abated significantly, confirming that quercetin attenuated AS by modulating the apelin signaling pathway (all P<0.05). CONCLUSION Quercetin alleviated AS lesions by up-regulation the apelin signaling pathway.
Mice ; Animals ; Apelin ; Tissue Plasminogen Activator/metabolism* ; Quercetin/therapeutic use* ; AMP-Activated Protein Kinases/metabolism* ; Sirtuin 1/metabolism* ; Signal Transduction/physiology* ; Atherosclerosis/metabolism* ; Apolipoproteins E

Mulberry (Morus alba L.) leaf is a well-established traditional Chinese botanical and culinary resource. It has found widespread application in the management of diabetes. The bioactive constituents of mulberry leaf, specifically mulberry leaf flavonoids (MLFs), exhibit pronounced potential in the amelioration of type 2 diabetes (T2D). This potential is attributed to their ability to safeguard pancreatic β cells, enhance insulin resistance, and inhibit α-glucosidase activity. Our antecedent research findings underscore the substantial therapeutic efficacy of MLFs in treating T2D. However, the precise mechanistic underpinnings of MLF's anti-T2D effects remain the subject of inquiry. Activation of brown/beige adipocytes is a novel and promising strategy for T2D treatment. In the present study, our primary objective was to elucidate the impact of MLFs on adipose tissue browning in db/db mice and 3T3-L1 cells and elucidate its underlying mechanism. The results manifested that MLFs reduced body weight and food intake, alleviated hepatic steatosis, improved insulin sensitivity, and increased lipolysis and thermogenesis in db/db mice. Moreover, MLFs activated brown adipose tissue (BAT) and induced the browning of inguinal white adipose tissue (IWAT) and 3T3-L1 adipocytes by increasing the expressions of brown adipocyte marker genes and proteins such as uncoupling protein 1 (UCP1) and beige adipocyte marker genes such as transmembrane protein 26 (Tmem26), thereby promoting mitochondrial biogenesis. Mechanistically, MLFs facilitated the activation of BAT and the induction of WAT browning to ameliorate T2D primarily through the activation of AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) signaling pathway. These findings highlight the unique capacity of MLF to counteract T2D by enhancing BAT activation and inducing browning of IWAT, thereby ameliorating glucose and lipid metabolism disorders. As such, MLFs emerge as a prospective and innovative browning agent for the treatment of T2D.
Mice ; Animals ; Adipose Tissue, Brown ; Sirtuin 1/pharmacology* ; Diabetes Mellitus, Type 2/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Morus/metabolism* ; Flavonoids/metabolism* ; Prospective Studies ; Signal Transduction ; Adipose Tissue, White ; Plant Leaves ; Uncoupling Protein 1/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism*

This study aims to explore the molecular mechanism of Lycium barbarum polysaccharides(LBP) in alleviating premature ovarian failure(POF) in mice via the 5'-adenosine monophosphate activated protein kinase(AMPK)/silent information regulator 1(Sirt1) signaling pathway. The POF mouse model was established by D-galactose(D-gal) injection at the back. Six groups were set up, including a normal control group, a model group, a LBP group, a 3-MA(autophagy inhibitor 3-methyladenine) group, an AMPK inhibitor group, and a LBPAMPK inhibitor group, with 15 mice in each group. After 28 continuous days of administration, enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of sex hormones [estradiol(E_2), luteinizing hormone(LH), and follicle-stimulating hormone(FSH)] in serum. The ovarian mass coefficient was measured. Senescence-associated β-Galactosidase(SA-β-Gal) staining and hematoxylin-eosin(HE) staining were performed for observing the state of ovarian senescence and the morphological changes of the ovary. Immunohistochemical method was used to measure the expression of the autophagy marker LC3-Ⅱ in ovarian tissue. Western blot was employed to measure the expression levels of the senescence marker p16~(INK4 a), autophagy markers(LC3-Ⅱ and Beclin-1), the autophagy substrate p62, lysosome-associated membrane protein 2(LAMP2), and the proteins in the AMPK/Sirt1 pathway and mammalian target of rapamycin complex 1(mTORC1)/UNC-51-like kinase 1 Ser757 site(Ulk1 Ser757) pathway. Compared with the normal control group, the modeling of POF decreased the ovarian granulosa cells and follicles, led to the ovarian aging and severe sex hormone secretion disorders, weakened ovarian autophagy activity, and down-regulated the expression of proteins in the AMPK/Sirt1 pathway(P<0.05). Compared with the model group, the treatment with LBP increased ovarian granulosa cells and follicles, alleviated aging and sex hormone disorders, increased autophagy activity, and activated the AMPK/Sirt1 pathway(P<0.05). Both 3-MA and AMPK inhibitor can inhibit autophagy and aggravate ovarian damage and aging in mice. AMPK inhibitor can partially attenuate the role of LBP in promoting autophagy activation and alleviating aging and ovarian tissue damage(P<0.05). LBP can alleviate the symptoms of POF induced by D-gal by promoting the activation of AMPK/Sirt1 pathway.
Animals ; Female ; Humans ; Mice ; AMP-Activated Protein Kinases/metabolism* ; Autophagy/drug effects* ; Follicle Stimulating Hormone/blood* ; Lycium/chemistry* ; Polysaccharides/therapeutic use* ; Primary Ovarian Insufficiency/drug therapy* ; Sirtuin 1/metabolism*

Silent information regulator 2-related enzyme 1 (SIRT1) is an aging-related protein activated with aging. Herein, we evaluated the role of SIRT1 in aging-related erectile dysfunction. The expression of SIRT1 was modulated in aged Sprague-Dawley rats following intragastric administration of resveratrol (Res; 5 mg kg^-1), niacinamide (NAM; 500 mg kg^-1) or Res (5 mg kg^-1) + tadalafil (Tad; phosphodiesterase-5 [PDE5] inhibitor; 5 mg kg^-1) for 8 weeks. Then, we determined erectile function by the ratio of intracavernosal pressure (ICP)/mean systemic arterial pressure (MAP). Cavernosal tissues were extracted to evaluate histological changes, cell apoptosis, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP), the superoxide dismutase (SOD)/3,4-methylenedioxyamphetamine (MDA) level, and the expression of SIRT1, p53, and forkhead box O3 (FOXO3a) using immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL), enzyme-linked immunosorbent assays, and western blot analysis. Compared with the control, Res treatment significantly improved erectile function, reflected by an increased content of smooth muscle and endothelium, NO/cGMP and SOD activity, and reduced cell apoptosis and MDA levels. The effect of Res was improved by adding Tad. In addition, the protein expression of SIRT1 was increased in the Res group, accompanied by decreased p53 and FOXO3a levels. In addition, inhibition of SIRT1 by NAM treatment resulted in adverse results compared with Res treatment. SIRT1 activation ameliorated aging-related erectile dysfunction, supporting the potential of SIRT1 as a target for erectile dysfunction treatment.
Animals ; Male ; Rats ; Cyclic GMP/metabolism* ; Erectile Dysfunction/metabolism* ; Nitric Oxide/metabolism* ; Penile Erection ; Penis/pathology* ; Phosphodiesterase 5 Inhibitors/pharmacology* ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Superoxide Dismutase/metabolism* ; Tumor Suppressor Protein p53/metabolism*

OBJECTIVES: Myocardial ischemia reperfusion injury (IRI) occurs occasionally in the process of ischemic heart disease. Sevoflurane preconditioning has an effect on attenuating IRI. Preserving the structural and functional integrity of mitochondria is the key to reduce myocardial IRI. Silent information regulator 3 (SIRT3), a class of nicotinamide adenine dinucleotide (NAD⁺) dependent deacetylases, is an important signal-regulating molecule in mitochondria. This study aims to explore the role of mitochondrial NAD⁺-SIRT3 pathway in attenuating myocardial IRI in rats by sevoflurane preconditioning. METHODS: A total of 60 male Sprague Dawley (SD) rats were randomly divided into 5 groups (n=12): A sham group (Sham group), an ischemia reperfusion group (IR group), a sevoflurane preconditioning group (Sev group, inhaled 2.5% sevoflurane for 30 min), a sevoflurane preconditioning+SIRT3 inhibitor 3-TYP group (Sev+3-TYP group, inhaled 2.5% sevoflurane for 30 min and received 5 mg/kg 3-TYP), and a 3-TYP group (5 mg/kg 3-TYP). Except for the Sham group, the IR model in the other 4 groups was established by ligating the left anterior descending coronary artery. The size of myocardial infarction was determined by double staining. Serum cardiac troponin I (cTnI) level was measured. The contents of NAD⁺ and ATP, the activities of mitochondrial complexes I, II, and IV, the content of MDA, the activity of SOD, and the changes of mitochondrial permeability were measured. The protein expression levels of SIRT3, SOD2, catalase (CAT), and voltage dependent anion channel 1 (VDAC1) were detected by Western blotting. The ultrastructure of myocardium was observed under transmission electron microscope. MAP and HR were recorded immediately before ischemia (T0), 30 min after ischemia (T1), 30 min after reperfusion (T2), 60 min after reperfusion (T3), and 120 min after reperfusion (T4). RESULTS: After ischemia reperfusion, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased (both P<0.01), and an obvious myocardial injury occurred, including the increase of myocardial infarction size and serum cTnI level (both P<0.01). Correspondingly, the mitochondria also showed obvious damage on energy metabolism, antioxidant function, and structural integrity, which was manifested as: the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level and mitochondrial permeability were increased (all P<0.01). Compared with the IR group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were increased in the Sev group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were decreased in the Sev group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were increased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were decreased in the Sev group (all P<0.01). Compared with the Sev group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased in the Sev+3-TYP group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were increased in the Sev+3-TYP group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were increased in the Sev+3-TYP group (all P<0.01). CONCLUSIONS Sevoflurane preconditioning attenuates myocardial IRI through activating the mitochondrial NAD⁺-SIRT3 pathway to preserve the mitochondrial function.
Adenosine Triphosphate/metabolism* ; Animals ; Male ; Mitochondria/metabolism* ; Myocardial Infarction/metabolism* ; Myocardial Reperfusion Injury/metabolism* ; NAD/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sevoflurane/metabolism* ; Sirtuin 3/metabolism* ; Voltage-Dependent Anion Channel 1/metabolism*