Down-regulation of XBP1s alleviates the senescence of renal tubular epithelial cells induced by hypoxia/reoxygenation through Sirt3/SOD2/mtROS signaling pathway / 器官移植

Objective To investigate the role and mechanism of spliced X-box binding protein 1 (XBP1s) in the senescence of primary renal tubular epithelial cells induced by hypoxia/reoxygenation (H/R). Methods Primary renal tubular epithelial cells were divided into the normal control group (NC group), H/R group, empty adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), empty adenovirus+H/R treatment group (Ad-shNC+H/R group) and targeted silencing XBP1s adenovirus+H/R treatment group (Ad-shXBP1s +H/R group), respectively. The expression levels of XBP1s in the NC, H/R, Ad-shNC and Ad-shXBP1s groups were measured. The number of cells stained with β-galactosidase, the expression levels of cell aging markers including p53, p21 and γH2AX, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined in the Ad-shNC, Ad-shNC+H/R and Ad-shXBP1s+H/R groups. Chromatin immunoprecipitation was employed to verify Sirtuin 3 (Sirt3) of XBP1s transcription regulation, and the expression levels of Sirt3 and downstream SOD2 after down-regulation of XBP1s were detected. Mitochondrial reactive oxygen species (mtROS) were detected by flow cytometry. Results Compared with the NC group, the expression level of XBP1s was up-regulated in the H/R group. Compared with the Ad-shNC group, the expression level of XBP1s was down-regulated in the Ad-shXBP1s group (both P<0.001). Compared with the Ad-shNC group, the number of cells stained with β-galactosidase was increased, the expression levels of p53, p21 and γH2AX were up-regulated, the levels of ROS, MDA and mtROS were increased, the SOD activity was decreased, the expression level of Sirt3 was down-regulated, and the ratio of Ac-SOD2/SOD2 was increased in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the number of cells stained with β-galactosidase was decreased, the expression levels of p53, p21 and γH2AX were down-regulated, the levels of ROS, MDA and mtROS were decreased, the SOD activity was increased, the expression level of Sirt3 was up-regulated and the ratio of Ac-SOD2/SOD2 was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Conclusions Down-regulation of XBP1s may ameliorate the senescence of primary renal tubular epithelial cells induced by H/R, which probably plays a role through the Sirt3/SOD2/mtROS signaling pathway.

Study on inhibitory effect and mechanism of hydrogen sulfide on the proliferation of cardiac fibroblasts / 中国药房

OBJECTIVE To investigate the inhibitory effect and the possible mechanism of hydrogen sulfide （H2S） on the proliferation of cardiac fibroblasts. METHODS The heart of neonatal SD rats was collected， and cardiac fibroblasts were separated with differential centrifugation. Using sodium hydrosulfide as the donor of H2S， the effects of H2S on the proliferation of cardiac fibroblasts induced by angiotensin Ⅱ （Ang Ⅱ）， hydroxyproline content and the expression of sirtuin 3 （SIRT3） protein were detected. After SIRT3 knockdown with siRNA technology， the effects of H2S on the proliferation of cardiac fibroblasts induced by Ang Ⅱ， hydroxyproline content， the expressions of collagen Ⅰ （Col Ⅰ）， collagen Ⅲ （Col Ⅲ ） and optic atrophy protein 1 （OPA1） were detected. RESULTS H2S could inhibit the proliferation of Ang Ⅱ-induced cardiac fibroblasts， reduce the content of hydroxyproline and increase the expression of SIRT3 （P＜0.05）. After down-regulating the expression of SIRT3 with siRNA technology， the inhibition of H2S on the proliferation of Ang Ⅱ-induced cardiac fibroblasts and the reduction of hydroxyproline content were both inhibited， and the effect of H2S on reducing the expression of Col Ⅰ and Col Ⅲ and enhancing the expression of OPA1 was also significantly weakened. CONCLUSIONS H2S inhibits the proliferation of Ang Ⅱ -induced cardiac fibroblasts through increasing the expression of SIRT3.

The Protective Effect of Kaempferol Against Ischemia/Reperfusion Injury Through Activating SIRT3 to Inhibit Oxidative Stress

Abstract Introduction: The objective of this study is to investigate the protective effect of kaempferol against ischemia/reperfusion (IR) injury and the underlying molecular mechanisms. Methods: H9C2 cells were pretreated with kaempferol for 24 hours and further insulted with IR injury. Cell vitality, reactive oxygen species (ROS) level, glutathione (GSH) level, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and sirtuin-3 (SIRT3), B-cell lymphoma 2 (Bcl2), and Bcl2-associated X protein (Bax) expressions were evaluated. Moreover, short interfering ribonucleic acid targeting SIRT3 was used to investigate the role of SIRT3 against IR mediated by kaempferol in vitro. IR mice models were also established to confirm the protective effects of kaempferol on IR in vivo. Results: After IR injury, H9C2 cells vitality was reduced, ROS levels, NADPH oxidase activity, and Bax expressions were increased, and GSH levels and Bcl2 expressions were decreased. After kaempferol pretreatment, the vitality of H9C2 cells was increased. The levels of ROS, NADPH oxidase activity, and Bax expression were decreased. In addition, levels of GSH and Bcl2 expression were enhanced. Furthermore, silencing SIRT3 attenuated the protective effect mediated by kaempferol, with increased ROS levels, NADPH oxidase activity, and Bax expression, along with reduced GSH level and Bcl2 expression. In vivo IR model showed that kaempferol could preserve IR-damaged cardiac function. Conclusion: Kaempferol has the capability of attenuating H9C2 cells IR injury through activating SIRT3 to inhibit oxidative stress.

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.

The role of down-regulation of Sirtuin 3 in skeletal muscle injury induced by iron excess / 中华内分泌代谢杂志

Objective:To explore the role of SIRT3 down-regulation in skeletal muscle injury through the changes of sirtuin 3 (SIRT3) expression in mouse skeletal muscle under iron excess in vitro or in vivo.Methods:Murine preosteoblast myoblast C2C12 cells were incubated in a medium supplemented with ferric ammonium citrate (FAC). The proliferation, apoptotic were assessed, the cell morphology was observed, and the expression of SIRT3 mRNA, protein and activity were detected. ICR mice were randomly divided into control group, FAC group and FAC+ deferrioxamine (deferoxamine, DFO) group. Normal saline was injected in the control group. FAC followed by injection of normal saline in the FAC group; and FAC followed by DFO in the FAC+ DFO group. The non-heme iron level, content of SIRT3 protein and in situ apoptosis were detected, morphology of skeletal muscle was observed.Results:Proliferation of C2C12 cells was inhibited, and the apoptotic rate were increased by FAC ( P<0.05). The mRNA, protein and activity of SIRT3 decreased by FAC ( P<0.05). The cells gradually shrank, and the length and number of myotubes were decreased by FAC. Both control and FAC+ DFO groups showed lower levels of non-heme iron in skeletal muscle compared with FAC group ( P<0.05). The levels of SIRT3 protein were decreased in FAC group compared with control group, while increased in FAC+ DFO group with FAC group ( P<0.05). The apoptotic indexes in control and FAC+ DFO groups were lower than that in FAC group. Compared with the control group, the disordered cell arrangement, fat deposition and inflammatory cell infiltration were presented in FCA group, and the change was alleviated in FAC+ DFO group. Conclusion:Iron excess can lead to the decrease of skeletal muscle mass in mice, and the mechanism may be related to the down-regulation of SIRT3 level.

Relationship between SIRT3 and mitochondrial function in mice with endotoxin-induced lung injury / 中华麻醉学杂志

Objective:To evaluate the relationship between silent information regulator 2 homologue 3 (SIRT3) and mitochondrial function in mice with endotoxin-induced lung injury.Methods:Twenty clean-grade healthy adult male wild C57BL/6 (SIRT3 + /+ ) mice, 20 SIRT3 knockout (SIRT3 -/-) mice, weighing 20-25 g, aged 6-8 weeks, were studied.SIRT3 + /+ mice and SIRT3 -/- mice were divided into 4 groups ( n=5 each) according to the random number table method: blank control group (group C, group SIRT3 -/-C), endotoxin-induced lung injury group (group L, group SIRT3 -/-L), endotoxin-induced lung injury plus resveratrol group (group L+ R, group SIRT3 -/-L+ R), and resveratrol group (group R, group SIRT3 -/-R). Resveratrol 15 mg/kg was intraperitoneally injected once a day for 7 consecutive days in L+ R, R, SIRT3 -/-L+ R and SIRT3 -/-R groups, while the equal volume of normal saline was injected in the rest groups.Lipopolysaccharid 15 mg/kg was injected via the tail vein to develop a mouse model of endotoxin-induced lung injury at 30 min after resveratrol injection on 7th day, in L+ R and SIRT3 -/-L+ R groups and at the corresponding time points in L and SIRT3 -/-L groups, while the equal volume of normal saline was injected in the other groups.Blood samples were collected from the orbital venous plexus at 12 h after injection of normal saline or lipopolysaccharid for determination of serum total oxidation state (TOS) and total antioxidant state (TAS) levels by the xylenol orange method and ABTS colorimetric method, and the oxidative stress index (OSI) was calculated.After the mice were sacrificed, the lung tissues were taken for microscopic examination of the pathological changes which were scored and for determination of the mitochondrial membrane potential (MMP) (by JC-1 method), cellular oxygen consumption rate (OCR) (by the specific fluorescent probe method), and expression of SIRT3 (by Western blot). Results:Compared with group C or group SIRT3 -/-C, the lung injury score, serum TOS concentration and OSI were significantly increased, TAS concentration, MMP and OCR were decreased, and SIRT3 expression was down-regulated in L, L+ R, SIRT3 -/-L and SIRT3 -/-L+ R groups ( P<0.05). Compared with group L, the lung injury score, serum TOS concentration and OSI were significantly decreased, TAS concentration, MMP and OCR were increased, and SIRT3 expression was up-regulated in group L+ R, and lung injury score, serum TOS concentration and OSI were significantly increased, TAS concentration, MMP and OCR were decreased, and SIRT3 expression was down-regulated in group SIRT3 -/-L ( P<0.05). Compared with group L+ R, the lung injury score, serum TOS concentration and OSI were significantly increased, the TAS concentration, MMP and OCR were decreased, and the expression of SIRT3 was down-regulated in group SIRT3 -/- L+ R ( P<0.05). There was no significant difference in the indicators mentioned above between group SIRT3 -/-L+ R and group SIRT3 -/-L ( P>0.05). Conclusions:Down-regulation of SIRT3 expression can lead to impaired mitochondrial function, which is involved in the pathophysiological mechanism of endotoxin-induced lung injury.

Role of Sirt3 expression in peritoneal macrophages in dexmedetomidine-induced inhibition of inflammatory responses in septic mice / 中华麻醉学杂志

Objective:To evaluate the role of nicotinamide adenine dinucleotide-dependent deacetylase Sirtuin3 (Sirt3) expression in peritoneal macrophages in dexmedetomidine-induced inhibition of inflammatory responses in septic mice.Methods:Sixty-four healthy male C57BL/6 mice, aged 7 weeks, weighing about 20 g, were divided into 4 groups ( n=16 each) using a random number table method: sham operation group (S group), sepsis group (Sep group), dexmedetomidine group (DEX group), and dexmedetomidine plus Sirt3 inhibitor 3-TYP group (TYP group). Sepsis was induced by cecal ligation and puncture.In group DEX, normal saline 0.25 ml was intraperitoneally injected at 1 h before development of the model, and 30 min later dexmedetomidine 50 μg/kg (in 0.25 ml of normal saline) was intraperitoneally injected.In group S and group Sep, the equal volume of normal saline was intraperitoneally injected at 1 h and 30 min before development of the model, respectively.In group S, laparotomy and cecal extraction were performed without ligation and perforation.Peritoneal lavage was obtained at 24 h after operation, and peritoneal macrophages were cultured for 1 h. The expression of Sirt3, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) mRNA in peritoneal macrophages was detected using quantitative real-time polymerase chain reaction.Western blot was used to detect the expression of Sirt3 in peritoneal macrophages.The concentrations of IL-1β and TNF-α in peritoneal fluid were measured by enzyme-linked immunosorbent assay.The 10-day survival of mice in each group was observed, and the survival rates were calculated. Results:Compared with group S, the expression of Sirt 3 protein and mRNA in peritoneal macrophages was significantly down-regulated, the expression of IL-1β and TNF-α mRNA was up-regulated, the concentrations of IL-1β and TNF-α in peritoneal lavage were increased, and the 10-day survival rates were decreased in Sep, DEX and TYP groups ( P<0.05). Compared with group Sep, the expression of Sirt3 protein and mRNA in peritoneal macrophages was significantly up-regulated, the expression of IL-1β and TNF-α mRNA was down-regulated, the concentrations of IL-1β and TNF-α in the peritoneal lavage were decreased, and the 10-day survival rate was increased in group DEX ( P<0.05). Compared with group DEX, the expression of Sirt3 protein and mRNA and IL-1β and TNF-α mRNA in peritoneal macrophages was significantly up-regulated, the concentrations of IL-1β and TNF-α in peritoneal lavage were increased, and the 10-day survival rate was decreased in group TYP ( P<0.05). Conclusions:Up-regulation of Sirt3 expression in peritoneal macrophages is involved in the process of dexmedetomidine-induced inhibition of inflammatory responses in septic mice.

Protective Effects of Gallic Acid on the MPTP-Mediated Dopaminergic Neurons Damage in Parkinson's Disease / 中国药学杂志

OBJECTIVE: To study the neuroprotective effects of gallic acid in animal models of Parkinson's disease (PD) and its related molecular mechanisms. METHODS: Thirty-six mice were randomly divided into control group, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP) group and gallic acid group. Mice in MPTP group and gallic acid group were given an intraperitoneal injection of 30 mg•kg-1 MPTP daily for 7 d. The mice in control group were given the same amount of normal saline at the same time. The mice in gallic acid group received oral administration of 200 mg•kg-1 gallic acid daily from the first day, gallic acid was administered continuously for 14 d, and mice in MPTP group and control group received the same amount of normal saline. After 7 d of drug administration, the behavioral function was evaluated by rod climbing test and suspension test. The expression of tyrosine hydroxylase(TH) in the substantia nigra and striatum were detected by immunohistochemistry. The number of apoptotic neurons in the substantia nigra were measured by TUNEL assay. SIRT3 mRNA levels were analyzed by qRT-PCR, and protein expression levels were detected by Western blot. RESULTS: Compared with the control group, the ability of motor coordination weakened, the TH expression levels decreased in the substantia nigra and striatum, the number of apoptotic neurons in the substantia nigra significantly increased, the SIRT3 mRNA and protein levels in the substantia nigra obviously declined, and the SOD2 protein expression also dramatically reduced in the MPTP group, the differences between the groups were all statistically significant (P<0.05). After treatment with gallic acid, the ability of motor coordination enhanced, the TH expression level elevated, the number of apoptotic neurons markedly reduced, SIRT3 mRNA and protein levels increased, and SOD2 protein expression also up-regulated in the gallic acid group. Compared with the MPTP group, the differences were all statistically significant (P<0.05). CONCLUSION: Gallic acid can improve the behavioral dysfunction and inhibit dopaminergic neurons damage in PD mice. The mechanism of action may be related to the up-regulation of SIRT3 gene expression.

Role of sirt3 in molecular mechanism of melatonin protecting dopaminergic neurons in Parkinson' s disease / 中国药理学通报

Aim To investigate the role of SIRT3 in the molecular mechanism of melatonin protecting do-paminergic neurons in Parkinson' s disease ( PD). Methods Forty-eight mice were randomly divided into control group, model group and treatment group. The mice in treatment group received intraperitoneal injection of melatonin (10 mg • kg"1) and MPTP (30 mg • kg ~1). The mice in model group only received intraperitoneal injection of MPTP (30 mg • kg~1 ) , and the mice in control group received the same a-mount of normal saline. Melatonin was administered continuously for 14 days. The expressions of TH and lba-1 in substantia nigra were analyzed by immunohis-tochemistry. The levels of oxidative stress ( ROS, MDA, SOD) and inflammatory factors (TNF-a, IL-lp) in the midbrain were measured by ELISA. SIRT3 mRNA level was analyzed by qRT-PCR, and protein expression level was detected by immunocytochemistry assay and Western blot. Results Compared to control group, the TH expression decreased and Iba-1 expression increased in the substantia nigra, the oxidative stress and inflammatory injury in the midbrain were significantly enhanced, the SIRT3 mRNA and protein levels in the substantia nigra obviously declined, the SOD2 protein expression was also dramatically reduced, and the iNOS protein expression was elevated in model group; the differences between the groups were all statistically significant ( P < 0. 05 ). After treatment with melatonin, the TH expression increased, Iba-1 expression decreased, oxidative stress and inflammatory injury markedly decreased, SIRT3 mRNA and protein levels were elevated, SOD2 protein expression was up-regulated, and iNOS protein expression was down-regulated in treatment group. Compared to model group, the differences were all statistically significant ( P < 0.05). Conclusions Melatonin can counteract the damage of dopaminergic neurons by up-regulating the expression of SIRT3 in PD animal model. Its mechanisms of action are related to inhibiting microglia activation, and alleviating oxidative stress and inflammation injury.

Effect of silencing sirtuin 3 on resveratrol-induced apoptois of human ovarian cancer SKOV3 cels / 吉林大学学报(医学版)

Objective: To investigate the effect of silencing sirtuin 3 (Sirt3) on the apoptosis of human ovarian cancer SKOV3 cells induced by resveratrol (Res), and to explore its mechanism of promoting apoptosis. Methods: The human ovarian cancer SKOC3 cells were cultured with different concentrations 0, 2. 5, 5. 0, 10.0, 20.0, 40.0 and 80.0 mg · L-1) of Res for 24 h. The survival rate of cells was measured by MTT assay. The SKOV3 cells were randomly divided into control group, Sirt3 inhibitory 3-1H-1, 2, 3-triazol-4-yl) pyridine 3-TYP group, Res group and 3-TYP+Res group. After 24 h of culture, the inhibitory rates of proliferation of the cells in various groups were detected by MTT assay; the nuclei were stained with Hoechst 33342, and the morphorgy nucleus was observed by laser confocal microscope; reactive oxygen species (ROS) probe was used to detect the intracellular ROS levels; Western blotting method was used to detect the expression levels of Sirt3, Bax, Bcl-2 and cleaved caspase-3 proteins in the cells in various groups. Results: The results of MTT assay showed that the survival rates of SKOV3 cells were significantly decreased with the increase of concentration of Res, and the median inhibitory concentration (IC50) was 42. 73 mg · L-1. Compared with control group, the inhibitory rates of proliferation of cells in Res group and 3-TYP+Res group were significantly decreased (P0.05); the protein expression levels of Sirt3 and Bcl-2 proteins in Res group were significantly decreased (P< 0.05), and the expression levels of Bax and cleaved caspase-3 proteins were significantly increased (P<0.05). Compared with Res group, the expression levels of Bax and cleaved caspase-3 proteins in 3-TYP + Res group were significantly increased (P<0.05), and the expression levels of Bcl-2 and Sirt3 proteins in 3-TYP+Res group were significantly decreased (P<0.05). Conclusion: Res can induce the apoptosis of SKOV3 cells, and the inhibition of Sirt3 expression by 3-TYP can enhance the effect of Res.

Effect of Biling Qutong Prescription on SIRT3 Protein Expression and URAT1 mRNA in Skeletal Muscle of Diabetic Gout Rats / 中国实验方剂学杂志

Objective: To observe the effect of phlegm and blood stasis on the expressions of sirtuin 3(SIRT3)protein and urate transporter 1(URAT1) mRNA in skeletal muscle of diabetic rats with gout. Method: The 40 healthy rats, excepting the normal group, the remaining groups were fed with high-fat diet combined with low-dose streptozotocin solution (40 mg·kg-1) once a day, with blood glucose "16.7 mmol·L-1" as the criterion for the diabetes model. After 4 days, the 5% sodium urate solution was injected into the joint cavity once to induce the gout model. After the successful modeling, the Biling group (10 g·kg-1), the indomethacin group (5 mg·kg-1) and the pioglitazone group (10 mg·kg-1) continued to be administered for 21 days. The normal group and the model group were given the same amount of normal saline. The expression of SIRT3 protein in skeletal muscle tissue was determined by Western blot, URAT1 mRNA expression in bone tissue was detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR),and blood was collected to measure blood glucose (GLU), blood uric acid (UA) and C-reactive protein (CRP). Result: Compared with the normal group, GLU, UA and CRP in the model group were significantly increased (PPPPPPPPConclusion: Biling Qutong prescription with effects in purging turbidity, detoxifying and dredging collaterals can significantly reduce the content of serum inflammatory factor CRP, significantly increase the protein expression of SIRT3 in skeletal muscle tissue of model rats, lower the content of URAT1 mRNA, reduce the blood glucose and blood uric acid levels in diabetic gout rats, and protect joints.

Role of SIRT3 in dysfunction of energy metabolism induced by deoxycho-lic acid in human colon NCM460 cells / 中国病理生理杂志

AIM: To investigate the effect of deoxycholic acid (DCA) on the energy metabolism in human normal colon epithelial NCM460 cells.METHODS: NCM460 cells was treated with DCA at 10, 30 and 100 μmol/L for 5 d, or DCA at 100 μmol/L for 3, 5 and 7 d.After treated with DCA at 100 μmol/L for 3 d, the cells were treated with resveratrol, the activator of sirtuin 3 (SIRT3), for the next 4 d.Adenosine triphosphate (ATP) production in the mitochondria and lactate acid level were detected.The protein expression of SIRT3 was determined by Western blot.RESULTS: DCA inhibited the ATP production, increased lactate acid level, and downregulated the protein expression of SIRT3 in a dose-and time-dependent manner.Resveratrol at 10 μmol/L reversed the effects of DCA on the NCM460 cells.CONCLUSION: DCA induces the dysfunction of energy metabolism in NCM460 cells, and the mechanism may be related with SIRT3.

Poly(ADP-ribose) polymerase 1 contributes to oxidative stress through downregulation of sirtuin 3 during cisplatin nephrotoxicity / 대한해부학회지

Enhanced oxidative stress is a hallmark of cisplatin nephrotoxicity, and inhibition of poly(ADP-ribose) polymerase 1 (PARP1) attenuates oxidative stress during cisplatin nephrotoxicity; however, the precise mechanisms behind its action remain elusive. Here, using an in vitro model of cisplatin-induced injury to human kidney proximal tubular cells, we demonstrated that the protective effect of PARP1 inhibition on oxidative stress is associated with sirtuin 3 (SIRT3) activation. Exposure to 400 µM cisplatin for 8 hours in cells decreased activity and expression of manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase (GPX), and SIRT3, while it increased their lysine acetylation. However, treatment with 1 µM PJ34 hydrochloride, a potent PARP1 inhibitor, restored activity and/or expression in those antioxidant enzymes, decreased lysine acetylation of those enzymes, and improved SIRT3 expression and activity in the cisplatin-injured cells. Using transfection with SIRT3 double nickase plasmids, SIRT3-deficient cells given cisplatin did not show the ameliorable effect of PARP1 inhibition on lysine acetylation and activity of antioxidant enzymes, including MnSOD, catalase and GPX. Furthermore, SIRT3 deficiency in cisplatin-injured cells prevented PARP1 inhibition-induced increase in forkhead box O3a transcriptional activity, and upregulation of MnSOD and catalase. Finally, loss of SIRT3 in cisplatin-exposed cells removed the protective effect of PARP1 inhibition against oxidative stress, represented by the concentration of lipid hydroperoxide and 8-hydroxy-2'-deoxyguanosine; and necrotic cell death represented by a percentage of propidium iodide–positively stained cells. Taken together, these results indicate that PARP1 inhibition protects kidney proximal tubular cells against oxidative stress through SIRT3 activation during cisplatin nephrotoxicity.

The influence of E-cadherin/β-catenin on the glycolysis effect in PANC-1 cells / 中国癌症杂志

Background and purpose:Lower expression of E-cadherin is associated with metastasis of cancer cells, however, the correlation between E-cadherin and glucose metabolism has seldom been reported. This article studied the correlation between E-cadherin and glycolysis effect in PANC-1 cells.Methods:Through treatment of transforming growth factor β (TGF-β) in PANC-1 cells to decrease E-cadherin expression, knock-down the gene of E-cadherin interaction protein β-catenin, and overexpressing of E-cadherin, the effects of E-cadherin on the glucose uptake and lactate production ability and on the expression of key glycolytic genes were assessed.Results:E-cadherin negatively regulated the glycolytic effect of PANC-1 cells by inhibiting glucose uptake and lactate production (P<0.05). Moreover, E-cadherin interacting partner β-catenin signiifcantly promoted glucose metabolism transformation in PANC-1 cells (P<0.05). Moreover, key glycolysis regulator sirtuin 3 (SIRT3) could lower E-cadherin expression.Conclusion:Lower expression of E-cadherin induced the transformation of glucose metabolism transformation in PANC-1 cells and manipulation of E-cadherin expression level could change the glycolysis effect. Moreover, through maneuver glycolysis process could inhibit high metastatic potential of pancreatic cancer cells.

Expression of SIRT1 and SIRT3 varies according to age in mice / 대한해부학회지

Sirtuins (SIRTs) are involved in multiple cellular processes including those related to aging, cancer, and a variety of cellular functions including cell cycle progression, DNA repair, and cellular proliferation. SIRTs have been shown to extend the yeast life span, although there is presently little known about SIRT expression in the organs of mice. In the present study, we were especially interested in identifying differences in SIRT expression between young mice and aged mice. Specifically, we investigated the expression of SIRT1 and SIRT3 in the kidney, lung, skin, adipose tissue, and spleens of 6-month-old and 24-month-old mice using immunohistochemical staining. Compared with that in younger mice, the expression of SIRT1 in 24-month-old rats was increased in kidney, lung, and spleen tissue, while that of SIRT3 was decreased in adipose, kidney, and lung tissue. The results of our study suggest that aging is associated with altered patterns of expression of SIRT1 and SIRT3. In addition, we noted that the expression patterns of SIRT1 and SIRT3 varied by organ. Taken together, the results of this study suggest the possibility that SIRTs may be involved in diseases associated with aging.

LSD1 negatively regulates the expression of tumor suppressor gene SIRT3 in pancreatic cancer cell line PANC-1 / 中国癌症杂志

Background and purpose: Lysine specific demethylase 1(LSD1) is an important chromatin modifier. It epigenetically regulates gene expression pattern through chromatin modification and participates in maintenance of tumor malignant properties, such as oncogenesis, development, invasion, migration and metabolic transformation. SIRT3 (sirtuin 3) is a mitochondria localized tumor suppressor and regulates tumor metabolic transformation and oxidative stress. The correlation between LSD1 and SIRT3 has never been reported before. This study aimed to elucidate the correlation between LSD1 and SIRT3 with gene transcriptional regulation methods. Methods: RNA interference technique, co-immunoprecipitation assay(CoIP), chromatin immune-precipitation assay(ChIP) and ifrelfy luciferase activity assay were employed to elucidate the correlation between LSD1 and SIRT3 in pancreatic cancer. Results:mRNA and protein levels of SIRT3 were signiifcantly elevated in LSD1 knock-down PANC-1 cells. LSD1 interacts with PGC-1α, an important regulator of SIRT3 gene expression. LSD1 and PGC-1αoccupied the same region in SIRT3 promoter region through ChIP analysis. Luciferase activity assay validated LSD1 as a negative regulator of PGC-1αin SIRT3 gene transcriptional regulation. Conclusion:LSD1, as an important tumor promoter, negatively regulates the expression of tumor suppressor gene SIRT3, these results provide important clues for the role that LSD1 plays in aberrant metabolism and oxidative stress.

Alpha-lipoic acid inhibits mitochondrial oxidative stress in the rat skeletal muscle with chronic hypoxia exposure / 中国组织工程研究

BACKGROUND: α-lipoic acid is named as “nature antioxidant” and “mitochondrial nutrition”. But it is unclear whether α-lipoic acid can be used to protect skeletal muscle with chronic hypoxia exposure, as wel as the relative mechanism. OBJECTIVE: To observe the effect of α-lipoic acid on the antioxidant enzymes and oxidative stress in rat skeletal muscle with chronic hypoxia exposure, and to investigate the relative signaling pathway of α-lipoic acid. METHODS: Thirty-six Sprague Dawley rats were randomly divided into three groups: normoxia control group, hypoxia control group, and hypoxia+α-lipoic acid group. Rats in the hypoxia control group were subjected to hypoxia exposure in normobaric hypoxic tent with 11.3% oxygen concentration. Rats in the hypoxia+α-lipoic acid group were induced by adding α-lipoic acid (0.25%) in the normal diet. Al the interventions were lasted for 4 weeks. RESULTS AND CONCLUSION: α-lipoic acid in hypoxia could markedly enhance the mitochondrial Sirtuin-3 expression, improve the mitochondrial adenosine triphosphate synthesis activity and membrane potential, up-regulate the mitochondrial state 3 respiratory rate, respiratory control ratio and ratio of phosphorus to oxygen, down-regulate the mitochondrial state 4 respiratory rate and promote and up-regulate the activity of mitochondrial antioxidant enzymes such as manganese superoxide dismutase, glutathione peroxidase and catalase, thus inhibiting mitochondrial H2O2 generation rate and reducing mitochondrial malondialdehyde level. The results indicated that α-lipoic acid could improve the efficiency of energy metabolism of chronic hypoxia skeletal muscle mitochondria and inhibit reactive oxygen generation, and it could inhibit the oxidative stress through improving antioxidant enzyme activity of mitochondria. The protection mechanism of α-lipoic acid on hypoxia skeletal muscle mitochondria may be related to the increasing of mitochondrial state 3 respiratory rate.

Mechanisms of neuroprotection of silent information regulation 2 homolog 3 against hypoxic injury in hypoxic preconditioning / 中华神经科杂志

Objective To investigate the mechanisms underlying neuroprotection of silent information regulation 2 homolog 3 ( SIRT3 ) against hypoxia via preconditioning.Methods PG12 cells were randomly divided into control,hypoxic preconditioning ( Hyp),Hyp with oxygen-glucose deprivation (OGD) and OGD.MTT assay and DAPI staining were used to evaluate cellular viability.MitoSOX Red was used to measure the production of mitochondrial superoxide.The protein expression of SIRT3,PGC-1α and MnSOD were assessed by Western blot.Recombinant SIRT3 was also given to further investigate its roles in hypoxic preconditioning.Results The preconditioned PC12 cells had a higher survival rate.When expressed as a percentage of the control group,MTT values following 6 h OGD were around 51.0% in the OGD group but around 74.7% in the Hyp + OGD group ( F = 56,P ＜ 0.01).Mitochondrial ROS after Hyp was less than the OGD group.Both Hyp + OGD and OGD increased the expression of SIRT3,PGC-1α and MnSOD proteins,and these increases were greater after Hyp + OGD.Similarly,the application of recombinant SIRT3 to OGD also further increased the expression of these proteins.Conclusions Hypoxic preconditioning can protect PC12 cells against hypoxic injury.One possible mechanism of hypoxic preconditioning is via SIRT3 to upregulate PGC-la and,in turn,MnSOD to reduce generation of ROS.