SIRT1 Knockdown Research Articles

1,8-Cineole alleviates Nrf2-mediated redox imbalance and mitochondrial dysfunction in diabetes mellitus by targeting Sirt1

BackgroundType 2 diabetes mellitus (T2DM) is primarily attributed to impaired insulin secretion caused by β cell dysfunction. 1,8-Cineole is a key bioactive compound in the essential oil extracted from Fructus Alpiniae Zerumbet, which possesses anti-inflammatory and antioxidant properties. Nevertheless, it remains elusive about the protective effect and precise mechanisms of 1,8-Cineole against the β cell deterioration in DM. PurposeTo investigate the effect of 1,8-Cineole on β cell dysfunction in T2DM and the potential mechanism of its action. MethodsA mouse model of T2DM and a β cell model of high glucose induction were generated to analyze the pharmacological properties of 1,8-Cineole. Proteomic and network pharmacological analyses were conducted to identify the crucial pathways involved in T2DM. Resveratrol [a Sirtuin1 (Sirt1) agonist] and Sirt1 knockdown were used to ascertain the mechanism of 1,8-Cineole in T2DM. The binding affinity of 1,8-Cineole to Sirt1 was assessed with molecular docking, surface plasmon resonance, immunoprecipitation assay, and cellular thermal shift assay. ResultsFirst, dysregulated crucial pathways in T2DM were screened out, including redox imbalance and mitochondrial dysfunction. Subsequently, 1,8-Cineole was found to activate Sirt1 and nuclear factor E2-related factor 2 (Nrf2) to repress oxidative stress in both T2DM mice and high glucose-induced β cells, thereby relieving mitochondrial dysfunction and apoptosis. Furthermore, 1,8-Cineole specifically targeted Sirt1 and favored the direct interaction between Sirt1 and Nrf2, ultimately restoring β cell function. ConclusionsOur findings provide the first evidence that 1,8-Cineole directly binds to Sirt1 protein and enhances its stability, therefore rectifying impaired oxidative homeostasis, and then suppressing mitochondrial dysfunction and apoptosis in T2DM, indicating that 1,8-Cineole may be a potential candidate drug for DM treatment.

Abstract 4134837: Checkpoint Kinase 1 Attenuates Myocardial Ischemia-Reperfusion Injury Through Maintaining SIRT1-Dependent Mitochondrial Homeostasis

Background: Mitochondrial dysfunction is linked to myocardial ischemia-reperfusion (I/R) injury. Checkpoint kinase 1 (CHK1) could facilitate cardiomyocyte proliferation and cardiac repair post myocardial infarction, however, its role on mitochondrial function in I/R injury remains unknown. Research Questions: The purpose of this study is to explore the potential effects and mechanisms of CHK1 on mitochondrial homeostasis during myocardial I/R injury. Methods: To investigate the role of CHK1 on mitochondrial function following I/R injury, cardiomyocyte-specific knockout/knock-in mouse models were generated. Mass spectrometry-proteomics analysis and protein co-immunoprecipitation assays were conducted to dissect the molecular mechanism of CHK1. The interaction between CHK1 and SIRT1 was explored using truncated plasmids. Results: CHK1 was downregulated in myocardium post I/R and neonatal mouse cardiomyocytes (NMCMs) post oxygen-glucose deprivation/re-oxygenation (OGD/R). In vivo , CHK1 overexpression protected against myocardial I/R injury, while heterogenous CHK1 knockout exacerbated cardiomyopathy. In vitro , CHK1 inhibited OGD/R-induced cardiomyocyte apoptosis and bolstered cardiomyocyte survival. Mechanistically, CHK1 attenuated oxidative stress and preserved mitochondrial metabolism in cardiomyocytes under I/R. Moreover, disrupted mitochondrial homeostasis in I/R myocardium was restored by CHK1 through the promotion of mitochondrial biogenesis and mitophagy. Through mass spectrometry analysis following co-immunoprecipitation, SIRT1 was identified as a direct target of CHK1. The 266-390 domain of CHK1 interacted with the 160-583 domain of SIRT1. Importantly, CHK1 phosphorylated SIRT1 at Thr530 residue, thereby inhibiting SMURF2-mediated degradation of SIRT1. CHK1 Δ390 amino acids (aa) mutant functioned similarly to full-length CHK1 in scavenging ROS and maintaining mitochondrial dynamics. Consistently, cardiac-specific SIRT1 knockdown partially attenuated the protective role of CHK1 in I/R injury. Conclusions: Our findings revealed that CHK1 mitigates I/R injury and restores mitochondrial dynamics in cardiomyocytes through a SIRT1-dependent mechanism.

SIRT2 deacetylates and decreases the expression of FOXM1 in colon cancer.

New FOXM1-specific inhibitors with the potential to be used for therapeutic purposes are under extensive research. We hypothesized that deacetylation of FOXM1 would decrease protein expression, thus providing novel therapeutic management of colon cancers. Immunostaining was used to determine FOXM1 and SIRT2 expressions in human colon cancer tissue microarrays (n = 90) from Stage I to Stage IV. SIRT2-FOXM1 interaction was evaluated in colon cancer cells using immunoprecipitation. Deacetylation of FOXM1 via SIRT2 was determined using in vitro deacetylation assays. FOXM1 could be hyper-acetylated when p300 and pCAF histone acetyltransferases were administered alongside deacetylase inhibitors. We detected that SIRT2 and FOXM1 physically interacted, and SIRT2 deacetylated FOXM1 in vitro. SIRT2 overexpression led to a significant decrease while knockdown of SIRT2 increased the FOXM1 expression in HCT116 human colon carcinoma cells. In the analysis of 90 human colorectal cancer samples, high SIRT2 expression was observed in about 49% of colorectal cancer, intermediate in 29%, and low or no staining in 22%. Strong SIRT2 expression was found to be negatively associated with the FOXM1 staining in our clinical cohort. This study reveals a molecular interaction and association between SIRT2 and FOXM1 expression in colon cancer cell lines and human colon cancer samples, and suggests that targeting SIRT2 activity using small molecule modulators may be a promising therapeutic approach for colorectal cancer.

Jiangu Recipe Suppresses ER Stress-Induced Apoptosis and Inhibits Extracellular Matrix Degradation in Chondrocytes through Upregulating SIRT1 Expression.

This study aimed to explore the effects of Jiangu Recipe (JGR) on chondrocyte responses under tert-Butyl hydroperoxide (TBHP)-induced oxidative stress, specifically focusing on apoptosis and extracellular matrix (ECM) degradation. Chondrocytes were treated with varying JGR concentrations, and cell viability was assessed. The impact of JGR on TBHP-induced apoptosis and protein expression levels of apoptosis- related molecules (Bcl-2, Bax, and cleaved caspase-3) and ECM components (Collagen II, Aggrecan, MMP-13) was evaluated. JGR exhibited protective effects against oxidative stress in chondrocytes. Moreover, it maintained cell viability under tert-butyl hydroperoxide (TBHP) induction, suppressing apoptosis (Bax, cleaved caspase-3) and enhancing anti-apoptotic Bcl-2. JGR also attenuated extracellular matrix (ECM) degradation, promoting Collagen II and Aggrecan while reducing MMP-13 expression. Investigating endoplasmic reticulum (ER) stress, it was found that JGR downregulated TBHP-induced GRP78, CHOP, ATF4, p-PERK, and p-eIF2α, thus indicating ER stress modulation. SIRT1 played a key role, as JGR upregulated SIRT1, mitigating TBHP-induced downregulation. SIRT1 knockdown reversed JGR's protective effects, highlighting its crucial role in JGR-mediated responses. Our findings suggest that JGR mitigated TBHP-induced chondrocyte apoptosis and ECM degradation, highlighting its potential therapeutic application in osteoarthritis. Mechanistically, our study highlights that SIRT1 plays a crucial role in mediating the protective effects of JGR against ER stress-induced chondrocyte apoptosis and ECM degradation, providing a foundation for further clinical exploration in managing osteoarthritic conditions.

Quercetin inhibits cardiomyocyte apoptosis via Sirt3/SOD2/mitochondrial reactive oxygen species during myocardial ischemia–reperfusion injury

BackgroundMyocardial ischemia/reperfusion injury (MI/RI) can lead to impaired cardiac function. Quercetin (Que) has a positive effect and improves MI/RI. Sirtuin-3 (Sirt3) is a deacetylase that ameliorates oxidative stress and is associated with MI/RI. This study aimed to investigate the molecular mechanism by which Que protects cardiac function against MI/RI through the Sirt3 signaling pathway. MethodsWe conducted experiments by constructing hypoxia/reoxygenation (H/R) cardiomyocytes and MI/RI rat models. H9C2 cells were transfected with siRNA-Sirt3. Cardiomyocyte apoptosis was examined by TUNEL and Western blotting. The oxidative stress index was also determined. Mitochondrial reactive oxygen species (ROS) activity assays, ATP assays and mitochondrial membrane potential assays were performed. Evans Blue/TTC staining was used to examine surviving myocardial tissue. ResultsIn the constructed H/R cells and MI/RI animal models, it was found that myocardial cell apoptosis increased (Bcl-2 expression was downregulated; Bax and cleaved caspase-3/8/9 expression were upregulated). In addition, oxidative stress levels increased (MDA levels increased; SOD, CAT, GSH-Px levels decreased), myocardial tissue was damaged (LDH, CK content increased), Sirt3 expression was downregulated, acetylation levels of superoxide dismutase 2 (SOD2) increased (AC-SOD2), and mitochondrial ROS increased. Que treatment alleviated the effects of MI/RI on cardiomyocytes and rats. Sirt3 expression and activity were upregulated, SOD2 acetylation was decreased, and mitochondrial ROS production was reduced by Que treatment. After Sirt3 was knocked down, we found that AC-SOD2 expression was upregulated and mitochondrial ROS were increased in H/R cardiomyocytes, further increased the degree of injury, while Que treatment attenuated the effect of Sirt3 knockdown on H/R cardiomyocytes. ConclusionQue inhibits cardiomyocyte apoptosis, reduces oxidative stress levels, protects mitochondrial function and prevents the impairment of cardiac function during MI/RI via the Sirt3/SOD2/mitochondrial ROS pathway.

CHK1 attenuates cardiac dysfunction via suppressing SIRT1-ubiquitination

BackgroundMitochondrial dysfunction is linked to myocardial ischemia-reperfusion (I/R) injury. Checkpoint kinase 1 (CHK1) could facilitate cardiomyocyte proliferation, however, its role on mitochondrial function in I/R injury remains unknown. MethodsTo investigate the role of CHK1 on mitochondrial function following I/R injury, cardiomyocyte-specific knockout/overexpression mouse models were generated. Adult mouse cardiomyocytes (AMCMs) were isolated for in vitro study. Mass spectrometry-proteomics analysis and protein co-immunoprecipitation assays were conducted to dissect the molecular mechanism. ResultsCHK1 was downregulated in myocardium post I/R and AMCMs post oxygen-glucose deprivation/re‑oxygenation (OGD/R). In vivo, CHK1 overexpression protected against I/R induced cardiac dysfunction, while heterogenous CHK1 knockout exacerbated cardiomyopathy. In vitro, CHK1 inhibited OGD/R-induced cardiomyocyte apoptosis and bolstered cardiomyocyte survival. Mechanistically, CHK1 attenuated oxidative stress and preserved mitochondrial metabolism in cardiomyocytes under I/R. Moreover, disrupted mitochondrial homeostasis in I/R myocardium was restored by CHK1 through the promotion of mitochondrial biogenesis and mitophagy. Through mass spectrometry analysis following co-immunoprecipitation, SIRT1 was identified as a direct target of CHK1. The 266–390 domain of CHK1 interacted with the 160–583 domain of SIRT1. Importantly, CHK1 phosphorylated SIRT1 at Thr530 residue, thereby inhibiting SMURF2-mediated degradation of SIRT1. The role of CHK1 in maintaining mitochondrial dynamics control and myocardial protection is abolished by SIRT1 inhibition, while inactivated mutation of SIRT1 Thr530 fails to reverse the impaired mitochondrial dynamics following CHK1 knockdown. CHK1 Δ390 amino acids (aa) mutant functioned similarly to full-length CHK1 in scavenging ROS and maintaining mitochondrial dynamics. Consistently, cardiac-specific SIRT1 knockdown attenuated the protective role of CHK1 in I/R injury. ConclusionsOur findings revealed that CHK1 mitigates I/R injury and restores mitochondrial dynamics in cardiomyocytes through a SIRT1-dependent mechanism.

Sirt1 Mitigates Hepatic Lipotoxic Injury Induced by High-Fat-Diet in Fish Through Ire1α Deacetylation

BackgroundSilent information regulator protein 1 (Sirt1) is crucial in regulating lipid metabolism, but its specific role and mechanism in fish hepatic lipotoxic injury remain undefined. ObjectivesThis study aimed to elucidate the regulatory role of Sirt1 and the underlying mechanisms in dietary lipid-induced hepatic lipotoxic injury in a marine teleost black seabream. MethodsBlack seabream were fed a control diet (12% lipid level), high-fat diet (HFD) [18% lipid level, oleic acid (OA)-rich], or HFD supplemented with 0.25%, 0.50%, or 1.00% resveratrol (RSV) for 8 wk. The cultured hepatocytes were stimulated by OA (200 μM), OA supplemented with RSV (20 μM), or transfection with sirt1-small interfering RNA (sisirt1). Biochemical indices, gene expression (qPCR), histology, transmission electron microscope, immunofluorescence, Western blot, flow cytometry, and immunoprecipitation assays were conducted to evaluate hepatic lipid deposition, lipid metabolism, endoplasmic reticulum stress, inflammation and apoptosis, and determine protein interactions between Sirt1 and Ire1α. ResultsIn vivo, RSV supplementation increased mRNA and protein expression levels of sirt1 (236.2% ± 16.1% and 53.1% ± 14.3%) and downregulated the mRNA and phosphorylated protein expression levels of ire1α/Ire1α (46.0% ± 7.6% and 38.6% ± 7.0%), jnk/Jnk (57.6% ± 7.3% and 122.1%), and nuclear factor κ B (nf-κb/Nf-κb) p65 (41.7% ± 7.1% and 24.6% ± 0.8%) compared with the HFD group. Similar patterns were found in the in vitro experiments; however, after knockdown of sirt1, although the cells were incubated with RSV, the expression levels of ire1α/ Ire1α, jnk/Jnk, and nf-κb/Nf-κb p65 showed no significant differences compared with the OA treatment. Moreover, we found that mutation of K61 to arginine to mimic Ire1α deacetylation confers protection against Ire1α-mediated OA-rich HFD-induced inflammation and apoptosis. ConclusionsThe findings revealed that Sirt1 protects against OA-rich HFD-induced hepatic lipotoxic injury via the deacetylation of Ire1α on K61, hence reducing Ire1α autophosphorylation level, and suppressing Jnk and Nf-κb p65 activation. This mechanism is elucidated for the first time in fish.

Unlocking mitochondrial dysfunction-associated senescence (MiDAS) with NAD+ – A Boolean model of mitochondrial dynamics and cell cycle control

The steady accumulation of senescent cells with aging creates tissue environments that aid cancer evolution. Aging cell states are highly heterogeneous. 'Deep senescent' cells rely on healthy mitochondria to fuel a strong proinflammatory secretome, including cytokines, growth and transforming signals. Yet, the physiological triggers of senescence such as reactive oxygen species (ROS) can also trigger mitochondrial dysfunction, and sufficient energy deficit to alter their secretome and cause chronic oxidative stress – a state termed Mitochondrial Dysfunction-Associated Senescence (MiDAS). Here, we offer a mechanistic hypothesis for the molecular processes leading to MiDAS, along with testable predictions. To do this we have built a Boolean regulatory network model that qualitatively captures key aspects of mitochondrial dynamics during cell cycle progression (hyper-fusion at the G1/S boundary, fission in mitosis), apoptosis (fission and dysfunction) and glucose starvation (reversible hyper-fusion), as well as MiDAS in response to SIRT3 knockdown or oxidative stress. Our model reaffirms the protective role of NAD+ and external pyruvate. We offer testable predictions about the growth factor- and glucose-dependence of MiDAS and its reversibility at different stages of reactive oxygen species (ROS)-induced senescence. Our model provides mechanistic insights into the distinct stages of DNA-damage induced senescence, the relationship between senescence and epithelial-to-mesenchymal transition in cancer and offers a foundation for building multiscale models of tissue aging.

Rhein targets macrophage SIRT2 to promote adipose tissue thermogenesis in obesity in mice

Rhein, a component derived from rhubarb, has been proven to possess anti-inflammatory properties. Here, we show that rhein mitigates obesity by promoting adipose tissue thermogenesis in diet-induced obese mice. We construct a macrophage-adipocyte co-culture system and demonstrate that rhein promotes adipocyte thermogenesis through inhibiting NLRP3 inflammasome activation in macrophages. Moreover, clues from acetylome analysis identify SIRT2 as a potential drug target of rhein. We further verify that rhein directly interacts with SIRT2 and inhibits NLRP3 inflammasome activation in a SIRT2-dependent way. Myeloid knockdown of SIRT2 abrogates adipose tissue thermogenesis and metabolic benefits in obese mice induced by rhein. Together, our findings elucidate that rhein inhibits NLRP3 inflammasome activation in macrophages by regulating SIRT2, and thus promotes white adipose tissue thermogenesis during obesity. These findings uncover the molecular mechanism underlying the anti-inflammatory and anti-obesity effects of rhein, and suggest that rhein may become a potential drug for treating obesity.

Loss of NAMPT and SIRT2 but not SIRT1 attenuate GLO1 expression and activity in human skeletal muscle

Glyoxalase I (GLO1) is the primary enzyme for detoxification of the reactive dicarbonyl methylglyoxal (MG). Loss of GLO1 promotes accumulation of MG resulting in a recapitulation of diabetic phenotypes. We previously demonstrated attenuated GLO1 protein in skeletal muscle from individuals with type 2 diabetes (T2D). However, whether GLO1 attenuation occurs prior to T2D and the mechanisms regulating GLO1 abundance in skeletal muscle are unknown. GLO1 expression and activity were determined in skeletal muscle tissue biopsies from 15 lean healthy individuals (LH, BMI: 22.4 ± 0.7) and 5 individuals with obesity (OB, BMI: 32.4 ± 1.3). GLO1 protein was attenuated by 26 ± 0.3 % in OB compared to LH skeletal muscle (p = 0.019). Similar reductions for GLO1 activity were observed (p = 0.102). NRF2 and Keap1 expression were equivocal between groups despite a 2-fold elevation in GLO1 transcripts in OB skeletal muscle (p = 0.008). GLO1 knock-down (KD) in human immortalized myotubes promoted downregulation of muscle contraction and organization proteins indicating the importance of GLO1 expression for skeletal muscle function. SIRT1 KD had no effect on GLO1 protein or activity whereas, SIRT2 KD attenuated GLO1 protein by 28 ± 0.29 % (p < 0.0001) and GLO1 activity by 42 ± 0.12 % (p = 0.0150). KD of NAMPT also resulted in attenuation of GLO1 protein (28 ± 0.069 %, p = 0.003), activity (67 ± 0.09 %, p = 0.011) and transcripts (50 ± 0.13 %, p = 0.049). Neither the provision of the NAD+ precursors NR nor NMN were able to prevent this attenuation in GLO1 protein. However, NR did augment GLO1 specific activity (p = 0.022 vs NAMPT KD). These perturbations did not alter GLO1 acetylation status. SIRT1, SIRT2 and NAMPT protein levels were all equivocal in skeletal muscle tissue biopsies from individuals with obesity and lean individuals. These data implicate NAD+-dependent regulation of GLO1 in skeletal muscle independent of altered GLO1 acetylation and provide rationale for exploring NR supplementation to rescue attenuated GLO1 abundance and activity in conditions such as obesity.

Abstract Tu103: SIRT2 Inhibition Decreases Glycolysis and Attenuates Hypertrophic Response in H9c2 Cardiomyocytes

Introduction: Myocardial glycolysis increases in hypertrophic and failing hearts. Hyperacetylation also occurs in the failing heart, and many glycolytic enzymes are known to be subject to acetylation. However, it is generally considered that acetylation has inhibitory effects on glycolysis. As a result, it is not clear whether acetylation changes directly contribute to glycolysis changes in cardiac hypertrophy. We therefore determined whether changes in the acetylation of glycolytic enzymes and the activity of the cytosolic deacetylase SIRT2 regulate cardiac glycolysis. Methods: Glycolysis rates were directly measured in rat heart-derived H9c2 cardiomyocytes perfused with 5 mM [5- 3 H] glucose, 0.8 mM palmitate, and 4% bovine serum albumin. Before these metabolic measurements, H9c2 cells were treated with either a SIRT2 inhibitor (10 µM AGK2) or a vehicle for 24 hours. In separate experiments, SIRT2 was also knocked down in H9c2 cells using siRNA, followed by glycolysis rate determinations. The impact of SIRT2 inhibition or SIRT2 knockdown on the acetylation status of glycolytic enzyme was also assessed. Furthermore, the effects of SIRT2 inhibition on hypertrophic signalling were assessed by treating H9c2 cells with phenylephrine. Results: SIRT2 inhibition markedly decreased glycolysis rates in H9c2 cells compared to vehicle-treated cells (524±108 vs 2631±372 nmol . g dry wt -1. min -1 , p&lt;0.05). Similarly, SIRT2 knockdown resulted in a significant reduction in glycolysis rates compared to scrambled siRNA-treated H9c2 cells (745±31 vs 1659±168 nmol . g dry wt -1. min -1 , p&lt;0.05). The decrease in SIRT2 was accompanied by an increase in the acetylation status of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GPDH). Moreover, a trend towards increased phosphofructokinase (PFK) acetylation was also observed in SIRT2 knockdown H9c2 cells compared to scrambled siRNA-treated cells. Lastly, AGK2 treatment also attenuated phenylephrine-mediated hypertrophic responses in H9c2 cells. Conclusions: Increased acetylation of glycolytic enzymes is associated with a decrease in glycolysis, and SIRT2 inhibition or deletion in H9c2 cells significantly decreases glycolysis rates and attenuates hypertrophy. SIRT2 may therefore contribute to the increased glycolysis seen in hypertrophy and heart failure.

Specnuezhenide ameliorates hepatic fibrosis via regulating SIRT6-Mediated inflammatory signaling cascades

Ethnopharmacological relevanceLigustrum lucidum W.T. Aiton is a traditional Chinese medicine that has long been used with high hepatoprotective therapeutic and condition value. Specnuezhenide (SP), the standard prominent secoiridoid compound of Fructus Ligustri Lucidi may ameliorate hepatic inflammation in chronic liver diseases. Aim of the studyRegulating inflammation through SIRT6-P2X7R axis has caused the emergence of novel molecular mechanism strategies for reversing hepatic fibrosis. This study focused on the mechanism of SP in modulating the liver inflammatory microenvironment in hepatic fibrosis. Materials and methodsC57BL/6 mice with hepatic fibrosis were stimulated with thioacetamide (TAA) prior to administration of SP. Hepatic stellate cells (HSCs) or normal mouse primary hepatocytes were exposed to transforming growth factor-β (TGF-β) treatment. Meanwhile, normal mouse bone marrow-derived macrophages (BMDMs) were treated with lipopolysaccharide/adenosine triphosphate (LPS/ATP), aiming to obtain the conditioned medium. HSCs and hepatocytes were transfected with SIRT6 knockdown vector (siRNA-SIRT6) to estimate the impact of SP on the SIRT6-P2X7R/NLRP3 signaling pathway. ResultsSP suppressed the HSCs extracellular matrix (ECM) deposition as well as pro-inflammatory cytokine levels induced by the medium of BMDMs or TGF-β. In addition, SP also significantly up-regulated SIRT6, inhibited P2X7R-NLRP3 inflammasome in HSCs and hepatocytes, and functioned as MDL-800 (a SIRT6 agonist). SP reduced the hepatocytes pyroptosis and further prevented the occurrence of inflammatory response in the liver. SP could inhibit the activation of BMDMs and impede IL-1β and IL-18 from entering extracellular regions. Moreover, deficiency of SIRT6 in HSCs or hepatocytes reduced SP's regulation of P2X7R suppression. For TAA-treated mice, SP mitigated histopathological changes, ECM accumulation, EMT process, and NETs formation in hepatic fibrosis. ConclusionsTherefore, SP decreased inflammatory response via SIRT6-P2X7R/NLRP3 pathway and suppressed fibrillogenesis. These findings supported SP as the novel candidate to treat hepatic fibrosis.

Ropivacaine prompts ferroptosis to enhance the cisplatin-sensitivity of human colorectal cancer through SIRT1/Nrf2 signaling pathway

The ineffectiveness of cisplatin therapy in treating colorectal cancer (CRC) is attributed to an increase of resistance. It's necessary to investigate adjunctive agents capable of enhancing drug efficacy. Previous studies have shown that ropivacaine inhibit the growth of various cancer cells, but its impact on cisplatin resistance in tumors is not well understood. This study was to illustrate the impact and mechanism of ropivacaine enhanced cisplatin-sensitivity of CRC. Cisplatin alone treatment resulted in the elevation of reactive oxygen species (ROS) and intracellular Fe2+ levels, as well as a reduction in mitochondrial membrane potential (MMP) in cisplatin-sensitive LOVO cells, while these effects were mitigated in the cisplatin-resistant LOVO/DDP cells. The co-administration of ropivacaine with cisplatin inhibited cell viability and cell migration, decreased MMP, and promoted ROS accumulation and apoptosis in both LOVO cells and LOVO/DDP cells. And they upregulated the levels of ferroptosis makers and downregulated the expression of anti-ferroptosis proteins. However, this effect was reversed by ferroptosis inhibitor ferrostatin-1 or liproxstatin-1. Furthermore, we o demonstrated that the co-administration of ropivacaine and cisplatin resulted in a decrease in SIRT1 expression, and SIRT1 knockdown in LOVO/DDP cells enhanced the ferroptosis and the anti-tumor properties of ropivacaine, while also inhibiting the activation of the Nrf2/Keap1 pathway. The above results suggested that ropivacaine increased the sensitivity of CRC cells to cisplatin by promoting ferroptosis through the inhibition of SIRT1 expression, which proposes a therapeutic approach for overcoming cisplatin resistance in CRC.

SIRT1 Promotes Chemoradiotherapy Resistance in Esophageal Squamous Cell Carcinoma

Introduction: Identifying accurate biomarkers for predicting response to chemoradiotherapy (CRT) in patients with esophageal squamous cell carcinoma (ESCC) is a critical challenge. The protein SIRT1, recognized for its implications in longevity, has been associated with tumor promotion in ESCC. However, data regarding its correlation with CRT sensitivity remain unreported. Therefore, in this study, we aimed to investigate the relationship between SIRT1 expression and CRT sensitivity and concurrently assess the effect of SIRT1 knockdown on CRT sensitivity in ESCC. Methods: This study included 73 patients who underwent radical esophagectomy after CRT. SIRT1 expression in pre-treatment endoscopic biopsies was assessed through immunostaining, followed by a comparative analysis of CRT effects on surgical specimens. Small interfering RNA was used to attenuate SIRT1 expression in TE5 and TE10 cells, which were then subjected to cisplatin treatment at varying doses and concentrations and irradiation with X-rays, respectively. Results: High SIRT1 tissue expression was significantly associated with CRT resistance. Multivariate analysis identified high SIRT1 expression as an independent biomarker for poor CRT response. In TE-5 and TE-10 cells, SIRT1 knockdown significantly decreased cell viability and increased sensitivity to cisplatin and radiation treatment compared to that of the negative control. Conclusion: Our study results demonstrate the potential of SIRT1 as a predictive biomarker for CRT response in ESCC, highlighting the heightened sensitivity to CRT upon the transcriptional inactivation of SIRT1. Targeting SIRT1 emerges as a promising strategy for enhancing the efficacy of CRT for ESCC.

Investigation on Post Translational Modification of GSK3 Beta Upon SIRT6 Silencing in Non-Small Cell Lung Cancer Cell Lines

SIRT6 aberration has been widely reported in a variety of serious human disorders during the past few decades.This work is to analyse the SIRT6 knockdown effect on the stability of a non-small cell lung cancer cell lines expressing GSK-3Beta.The main objectives includes Silencing of SIRT6 in A549 and H460 cell lines by using (SIRT6 siRNA 1, SIRT6 siRNA 2) and investigation on the phosphorylation status of GSK-3beta.Methods used in this work are In vitro experiments with human NSCLC cells have been performed. Western Blot was performed to explore the key events in the regulation of GSK-3β by silencing SIRT6 in NSCLC. The findings of this study suggest that silencing of SIRT6 significantly promotes the phosphorylation status of GSK-3β and destabilizes it.

MKK3 depletion attenuates intestinal injury after traumatic hemorrhagic shock by restoring mitochondrial function.

Traumatic hemorrhagic shock (THS) is a complex pathophysiological process resulting in multiple organ failure. Intestinal barrier dysfunction is one of the mechanisms implicated in multiple organ failure. The present study aimed to explore the regulatory role of mitogen-activated protein kinase kinase 3 (MKK3) in THS-induced intestinal injury and to elucidate its potential mechanism. Rats were subjected to trauma and hemorrhage to establish a THS animal model. MKK3-targeted lentiviral vectors were injected via the tail vein 72h before modeling. Twelve hours post-modeling, the mean arterial pressure (MAP) and heart rate (HR) were monitored, and histological injury to the intestine was assessed via H&E staining and transmission electron microscopy. Mitochondrial function and mitochondrial reactive oxygen species (ROS) were evaluated. IEC-6 cells were exposed to hypoxia to mimic intestinal injury following THS in vitro. MKK3 deficiency alleviated intestinal injury and restored mitochondrial function in intestinal tissues from THS-induced rats and hypoxia-treated IEC-6 cells. In addition, MKK3 deficiency promoted Sirt1/PGC-1α-mediated mitochondrial biogenesis and restricted Pink1/Parkin-mediated mitophagy in the injured intestine and IEC-6 cells. Furthermore, the protective effect of MKK3 knockdown against hypoxia-induced mitochondrial damage was strengthened upon simultaneous LC3B/Pink1/Parkin knockdown or weakened upon simultaneous Sirt1 knockdown. MKK3 deficiency protected against intestinal injury induced by THS by promoting mitochondrial biogenesis and restricting excessive mitophagy.

Forsythoside B alleviates cerebral ischemia-reperfusion injury via inhibiting NLRP3 inflammasome mediated by SIRT1 activation.

The inflammatory response is a key factor in the pathogenesis of cerebral ischemia/reperfusion injury (CIRI), and anti-inflammatory interventions may offer a promising therapeutic strategy. Forsythoside B (FB) is a phenylethanoid glycoside isolated from Forsythiae fructus, which has been reported to have anti-inflammatory effects. However, the mechanism of the neuroprotective effect of FB on CIRI remains unclear. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). FB was administered intraperitoneally for 3 days prior to MCAO/R. Cerebral infarct volume and neurological deficit score were used as indices to evaluate MCAO/R injury. The serum levels of inflammatory factors and antioxidant enzymes were measured. The activation of silent information regulator 2 homolog 1 (Sirt1) and the inhibition of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) pathway were assessed through western blot and immunohistochemistry analysis. Furthermore, the rats were treated with Sirt1 shRNA 3 days before MCAO/R by stereotactical injection into the ipsilateral hemispheric region to assess the impact of Sirt1 knockdown on the protection of FB during MCAO/R. FB reduced cerebral infarct volume and neurological deficit score in MCAO/R rats. FB reduced pathological changes and cell apoptosis in the hippocampal CA1 region and cortex on the ischemic side of rats. FB inhibited the serum levels of inflammatory factors and increased the activities of antioxidant enzymes. Further study showed that FB inhibited the activation of the NLRP3 pathway and induced Sirt1 activation. FB demonstrated neuroprotective and anti-inflammatory effects by inhibiting the NLRP3 pathway through Sirt1 activation in CIRI.

Sirtuin1 (sirt1) regulates the glycolysis pathway and decreases cisplatin chemotherapeutic sensitivity to esophageal squamous cell carcinoma

ABSTRACT We aimed to evaluate the influence of sirtuin1 (sirt1) on the ESCC chemotherapeutic sensitivity to cisplatin. We used ESCC cell ablation sirt1 for establishing a xenograft mouse tumor model. The tumor volume was then detected. sirt1 was over-expressed significantly in ESCC patients and cells. Moreover, sirt1 knockdown raised ESCC sensitivity to cisplatin. Besides, glycolysis was associated with ESCC cell chemotherapy resistance to cisplatin. Furthermore, sirt1 increased ESCC cells’ cisplatin chemosensitivity through HK2. Sirt1 enhanced in vivo ESCC chemosensitivity to cisplatin. Overall, these findings suggested that sirt1 knockdown regulated the glycolysis pathway and raised the ESCC chemotherapeutic sensitivity.

Cardioprotective effect of Cinnamamide derivative compound 10 against myocardial ischemia-reperfusion through regulating cardiac autophagy via Sirt1

Background and purposeOur previous research discovered that cinnamamide derivatives are a new type of potential cardioprotective agents myocardial ischemia-reperfusion (MIR) injury, among which Compound 10 exhibits wonderful beneficial action in vitro. However, the exact mechanism of Compound 10 still needs to be elucidated. Experimental approachThe protective effect of Compound 10 was determined by detecting the cell viability and LDH leakage rate in H9c2 cells subjected to H2O2. Alterations of electrocardiogram, echocardiography, cardiac infarct area, histopathology and serum myocardial zymogram were tested in MIR rats. Additionally, the potential mechanism of Compound 10 was explored through PCR. Network pharmacology and Western blotting was conducted to monitor levels of proteins related to autophagic flux and mTOR, autophagy regulatory substrate, induced by Compound 10 both in vitro and in vivo, as well as expressions of Sirtuins family members. Key resultsCompound 10 significantly ameliorated myocardial injury, as demonstrated by increased cell viability, decreased LDH leakage in vitro, and declined serum myocardial zymogram, ST elevation, cardiac infarct area and improved cardiac function and microstructure of heart tissue in vivo. Importantly, Compound 10 markedly enhanced the obstruction of autophagic flux and inhibited excessive autophagy initiation against MIR by decreased ATG5, Rab7 and increased P-mTOR and LAMP2. Furthermore, Sirt1 knockdown hindered Compound 10’s regulation on mTOR, leading to interrupted cardiac autophagic flux. Conclusions and implicationsCompound 10 exerted cardioprotective effects on MIR by reducing excessive autophagy and improving autophgic flux blockage. Our work would take a novel insight in seeking effective prevention and treatment strategies against MIR injury.

SIRT1 mediates the excitability of spinal CaMKIIα-positive neurons and participates in neuropathic pain by controlling Nav1.3.

Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.