SIRT1 Inhibitor Research Articles

Myricetin alleviates H2O2-induced senescence and apoptosis in rat nucleus pulposus-derived mesenchymal stem cells.

Transplantation of mesenchymal stem cells (MSCs) has been reported to be a novel promising target for the regeneration of degenerated intervertebral discs (IVDs). However, the culture and survival limitations of MSCs remain challenging for MSC-based biological therapy. Myricetin, a common natural flavonoid, has been suggested to possess antiaging and antioxidant abilities. Therefore, we investigated the biological function of myricetin, and its related mechanisms involving cell senescence in intervertebral disc degeneration (IDD). The nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated from 4-month-old Sprague-Dawley (SD) rats and identified by examining surface markers and multipotent differentiation. Rat NPMSCs were cultured in an MSC culture medium or culture medium with different concentrations of H2O2. Myricetin or the combination of myricetin and EX527 were added to the culture medium to investigate the effects of myricetin. Cell viability was evaluated by cell counting kit-8 assays (CCK-8). The apoptosis rate was determined using Annexin V/PI dual staining. The mitochondrial membrane potential (MMP) was analyzed by a fluorescence microscope after JC-1 staining. The cell senescence was determined by SA-β-Gal staining. MitoSOX green was used to selectively estimate mitochondrial reactive oxygen species (ROS) Apoptosis-associated proteins (Bax, Bcl2, and cleaved caspase-3), senescence markers (p16, p21, and p53), and SIRT1/PGC-1α signaling pathway-related proteins (SIRT1 and PGC-1α) were evaluated by western blotting. The cells isolated from nucleus pulposus (NP) tissues met the criteria for MSCs. Myricetin showed no cytotoxicity up to a concentration of 100 μM in rat NPMSCs cultured for 24 h. Myricetin pretreatment exhibited protective effects against H₂O₂-induced apoptosis. Myricetin could also alleviate H₂O₂-induced mitochondrial dysfunctions of increased mitochondrial ROS production and reduced MMP. Moreover, myricetin pretreatment delayed rat NPMSC senescence, as evidenced by decreased exppression of senescence indicators. Pretreatment of NPMSCs with 10 μM EX527, a selective inhibitor of SIRT1, prior to exposure to 100 μM H2O2, reversed the inhibitory effects of myricetin on cell apoptosis. Myricetin could affect the SIRT1/PGC-1α pathway to protect mitochondrial functions and alleviate cell senescence in H₂O₂-treated NPMSCs.

Dihydroisotanshinone I and BMAL-SIRT1 Pathway in an In Vitro 6-OHDA-Induced Model of Parkinson’s Disease

Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from Danshen, as well as the corresponding mechanisms in an in vitro-based 6-OHDA-induced Parkinson’s disease (PD) model. SH-SY5Y human neuroblastoma cell lines were pretreated with 6-hydroxydopamine (6-OHDA) and challenged with DT. Subsequently, the cell viability and levels of reactive oxygen species (ROS) and caspase-3 were analyzed. The effect of DT on the 6-OHDA-treated SH-SY5Y cells and the expression of the core circadian clock genes were measured using a real-time quantitative polymerase chain reaction. Our results indicated that DT attenuated the 6-OHDA-induced cell death in the SH-SY5Y cells and suppressed ROS and caspase-3. Moreover, DT reversed both the RNA and protein levels of BMAL1 and SIRT1 in the 6-OHDA-treated SH-SY5Y cells. Additionally, the SIRT1 inhibitor attenuated the effect of DT on BMAL1 and reduced the cell viability. The DT and SIRT1 activators activated SIRT1 and BMAL1, and then reduced the death of the SH-SY5Y cells damaged by 6-OHDA. SIRT1 silencing was enhanced by DT and resulted in a BMAL1 downregulation and a reduction in cell viability. In conclusion, our investigation suggested that DT reduces cell apoptosis, including an antioxidative effect due to a reduction in ROS, and regulates the circadian genes by enhancing SIRT1 and suppressing BMAL1. DT may possess novel therapeutic potential for PD in the future, but further in vivo studies are still needed.

Dexmedetomidine alleviates anxiety-like behaviors in female mice with musculoskeletal pain through SIRT1/p53 axis

BackgroundMusculoskeletal pain is the most common form of chronic pain. Anxiety increases pain intensity and appears to have a major impact on the prevalence and also disability of musculoskeletal pain in women. We examined the effect of dexmedetomidine (DEX) on anxiety-like behaviors associated with musculoskeletal pain and the underlying molecular mechanism in female mice. MethodsMusculoskeletal pain was induced by injection of acidified saline into the gastrocnemius muscle in adult female mice, and the von Frey filament test is used to measure mechanical sensitivity. DEX and EX527 (SIRT1 inhibitor) were administered after modelling. Behavioral tests were used for anxiety and motor activity tests. SIRT1, p53 and acetyl-p53 were quantified by Western blot. ResultsAdult female mice with musculoskeletal pain exhibit increased fear-like behavior by reducing SIRT1 expression in the medial prefrontal cortex (mPFC). While administration of DEX was able to alleviate mechanical hypersensitivity and anxiety-like behaviors by blocking SIRT1 decline and acetyl-p53 upregulation in mPFC, EX527 inhibited acetyl-p53 rise and reversed the antinociceptive and anxiolytic effects of DEX. ConclusionDEX may alleviate anxiety-like behaviors in mice with musculoskeletal pain via the SIRT1/p53 axis. These results suggest that DEX may have a potential therapeutic role in musculoskeletal pain-induced anxiety.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

Mitigation of triptolide-induced testicular Sertoli cell damage by melatonin via regulating the crosstalk between SIRT1 and NRF2

Triptolide (TP) is an important active compound from Tripterygium wilfordii Hook F (TwHF), however, it is greatly limited in clinical practice due to its severe toxicity, especially testicular injury. Melatonin is an endogenous hormone and has beneficial effects on the reproductive system. However, whether triptolide-induced testicular injury can be alleviated by melatonin and the underlying mechanism are not clear. In this study, we aimed to explore whether triptolide-induced testicular Sertoli cells toxicity can be mitigated by melatonin and the underlying mechanisms involved. Cell apoptosis was assessed by flow cytometry, western blot, immunofluorescence and immunohistochemistry. Fluorescent probe Mito-Tracker Red CMXRos was used to observe the mitochondria morphology. Mitochondrial membrane potential and Ca2+ levels were used to investigate mitochondrial function by confocal microscope and flow cytometry. The expression levels of SIRT1/Nrf2 pathway were detected by western blot, immunofluorescence and immunohistochemistry. Small interfering RNA of NRF2 and SIRT1 inhibitor EX527 was used to confirm the role of SIRT1/NRF2 pathway in the mitigation of triptolide-induced Sertoli cell damage by melatonin. Co-Immunoprecipitation assay was used to determine the interaction between SIRT1 and NRF2. Triptolide-induced dysfunction of testicular Sertoli cells was significantly improved by melatonin treatment. Specifically, triptolide-induced oxidative stress damage and changes of mitochondrial morphology, mitochondrial membrane potential, and BTB integrity were alleviated by melatonin. Mechanistically, triptolide inhibited SIRT1 and then reduced the activation of NRF2 pathway via regulating the interaction between SIRT1 and NRF2, thereby downregulating the downstream antioxidant genes, which was reversed by melatonin. Nevertheless, knockdown of NRF2 or inhibition of SIRT1 abolished the protective effect of melatonin. Triptolide-induced testicular Sertoli cell damage could be alleviated by melatonin via regulating the crosstalk between SIRT1 and NRF2, which is helpful for developing a new strategy to alleviate triptolide-induced toxicity.

SIRT2 inhibition attenuates the vasculopathy and vision impairment via Akt signaling in retinopathy of prematurity

Despite decades of researches, the underlying mechanism of retinopathy of prematurity (ROP) remains unclear. The role of Sirt2, which is involved in both angiogenesis and inflammation, both pivotal in ROP, was investigated in an animal model of ROP known as oxygen-induced retinopathy (OIR). Our study found that Sirt2 was overexpressed and colocalized with microglia in OIR. Furthermore, it demonstrated that the level of Sirt2 was upregulated in hypoxia microglia BV-2 in vitro. Subsequently, our results elucidated that administration of the Sirt2 antagonist AGK2 attenuated the avascular and neovascular area and downregulated the expression of IGF-1. The phosphorylation of Akt and the expression of IGF-1 were upregulated in hypoxia BV-2 and conditional media collected from BV-2 under hypoxia promoted the migration and tube formation of retinal capillary endothelial cells, which were suppressed with AGK2. Notably, our findings are the first to demonstrate the deleterious role of Sirt2 in ROP, as Sirt2 inhibition led to the downregulation of Akt/IGF-1 and ameliorated vasculopathy, ultimately improving visual function. These results suggest that Sirt2 may be a promising therapeutic target for ROP.

Through regulation of the SIRT1 pathway plant sterol ester of α-linolenic acid inhibits pyroptosis thereby attenuating the development of NASH in mice

Increasing evidence demonstrated that pyroptosis and subsequent inflammation played an important role in the pathological process of non-alcoholic steatohepatitis (NASH). Plant sterol ester of α-linolenic acid (PS-ALA) was beneficial for non-alcoholic fatty liver disease, but the underlying mechanisms are still not fully understood. This study aims to investigate whether PS-ALA can protect against proptosis via regulating SIRT1. Thirty male C57BL/6J mice were fed a normal diet, a high-fat and high-cholesterol diet (HFCD), or a HFCD supplemented with either 1.3%ALA, 2%PS, or 3.3% PS-ALA for 24 weeks. Hepatocytes were treated with oleic acid and cholesterol (OA/Cho) with or without PS-ALA. We found that PS-ALA ameliorated NASH in HFCD-fed mice. In addition, PS-ALA decreased the expression of NLRP3 and ASC and reduced the co-localization of NLRP3 and cleave-Caspase-1. Also, PS-ALA protected against pyroptosis as evidenced by decreased co-localization of GSDMD and propidium iodide (PI) positive cells. Mechanistically, we revealed that the inhibitory action of PS-ALA on the pyroptosis was mediated by SIRT1. This was demonstrated by the fact that silencing SIRT1 with small interfering RNA or inhibition of SIRT1 with its inhibitor abolished the inhibition effect of PS-ALA on the expression of NLRP3 and GSDMD cleavage. Collectively, the data from the present study reveals a novel mechanism that PS-ALA inhibits pyroptosis and it triggered inflammation via stimulating SIRT1, which provides new insights into the beneficial effect of PS-ALA on NASH.

Melatonin promotes parthenogenetic development of vitrified-warmed mouse MII oocytes, potentially by reducing oxidative stress through SIRT1

Previous studies have demonstrated that melatonin could ameliorate oxidative stress during the cryopreservation of mouse MII oocytes and their in vitro culture after parthenogenetic activation. However, the underlying molecular mechanism remained poorly understood. This study was conducted to investigate whether melatonin could modulate the oxidative stress in the parthenogenetic 2-cell embryos derived from vitrified-warmed oocytes through SIRT1. The results showed that the reactive oxygen species levels increased, the glutathione levels and SIRT1 expression decreased significantly in parthenogenetic 2-cell embryos derived from cryopreserved oocyte, and the parthenogenetic blastocyst formation rates significantly decreased when compared to those derived from control oocytes. These unfavorable phenomena were prevented by the addition of either 10−9 mol/L melatonin or 10−6 mol/L SRT-1720 (SIRT1 agonist), and it was restored by the supplementation of 10−9 mol/L melatonin in combination with 2 × 10−5 mol/L EX527 (SIRT1 inhibitor). Therefore, the findings from the present study concluded that melatonin may reduce oxidative stress via regulating SIRT1, and potentially promote the parthenogenetic development of vitrified-warmed mouse MII oocytes.

Activated SIRT1 contributes to DPT-induced glioma cell parthanatos by upregulation of NOX2 and NAT10.

Parthanatos is a type of programmed cell death dependent on hyper-activation of poly (ADP-ribose) polymerase 1 (PARP-1). SIRT1 is a highly conserved nuclear deacetylase and often acts as an inhibitor of parthanatos by deacetylation of PARP1. Our previous study showed that deoxypodophyllotoxin (DPT), a natural compound isolated from the traditional herb Anthriscus sylvestris, triggered glioma cell death via parthanatos. In this study, we investigated the role of SIRT1 in DPT-induced human glioma cell parthanatos. We showed that DPT (450 nmol/L) activated both PARP1 and SIRT1, and induced parthanatos in U87 and U251 glioma cells. Activation of SIRT1 with SRT2183 (10 μmol/L) enhanced, while inhibition of SIRT1 with EX527 (200 μmol/L) or knockdown of SIRT1 attenuated DPT-induced PARP1 activation and glioma cell death. We demonstrated that DPT (450 nmol/L) significantly decreased intracellular NAD+ levels in U87 and U251 cells. Further decrease of NAD+ levels with FK866 (100 μmol/L) aggravated, but supplement of NAD+ (0.5, 2 mmol/L) attenuated DPT-induced PARP1 activation. We found that NAD+ depletion enhanced PARP1 activation via two ways: one was aggravating ROS-dependent DNA DSBs by upregulation of NADPH oxidase 2 (NOX2); the other was reinforcing PARP1 acetylation via increase of N-acetyltransferase 10 (NAT10) expression. We found that SIRT1 activity was improved when being phosphorylated by JNK at Ser27, the activated SIRT1 in reverse aggravated JNK activation via upregulating ROS-related ASK1 signaling, thus forming a positive feedback between JNK and SIRT1. Taken together, SIRT1 activated by JNK contributed to DPT-induced human glioma cell parthanatos via initiation of NAD+ depletion-dependent upregulation of NOX2 and NAT10.

Abstract A014: NFI-family transcriptional factors direct AR cistrome redistribution during prostate cancer tumorigenesis

Abstract Previous studies found AR cistrome reprogramming during transition from normal prostate to PCa, and progression to CRPC. AR binding sites (bs) in PCa were reported to be enriched for FOXA1 and HOXB13, in comparison to normal prostate where these are not enriched. However, mechanisms that may drive FOXA1 and HOXB13 to these sites and initiate this neoplastic transformation remain unclear. To address this, we first asked if there are other transcription factors (TFs) involved in tumor evolution. A motif enrichment analysis using patient data set (GSE130408) showed significant enrichment of NFI and NFI half motifs at AR bs in localized hormone sensitive PCa in comparison to normal prostate (0.07 vs 0.04, p=0.0001; and 0.27 vs 0.21, p=0.0044, respectively). Notably, NFI TFs were shown previously to be associated with AR bs in PCa cells, and to modulate AR-target gene expression and changes in chromatin accessibility. This suggested that NFI TFs may contribute to AR cistrome redistribution that drives PCa development. Next, we generated a CRPC cell model resistant to enzalutamide (ENZ), and found that AR activity in these cells was driven by the ARv7 splice variant. Notably, ATAC-seq identified in these cells a subset of sites with greatly increased chromatin accessibility, and ChIP-seq showed that these ATAC-up sites were associated with ARv7 binding and increased H3K27acetylation (H3K27ac, the mark of active enhancers). Significantly, these sites also were enriched for NFI and NFI half motifs (0.04, p<0.0001 and 0.1, p<0.0001, respectively). Notably, while AR is known as a transcriptional activator, it also has repressive functions. To study AR-direct repression, we examined the redistribution of H3K27ac mark in response to AR-inhibition (ENZ) or stimulation (DHT). We identified subsets of AR-bound regions with increased H3K27ac after ENZ that corresponded to regions with decreased H3K27ac after DHT. These regions were highly enriched for NFI and NFI-half motifs (0.04, p<0.0001 and 0.14, p<0.0001, respectively). As expected, these regions also were associated with DHT-repressed genes including UGT2B15, physiologically involved in steroid hormones metabolism. Notably, depletion of NFI-B and X (the most expressed NFI-family members in PCa) resulted in increased H3K27ac levels at UGT2B15, and impaired the suppressive effect of DHT on UGT2B15 expression. RNA-seq after NFIB and X knockdown then showed 212 up- and 119 downregulated genes, indicating NFI is a modulator of both AR-activated and repressed genes. Ingenuity Pathway Analysis demonstrated contribution of NFIs to androgen signaling and maintaining the activity of Sirtuin (SIRT) pathways. SIRTs possess deacetylase activity, including deacetylation of histones, and inhibition of SIRT1 resulted in dramatically increased UGT2B15 expression. Collectively, these data support a role for NFI TFs in directing the AR cistrome during PCa tumorigenesis, and in modulating its transcriptional activation versus repressive functions, with the latter being mediated by recruitment of SIRT1. Citation Format: Larysa Poluben, Joshua Russo, Olga Voznesensky, Mannan Nouri, Anastasia Stavridi, Xintao Qiu, Matthew Freedman, Henry W. Long, Steven P. Balk. NFI-family transcriptional factors direct AR cistrome redistribution during prostate cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A014.

Medical Prospect of Melatonin in the Intervertebral Disc Degeneration through Inhibiting M1-Type Macrophage Polarization via SIRT1/Notch Signaling Pathway.

The study aims to explore the medical prospect of melatonin (MLT) and the underlying therapeutic mechanism of MLT-mediated macrophage (Mφ) polarization on the function of nucleus pulposus (NP) in intervertebral disc degeneration (IDD). RAW 264.7 Mφs were induced by lipopolysaccharide (LPS) to simulate Mφ polarization and the inflammatory reaction of Mφs with or without MLT were detected. Conditioned medium (CM) collected from these activated Mφs with or without MLT treatment were further used to incubate NP cells. The oxidative stress, inflammation and extracellular matrix (ECM) metabolism in NP cells were determined. Then, the changes in SIRT1/Notch signaling were detected. The agonist (SRT1720) and inhibitor (EX527) of SIRT1 were used to further explore the association among MLT. The interaction between SIRT1 and NICD was detected by immunoprecipitation (IP). Finally, puncture-induced rat IDD models were established and IDD degrees were clarified by X-ray, MRI, H&E staining and immunofluorescence (IF). The results of flow cytometry and inflammation detection indicated that LPS could induce M1-type Mφ polarization with pro-inflammatory properties. MLT significantly inhibited the aforementioned process and inhibited M1-type Mφ polarization, accompanied by the alleviation of inflammation. Compared with those without MLT, the levels of oxidative stress, pro-inflammatory cytokines and ECM catabolism in NP cells exposed to CM with MLT were markedly downregulated in a dose-dependent manner. The inhibition of SIRT1 and the enhancement of Notch were observed in activated Mφs and they can be reversed after MLT treatment. This prediction was further confirmed by using the SRT1720 and EX527 to activate or inhibit the signaling. The interaction between SIRT1 and NICD was verified by IP. In vivo study, the results of MRI, Pfirrmann grade scores and H&E staining demonstrated the degree of disc degeneration was significantly lower in the MLT-treated groups when compared with the IDD control group. The IF data showed M1-type Mφ polarization decreased after MLT treatment. MLT could inhibit M1-type Mφ polarization and ameliorate the NP cell injury caused by inflammation in vitro and vivo, which is of great significance for the remission of IDD. The SIRT1/Notch signaling pathway is a promising target for MLT to mediate Mφ polarization.

Honokiol prevents chronic cerebral hypoperfusion induced astrocyte A1 polarization to alleviate neurotoxicity by targeting SIRT3-STAT3 axis.

Alzheimer's Dementia (AD) and Vascular Dementia (VaD) are two main types of dementias for which no specific treatment is available. Chronic Cerebral Hypoperfusion (CCH) is a pathogenesis underlying AD and VaD that promotes neuroinflammatory responses and oxidative stress. Honokiol (HNK) is a natural compound isolated from magnolia leaves that can easily cross blood brain barrier and has anti-inflammatory and antioxidant effects. In the present study, the effects of HNK on astrocyte polarization and neurological damage in in vivo and in vitro models of chronic cerebral hypoperfusion were explored. We found that HNK was able to inhibit the phosphorylation and nuclear translocation of STAT3, A1 polarization, and reduce conditioned medium's neuronal toxicity of astrocyte under chronic hypoxia induced by cobalt chloride; STAT3 phosphorylation inhibitor C188-9 was able to mimic the above effects of HNK, suggesting that HNK may inhibit chronic hypoxia-induced A1 polarization in astrocytes via STAT3. SIRT3 inhibitor 3-TYP reversed, while Sirt3 overexpression mimicked the inhibitory effects of HNK on oxidative stress, STAT3 phosphorylation and nuclear translocation, A1 polarization and neuronal toxicity of astrocyte under chronic hypoxic conditions. For in vivo research, continuous intraperitoneal injection of HNK (1mg/kg) for 21 days ameliorated the decrease in SIRT3 activity and oxidative stress, inhibited astrocytic STAT3 nuclear translocation and A1 polarization, and prevented neuron and synaptic loss in the hippocampal of CCH rats. Besides, HNK application improved the spatial memory impairment of CCH rats, as assessed with Morris Water Maze. In conclusion, these results suggest that the phytochemical HNK can inhibit astrocyte A1 polarization via regulating SIRT3-STAT3 axis, thus improving CCH-induced neurological damage. These results highlight HNK as novel treatment for dementia with underlying vascular mechanisms.

Dietary Intervention with the Gut Microbial Metabolite Urolithin A Attenuates Lipopolysaccharide-Induced Neuroinflammation and Cognitive Deficits via the Sirt1/acetyl-NF-κB Signaling Pathway.

The gut microbial metabolite Urolithin A (UA) exhibits anti-inflammatory properties in vivo and in vitro. Lipopolysaccharide (LPS), which is present in abundance in the gut, induces chronic neuroinflammation and triggers behavioral abnormalities. However, the neuroprotective effects of UA and the underlying mechanisms implicate in an LPS-elicited neuroinflammation mouse model remain elusive. Daily administration of UA (200mg kg-1 d-1 ; i.g.) for 21 days significantly mitigates cognitive deficits following LPS exposure. UA prevents LPS-induced neural loss and synaptic injury in the hippocampus. UA administration substantially represses LPS-triggered glial cell activation and the production of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). Further study reveals that UA promotes Sirt1 expression and NF-κB p65 deacetylation. Importantly, all the beneficial effects of UA, including biochemical and neuropathological changes and cognitive function, are abrogated by 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527), a specific Sirt1 inhibitor. The findings indicate that UA ameliorates LPS-triggered neural/synaptic damage and cognitive deficits, which potentially contributes to the inhibition of neuroinflammation by promoting the Sirt1/acetyl-NF-κB signaling pathway.

SIRT1 downregulation provokes immune-inflammatory responses in hair follicle outer root sheath cells and may contribute to development of alopecia areata

BackgroundSilent information regulator 1 (SIRT1), a type III histone deacetylase, is involved in various cutaneous and systemic autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. However, little is known about the role of SIRT1 in the development of alopecia areata (AA). ObjectivesThis study investigated whether SIRT1 regulates the hair follicle immune system and is involved in AA pathogenesis. MethodsSIRT1 expression in human scalp tissue was analyzed using immunohistochemical staining, qPCR, and western blotting. The regulatory effect of SIRT1 was evaluated after stimulation with the double-stranded RNA mimic polyinosinic:polycytidylic acid (poly I:C) in hair follicle outer root sheath (ORS) cells and C3H/HeJ mice. ResultsSIRT1 expression was significantly reduced in the AA scalp compared to the normal scalp. SIRT1 inhibition upregulated MHC class I polypeptide-related sequence A and UL16 binding protein 3 in hair follicle ORS cells. SIRT1 inhibition also promoted the production of Th1 cytokines (IFN-γ and TNF-α), IFN-inducible chemokines (CXCL9 and CXCL10), and T cell migration in ORS cells. Conversely, SIRT1 activation suppressed the autoreactive inflammatory responses. The counteractive effect of the immune response by SIRT1 was mediated through the deacetylation of NF-κB and phosphorylation of STAT3. ConclusionSIRT1 downregulation induces immune-inflammatory responses in hair follicle ORS cells and may contribute to AA development.

A novel combination of metformin and resveratrol alleviates hepatic steatosis by activating autophagy through the cAMP/AMPK/SIRT1 signaling pathway.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent liver disorder that is associated with the accumulation of triglycerides (TG) in hepatocytes. Resveratrol (RSV), as a natural product, and metformin have been reported to have potential lipid-lowering effects for the treatment of NAFLD via autophagy, but the combined effects of both have not yet been studied. The current study aimed to investigate the role of autophagy in the lipid-lowering effects of RSV, alone and in combination with metformin, on the hepatic steatosis model of HepG2 cells and elucidate the mechanism of action. Triglyceride measurement and real-time PCR showed that RSV-metformin reduced lipid accumulation and the expression of lipogenic genes in palmitic acid (PA)-induced HepG2 cells. Additionally, the LDH release assay indicated that this combination protected HepG2 cells against PA-induced cell death through autophagy. The western blotting analysis revealed that RSV-metformin induced autophagy by reducing the expression of p62 and increasing LC3-I and LC3-II proteins. This combination also enhanced cAMP, phosphorylated AMP-activated protein kinase (p-AMPK), and Beclin-1 levels in HepG2 cells. Furthermore, SIRT1 inhibitor treatment inhibited autophagy induced by RSV-metformin, which indicated the autophagy induction is SIRT1-dependent. This study demonstrated for the first time that RSV-metformin reduced hepatic steatosis by triggering autophagy via the cAMP/AMPK/SIRT1 signaling pathway.

Effects of Deacetylase Inhibition on the Activation of the Antioxidant Response and Aerobic Metabolism in Cellular Models of Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by a dysfunctional DNA repair and an oxidative stress accumulation due to defective mitochondrial energy metabolism, not counteracted by endogenous antioxidant defenses, which appear down-expressed compared to the control. Since the antioxidant response lack could depend on the hypoacetylation of genes coding for detoxifying enzymes, we treated lymphoblasts and fibroblasts mutated for the FANC-A gene with some histone deacetylase inhibitors (HDACi), namely, valproic acid (VPA), beta-hydroxybutyrate (OHB), and EX527 (a Sirt1 inhibitor), under basal conditions and after hydrogen peroxide addition. The results show that VPA increased catalase and glutathione reductase expression and activity, corrected the metabolic defect, lowered lipid peroxidation, restored the mitochondrial fusion and fission balance, and improved mitomycin survival. In contrast, OHB, despite a slight increase in antioxidant enzyme expressions, exacerbated the metabolic defect, increasing oxidative stress production, probably because it also acts as an oxidative phosphorylation metabolite, while EX527 showed no effect. In conclusion, the data suggest that VPA could be a promising drug to modulate the gene expression in FA cells, confirming that the antioxidant response modulation plays a pivotal in FA pathogenesis as it acts on both oxidative stress levels and the mitochondrial metabolism and dynamics quality.

Echinacoside ameliorates 5-fluorouracil-induced endothelial injury and senescence through SIRT1 activation

Echinacoside (ECH) is a natural bioactive component with antioxidant, anti-inflammatory, anti-apoptosis, and anti-tumor properties. In the current study, we explore the ECH-mediated protective effect and underlying mechanism of 5-fluorouracil (5-FU)-induced endothelial injury and senescence in the Human umbilical vein endothelial cells (HUVECs). In HUVECs, Cell viability, Apoptosis and Senescence assays evaluated 5-fluorouracil-induced endothelial injury and senescence. Protein expressions were assessed using RT-qPCR and Western blotting. Our results showed that 5-FU-induced endothelial injury and endothelial cell senescence could be improved when treated with ECH in HUVECs. ECH treatment potentially attenuated oxidative stress and ROS production in HUVECs. In addition, the effect of ECH on autophagy markedly reduced the percentage of HUVECs with LC3-II dots and suppressed the Beclin-1 and ATG7 mRNA expression but enhanced the p62 mRNA expression. Besides, ECH treatment significantly increased migrated cells and suppressed the adhesion of THP-1 monocytes in HUVECs. Furthermore, ECH treatment activated the SIRT1 pathway, and its related proteins (SIRT1, p-AMPK and eNOS) expression increased. Nicotinamide (NAM), an inhibitor of SIRT1, significantly attenuated the ECH-induced decrease in the apoptotic rate, increased SA-β-gal-positive cells and significantly reversed the ECH-induced reduction of endothelial senescence. Our results demonstrated that ECH employed endothelial injury and senescence in HUVECs via activation of the SIRT1 pathway.

Zigui-Yichong-Fang protects against cyclophosphamide-induced premature ovarian insufficiency via the SIRT1/Foxo3a pathway

Ethnopharmacological relevanceZigui-Yichong-Fang (ZGYCF) is a traditional Chinese medicine prescription for the treatment of infertility and premature ovarian insufficiency (POI). It is clinically used to regulate hormone levels, improve ovarian reserve and increase pregnancy rate. However, the exact mechanism of action is not yet clear. Aims of the studyThis study aimed to explore the potential impact and mechanism of ZGYCF on POI, and provide a scientific basis for its clinical application. Materials and methodsUHPLC‒MS/MS was used to identify the main compounds of ZGYCF. Female 8-week-old C57BL/6N mice were randomized into four group containing the vehicle control (Veh) group, the cyclophosphamide (CTX) model group, the low-dose ZGYCF (CTX-ZG-L) group and the high-dose ZGYCF (CTX-ZG-H) group. A mouse POI model was induced with a single intraperitoneal injection of CTX, and the therapeutic effects of different doses of ZGYCF on POI were evaluated according to the ovarian weight coefficient, serum AMH, serum E2, ovarian histomorphology and follicle counts. After the dose screening experiment, the CTX-ZG-L group was renamed the CTX-ZG group and subjected to follow-up experiments. RNA-seq was used to explore the mechanism of POI and the therapeutic mechanism of ZGYCF on POI in Veh group, CTX group and CTX-ZG group. The mechanism of action of ZGYCF on POI were determined by measuring serum hormone level, histomorphology, follicle counts, protein expression and acetylation modification in groups of Veh, CTX, CTX-ZG and CTX-ZG-Nam (SIRT1 inhibitor). ResultsA total of 37 compounds in ZGYCF were identified. ZGYCF attenuated the morphological changes in ovarian tissue in POI model mice, increased serum AMH and E2 levels, reduced the damage to primordial follicles and other follicles at all stages, and protected ovarian reserve. RNA-seq results suggested that the genes expression of the PI3K signaling and apoptosis signaling pathways was increased in POI mice, while ZGYCF upregulated SIRT1 gene and the expression of estradiol, apoptosis inhibition and other signaling pathway genes. Immunohistochemical staining, TUNEL staining, Western blot analysis and immunoprecipitation results showed that in CTX group, SIRT1 expression and Foxo3a nuclei localization were decreased, while Ac-Foxo3a, p-AKT, p-Foxo3a and apoptotic markers were upregulated. After administration of ZGYCF, these conditions were reversed, however, after treatment with the SIRT1 inhibitor, the results were opposite to those of ZGYCF. ConclusionsAcetylated Foxo3a plays an important role in the occurrence of POI. ZGYCF improves the ovarian reserve of CTX-induced POI mice by activating SIRT1-mediated deacetylation of Foxo3a, and played a role in the treatment of POI. SIRT1 may be a novel target for ZGYCF to ameliorate POI.

Sirt1 protects against intervertebral disc degeneration induced by 1,25-dihydroxyvitamin D insufficiency in mice by inhibiting the NF-κB inflammatory pathway.

It has been demonstrated that vitamin D deficiency is associated with an increased risk of patients developing lumbar disc herniation. However, intervertebral disc degeneration caused by active vitamin D deficiency has not been reported. Thus, the purpose of this study was to e investigate the role and mechanism of 1,25-dihydroxyvitamin D (1,25(OH)2D) insufficiency in promoting intervertebral disc degeneration. The phenotypes of intervertebral discs were compared in wild-type mice and mice with heterozygous deletion of 1α-hydroxylase [1α(OH)ase+/-] at 8 mouths of age using iconography, histology and molecular biology. A mouse model that overexpressed Sirt1 in mesenchymal stem cells on a 1α(OH)ase+/- background (Sirt1Tg/1α(OH)ase+/-) was generated by crossing Prx1-Sirt1 transgenic mice with 1α(OH)ase+/- mice and comparing their intervertebral disc phenotypes with those of Sirt1Tg, 1α(OH)ase+/- and wild-type littermates at 8 months of age. A vitamin D receptor (VDR)-deficient cellular model was generated by knock-down of endogenous VDR using Ad-siVDR transfection into nucleus pulposus cells; VDR-deficient nucleus pulposus cells were then treated with or without resveratrol. The interactions between Sirt1 and acetylated p65, and p65 nuclear localization, were examined using co-immunoprecipitation, Western blots and immunofluorescence staining. VDR-deficient nucleus pulposus cells were also treated with 1,25(OH)2D3, or resveratrol or 1,25(OH)2D3 plus Ex527 (an inhibitor of Sirt1). Effects on Sirt1 expression, cell proliferation, cell senescence, extracellular matrix protein synthesis and degradation, nuclear factor-κB (NF-κB), and expression of inflammatory molecules, were examined, using immunofluorescence staining, Western blots and real-time RT-PCR. 1,25(OH)2D insufficiency accelerated intervertebral disc degeneration by reducing extracellular matrix protein synthesis and enhancing extracellular matrix protein degradation with reduced Sirt1 expression in nucleus pulposus tissues. Overexpression of Sirt1 in MSCs protected against 1,25(OH)2D deficiency-induced intervertebral disc degeneration by decreasing acetylation and phosphorylation of p65 and inhibiting the NF-κB inflammatory pathway. VDR or resveratrol activated Sirt1 to deacetylate p65 and inhibit its nuclear translocation into nucleus pulposus cells. Knockdown of VDR decreased VDR expression and significantly reduced the proliferation and extracellular matrix protein synthesis of nucleus pulposus cells, significantly increased the senescence of nucleus pulposus cells and significantly downregulated Sirt1 expression, and upregulated matrix metallopeptidase 13 (MMP13), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) expression; the ratios of acetylated and phosphorylated p65/p65 in nucleus pulposus cells were also increased. Treatment of nucleus pulposus cells with VDR reduction using 1,25(OH)2D3 or resveratrol partially rescued the degeneration phenotypes, by up-regulating Sirt1 expression and inhibiting NF-κB inflammatory pathway; these effects in nucleus pulposus cells were blocked by inhibition of Sirt1. Results from this study indicate that the 1,25(OH)2D/VDR pathway can prevent the degeneration of nucleus pulposus cells by inhibiting the NF-κB inflammatory pathway mediated by Sirt1.The Translational Potential of This Article: This study provides new insights into the use of 1,25(OH)2D3 to prevent and treat intervertebral disc degeneration caused by vitamin D deficiency.

Procyanidin B2 regulates the Sirt1/Nrf2 signaling pathway to improve random-pattern skin flap survival.

Random-pattern skin flaps have been widely used in the reconstruction of damaged tissues. Ischemia-reperfusion injury occurring in the distal regions of the flap is a common issue, which often leads to flap necrosis and restricts its clinical applications. Procyanidin B2 (PB2), a naturally occurring flavonoid in large quantities in various fruits, has been demonstrated to exhibit several significant pharmacological properties. However, the effect of PB2 on flap viability is not clearly known. Here, using Western blot analysis, immunohistochemistry, and immunofluorescence staining, we observed that PB2 significantly reduced oxidative stress and inflammation and enhanced angiogenesis. Mechanically, we provided evidence for the first time that the beneficial effects of PB2 occur through the activation of the Sirt1/Nrf2 signaling pathway. Moreover, co-administration of PB2 and EX527, a selective inhibitor of Sirt1, resulted in down-regulation of the expression of Sirt1, Nrf2, and downstream antioxidants. In summary, our study showed that PB2 might be a novel therapeutic strategy for improving the survival of random-pattern skin flaps.