Yanhao He

SIRT1 inhibits inflammatory response partly through regulation of NLRP3 inflammasome in vascular endothelial cells

Emerging evidence has indicated that vascular endothelial cells (ECs) not only form the barrier between blood and the vessel wall but also serve as conditional innate immune cells. Our previous study found that SIRT1, a class III histone deacetylase, inhibits the inflammatory response in ECs. Recent studies revealed that SIRT1 also participates in the modulation of immune responses. Although the NLRP3 inflammasome is known to be a crucial component of the innate immune system, there is no direct evidence demonstrating the anti-inflammatory effect of SIRT1 on ECs through the NLRP3 inflammasome. In this study, we observed that lipopolysaccharide (LPS) and adenosine triphosphate (ATP) triggered the activation of NLRP3 inflammasome in human umbilical vein ECs (HUVECs). Moreover, SIRT1 expression was reduced in HUVECs stimulated with LPS and ATP. SIRT1 activator inhibited the expression of monocyte chemotactic protein-1 (MCP-1) and C-reactive protein (CRP), whereas SIRT1 knockdown resulted in significant increases in MCP-1 and CRP levels in HUVECs stimulated with LPS and ATP. Importantly, the lack of SIRT1 enhanced NLRP3 inflammasome activation and subsequent caspase-1 cleavage. On the other hand, NLRP3 siRNA blocked the activation of the NLRP3 inflammasome in HUVECs stimulated with LPS plus ATP. Further study revealed that NLRP3 inflammasome blockade significantly reduced MCP-1 and CRP production in HUVECs. In vivo studies indicated that implantation of the periarterial carotid collar inhibited arterial SIRT1 expression in rabbits. Meanwhile, treatment with a SIRT1 activator decreased the expression levels of MCP-1 and CRP in collared arteries and the interleukin (IL)-1β level in serum. Taken together, these findings indicate that NLRP3 inflammasome activation promoted endothelial inflammation and that SIRT1 inhibits the inflammatory response partly through regulation of the NLRP3 inflammasome in ECs.

Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1.

Recent studies demonstrated that the ligand-activated transcription factor peroxisome proliferator-activated receptorα (PPARα) acts in association with histone deacetylase sirtuin 1 (SIRT1) in the regulation of metabolism and inflammation involved in cardiovascular diseases. PPARα activation also participates in the modulation of cell apoptosis. Our previous study found that SIRT1 inhibits the apoptosis of vascular adventitial fibroblasts (VAFs). However, whether the role of PPARα in apoptosis of VAFs is mediated by SIRT1 remains unknown. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on cell apoptosis and SIRT1 expression and related mechanisms in ApoE(-/-) mice and VAFs in vitro. We found that fenofibrate inhibited cell apoptosis in vascular adventitia and up-regulated SIRT1 expression in aorta of ApoE(-/-) mice. Moreover, SIRT1 activator resveratrol (RSV) further enhanced these effects of fenofibrate. In vitro study showed that activation of PPARα by fenofibrate inhibited TNF-α-induced cell apoptosis and cell cycle arrest in VAFs. Meanwhile, fenofibrate up-regulated SIRT1 expression and inhibited SIRT1 translocation from nucleus to cytoplasm in VAFs stimulated with TNF-α. Moreover, the effects of fenofibrate on cell apoptosis and SIRT1 expression in VAFs were reversed by PPARα antagonist GW6471. Importantly, treatment of VAFs with SIRT1 siRNA or pcDNA3.1(+)-SIRT1 showed that the inhibitory effect of fenofibrate on cell apoptosis in VAFs through SIRT1. On the other hand, knockdown of FoxO1 decreased cell apoptosis of VAFs compared with fenofibrate group. Overexpression of FoxO1 increased cell apoptosis of VAFs compared with fenofibrate group. Further study found that fenofibrate decreased the expression of acetylated-FoxO1 in TNF-α-stimulated VAFs, which was abolished by SIRT1 knockdown. Taken together, these findings indicate that activation of PPARα by fenofibrate inhibits cell apoptosis in VAFs partly through the SIRT1-mediated deacetylation of FoxO1.

Cordycepin alleviates airway hyperreactivity in a murine model of asthma by attenuating the inflammatory process

Cordycepin (Cor), which is a naturally occurring nucleoside derivative isolated from Cordyceps militaris, has been shown to exert excellent antiinflammatory activity in a murine model of acute lung injury. Thus, this study aimed to evaluate the antiasthmatic activity of Cor (10, 20, and 40 mg/kg) and to investigate the possible underlying molecular mechanisms. We found that Cor attenuated airway hyperresponsiveness, mucus hypersecretion, and ovalbumin (Ova)-specific immunoglobulin (Ig) E, and alleviated lung inflammation with decreased eosinophils and macrophages in the bronchoalveolar lavage (BAL) fluid. Notably, Cor reduced the upregulation of eotaxin, intercellular cell adhesion molecule-1 (ICAM-1), IL-4, IL-5, and IL-13 in the BAL fluid. Furthermore, Cor markedly blocked p38-MAPK and nuclear factor-kappaB (NF-κB) signalling pathway activation in the Ova-driven asthmatic mice. In conclusion, this study demonstrated that some of the antiasthmatic benefits of Cor attributable to diets and/or tonics may result from reductions in inflammatory processes and that these antiasthmatic properties involve the inhibition of Th2-type responses through the suppression of the p38-MAPK and NF-κB signalling pathways.

Protective effects of hydroxysafflor yellow A (HSYA) on alcohol-induced liver injury in rats

Hydroxysafflor yellow A (HSYA), the main active natural constituent extracted from Carthamus tinctorius L., has been widely used for the treatment of cerebrovascular and cardiovascular diseases. The aim of this study is to explore the effect of HSYA on alcohol-induced liver injury and the underlying mechanism. Male Sprague-Dawley rats were used to establish the liver injury model induced by alcohol. HSYA treatment ameliorated serum biochemical indicators by reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronan (HA), laminin (LN), and type III precollagen (III-C) in rats. HSYA efficiently increased the activity and messenger RNA (mRNA) of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in rat liver tissue compared with those of model group, which was obviously reduced by alcohol. HSYA also apparently decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in rat liver tissue compared with those of model group, which was obviously enhanced by alcohol. Histological studies demonstrated that HSYA substantially reduced the number of macro- and micro-vesicular steatosis, suppressed hepatic fibrogenesis and shrunk ballooning degeneration areas, ameliorated the severity of liver damage induced by long-term drinking, and finally improved the liver architecture. In addition, immunohistochemistry study indicated that the activation of transforming growth factor β1 (TGF-β1) stimulated by alcohol in rat liver tissue was significantly blocked by HSYA. Collectively, these data demonstrated that HSYA can effectively protect the liver of rats from long-term alcohol injury, which relates with the enhanced antioxidant capacity of liver tissues and inhibition of TGF-β1 expression.

Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells

NLRP3 inflammasome not only functions as a critical effector in innate immunity, but also triggers the production of proinflammatory cytokines involved in inflammation-associated diseases. Sirtuin 1 (SIRT1) plays an important role in the regulation of cellular inflammation. However, whether the activation of NLRP3 inflammasome is regulated by SIRT1 remains unknown. In this study, we investigated the regulatory effect of SIRT1 on NLRP3 inflammasome and the underlying mechanisms. We found that lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced the activation of NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs). Activation of SIRT1 inhibited NLRP3 inflammasome activation and subsequent caspase-1 cleavage as well as interleukin (IL)-1β secretion, whereas SIRT1 knockdown obviously enhanced the activation of NLRP3 inflammasome in HUVECs. Importantly, gene silencing of SIRT1 abrogated the inhibitory effect of SIRT1 activator on NLRP3 inflammasome formation and IL-1β production in HUVECs stimulated with LPS plus ATP. Further study indicated that cluster of differentiation 40 (CD40) may be involved in the regulation of NLRP3 inflammasome by SIRT1. In vivo studies indicated that implantation of the periarterial carotid collar increased the arterial expression levels of CD40 and CD40 Ligand (CD40L), but inhibited arterial SIRT1 expression in the rabbits. Moreover, treatment with SIRT1 activator decreased CD40 and CD40L levels in collared arteries. Meanwhile, serum IL-1β level, the marker of inflammasome activation, was also inhibited by SIRT1 activation. Taken together, these findings revealed a novel regulatory mechanism of NLRP3 inflammasome by SIRT1, which may be related to suppression of CD40.

Construction and characterization of a pure protein hydrogel for drug delivery application

Injectable hydrogels have a variety of applications, including regenerative medicine, tissue engineering and controlled drug delivery. In this paper, we reported on a pure protein hydrogel based on tetrameric recombinant proteins for the potential drug delivery application. This protein hydrogel was formed instantly by simply mixing two recombinant proteins (ULD-TIP1 and ULD-GGGWRESAI) through the specific protein-peptide interaction. The protein hydrogel was characterized by rheology and scanning electron microscopy (SEM). In vitro cytotoxicity test indicated that the developed protein hydrogel had no apparent cytotoxicity against L-929 cells and HCEC cells after 48h incubation. The formed protein hydrogels was gradually degraded after incubation in phosphate buffered solution (PBS, pH=7.4) for a period of 144h study, as indicated by in vitro degradation test. Encapsulation of model drug (sodium diclofenac; DIC) were achieved by simple mixing of drugs with hydrogelator and the entrapped drugs was almost completely released from hydrogels within 24h via a diffusion manner. As a conclusion, the simple and mild preparation procedure and good biocompatibility of protein hydrogel would render its good promising candidate for drug delivery applications.

Nicotinic acid inhibits vascular inflammation via the SIRT1-dependent signaling pathway.

Nicotinic acid (NA) has recently been shown to inhibit inflammatory response in cardiovascular disease. Sirtuin1 (SIRT1), a NAD(+)-dependent class III histone deacetylase, participates in the regulation of cellular inflammation. We hypothesized that dietary supplementation of NA could attenuate vascular inflammation via modulation of SIRT1 pathway. New Zealand White rabbits received chow or chow supplemented with 0.6% (wt/wt) NA for 2 weeks. Acute vascular inflammation was induced in the animals by placing a non-occlusive silastic collar around the left common carotid artery. At 24 h after collar implantation, the collar-induced production of C-reactive protein and monocyte chemotactic protein-1 was significantly suppressed in the NA-supplemented animals. Meanwhile, NA also decreased the expression of cluster of differentiation 40 (CD40) and CD40 ligand, but up-regulated SIRT1 expression, both in rabbits and in lipopolysaccharide-stimulated endothelial cells. Moreover, knockdown of SIRT1 reversed the inhibitory effect of NA on CD40 expression. Further study revealed that NA also decreased the expression of CD40 partly through mammalian target of rapamycin. These results indicate that NA protects against vascular inflammation via the SIRT1/CD40-dependent signaling pathway.

SIRT1 Regulates the Inflammatory Response of Vascular Adventitial Fibroblasts through Autophagy and Related Signaling Pathway

Background/Aims: Autophagy is a lysosomal degradation pathway that is essential for cellular survival, differentiation, and homeostasis. Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, plays a pivotal role in modulation of autophagy. Recent studies found that autophagy was involved in the regulation of inflammatory response. In this study, we aimed to determine the effect of SIRT1 on autophagy and inflammation, and whether autophagy can regulate the inflammatory response in vascular adventitial fibroblasts (VAFs). Methods: Cell autophagy was evaluated by fluorescence microscope and transmission electron microscopy. The expression of protein and mRNA were determined by Western blot analysis and real time-PCR. The production of cytokine was detected by ELISA. Results: TNF-α induced autophagy and increased SIRT1 expression in VAFs. SIRT1 activator resveratrol enhanced TNF-α-induced VAF autophagy. In contrast, SIRT1 knockdown attenuated VAF autophagy. Both the Akt inhibitor MK2206 and mTOR inhibitor rapamycin further increased TNF-α-induced VAF autophagy. Furthermore, SIRT1 knockdown increased Akt phosphorylation and inhibited the autophagy in VAFs. However, MK2206 attenuated the effect of SIRT1 knockdown on VAF autophagy. In addition, ingenuity pathway analysis showed that there is a relationship between cell autophagy and inflammation. We found that SIRT1 knockdown increased the expression of NLRP3 and interleukin (IL)-6 and promoted the production of IL-1β in VAFs. Further study showed that autophagy activation decreased the expression of NLRP3 and IL-6 and inhibited the production of IL-1β, whereas autophagy inhibition increased the inflammatory response of VAFs. More importantly, our study showed that autophagy was involved in the degradation of NLRP3 through the autophagy-lysosome pathway. Conclusion: SIRT1 not only regulates VAF autophagy through the Akt/mTOR signaling pathway but also suppresses the inflammatory response of VAFs through autophagy.

Divalproex sodium enhances the anti-leukemic effects of imatinib in chronic myeloid leukemia cells partly through SIRT1

Imatinib (IM) represents a breakthrough in the treatment of chronic myeloid leukemia (CML) by inhibiting the activity of Bcr-Abl tyrosine kinase. However, many patients exhibit resistance to IM in the clinic. Recent studies have indicated that sirtuin 1 (SIRT1), a class III histone deacetylase (HDAC), plays an important role in leukemogenesis. In addition, some HDAC inhibitors are being tested to determine their anti-cancer activities in clinical trials. Divalproex sodium (DVPX), a first-line treatment for epilepsy, is also a HDAC inhibitor. However, it is unclear whether the anti-leukemic effects of IM in combination with DVPX on CML cells are related to SIRT1. The aim of this study was to investigate the effects of IM in combination with DVPX on cell viability, apoptosis, and cell cycle arrest in CML cells and to explore the underlying mechanisms. It was found that DVPX enhanced IM-induced cell growth inhibition, apoptosis and cell cycle arrest in K562-S and K562-G cells. Surprisingly, the level of p-Bcr-Abl was similar in K562-S and K562-G cells. Moreover, IM combined with DVPX had no effects on the phosphorylation of Bcr-Abl and its downstream target STAT5. Further study revealed that SIRT1 expression was higher in K562-G cells compared with K562-S cells. DVPX enhanced the inhibitory effect of IM on SIRT1 expression in K562-S and K562-G cells. Furthermore, knockdown of SIRT1 promoted apoptosis of K562-G cells treated with IM and DVPX. These results indicate that DVPX may increase the sensitivity of CML cells to IM and reverse IM resistance by regulating SIRT1 expression.

SIRT1 inhibition promotes atherosclerosis through impaired autophagy

SIRT1, a highly conserved NAD+-dependent protein deacetylase, plays a pivotal role in the pathogenesis and therapy of atherosclerosis (AS). The aim of this study is to investigate the potential effects of SIRT1 on AS in ApoE-/- mice and the underlying mechanisms of autophagy in an ox-LDL-stimulated human monocyte cell line, THP-1. In vivo, the accelerated atherosclerotic progression of mice was established by carotid collar placement; then, mice were treated for 4 weeks with a SIRT1-specific inhibitor, EX-527. The atherosclerotic lesion size of EX-527-treated mice was greatly increased compared to that of the mice in the control group. Immunostaining protocols confirmed that the inhibition of SIRT1 during plaque initiation and progression enhanced the extent of intraplaque macrophage infiltration and impaired the autophagy process. In vitro cultured THP-1 macrophages exposed to ox-LDL were utilized to study the link between the SIRT1 function, autophagy flux, pro-inflammatory cytokine secretion, and foam cell formation using different methods. Our data showed that ox-LDL markedly suppressed SIRT1 protein expression and the autophagy level, while it elevated the MCP-1 production and lipid uptake. Additionally, the application of the SIRT1 inhibitor EX-527 or SIRT1 siRNA further attenuated ox-LDL-induced autophagy inhibition. In conclusion, our results show that the inhibition of SIRT1 promoted atherosclerotic plaque development in ApoE-/- mice by increasing the MCP-1 expression and macrophage accumulation. In particular, we demonstrate that blocking SIRT1 can exacerbate the acetylation of key autophagy machinery, the Atg5 protein, which further regulates the THP-1 macrophage-derived foam cell formation that is triggered by ox-LDL.SIRT1, a highly conserved NAD+-dependent protein deacetylase, plays a pivotal role in the pathogenesis and therapy of atherosclerosis (AS). The aim of this study is to investigate the potential effects of SIRT1 on AS in ApoE–/– mice and the underlying mechanisms of autophagy in an ox-LDL-stimulated human monocyte cell line, THP-1. In vivo, the accelerated atherosclerotic progression of mice was established by carotid collar placement; then, mice were treated for 4 weeks with a SIRT1-specific inhibitor, EX-527. The atherosclerotic lesion size of EX-527-treated mice was greatly increased compared to that of the mice in the control group. Immunostaining protocols confirmed that the inhibition of SIRT1 during plaque initiation and progression enhanced the extent of intraplaque macrophage infiltration and impaired the autophagy process. In vitro cultured THP-1 macrophages exposed to ox-LDL were utilized to study the link between the SIRT1 function, autophagy flux, pro-inflammatory cytokine secretion, and foam cell formation using different methods. Our data showed that ox-LDL markedly suppressed SIRT1 protein expression and the autophagy level, while it elevated the MCP-1 production and lipid uptake. Additionally, the application of the SIRT1 inhibitor EX-527 or SIRT1 siRNA further attenuated ox-LDL-induced autophagy inhibition. In conclusion, our results show that the inhibition of SIRT1 promoted atherosclerotic plaque development in ApoE–/– mice by increasing the MCP-1 expression and macrophage accumulation. In particular, we demonstrate that blocking SIRT1 can exacerbate the acetylation of key autophagy machinery, the Atg5 protein, which further regulates the THP-1 macrophage-derived foam cell formation that is triggered by ox-LDL.