Chlorogenic Acid Alleviates High Glucose-induced HK-2 Cell Oxidative Damage through Activation of KEAP1/NRF2/ARE Signaling Pathway.

OBJECTIVE: To investigate the alleviating effect of chlorogenic acid (CGA) on oxidative damage in high glucose (HG)-induced HK-2 cells and to explore its potential mechanisms. METHODS: We cultured the human proximal tubular cell line HK-2 and divided them into the control group and different concentrations of CGA groups (0, 5, 10, 25, 50, 100, 200 µM). The trypan blue dye test was used to detect CGA's potential cytotoxicity on HK-2 cells. Then, we treated HK-2 with HG and CGA; the Cell Counting Kit-8 (CCK-8) method was used to detect the cell viability of HK-2 cells in each group. Flow cytometry was employed to measure the apoptosis rate of cells. Western blot was performed to detect the expression of apoptosis proteins B-cell lymphoma-2 (BCL-2), BCL-2-associated X protein (BAX), cysteinyl aspartate specific proteinase (CASPASE)-9, and CASPASE-3. In addition, enzymatic activities, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and lipid peroxide (LPO), were measured with the corresponding detection kits. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay and flow cytometry were performed to detect reactive oxygen species (ROS) production. Western blot analysis and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were conducted to evaluate protein and mRNA expressions of the Kelch-like ECH-associated protein-1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Antioxidant Response Elements (ARE) signaling pathway. RESULTS: The outcomes showed that, in a dose-dependent way, CGA dramatically increased the vitality of HK-2 induced by HG. Furthermore, CGA significantly reduced the HG-stimulated HK-2 cell apoptosis, which may be linked to the promotion of BCL-2 and the suppression of BAX, cleaved-CASPASE-3, and cleaved-CASPASE-9 expression. In HK-2 cells, CGA reduced the formation of ROS generated by HG levels and markedly boosted the activity of the antioxidant enzymes SOD, GSH-Px, and CAT. Furthermore, compared with the HG group, CGA significantly raised NRF2 nuclear expression and downregulated NRF2 cytosolic expression and increased the mRNA expression of NRF2 and its target genes, heme oxygenase-1 (HO-1), KEAP1, and NAD(P)H dehydrogenase quinone 1 (NQO1). CONCLUSION: These results show that CGA might be useful in managing oxidative damage in HG-induced HK-2 cells.

Dye-based dual-emission Eu-MOF synthesized by Post-modification for the sensitive ratio fluorescence visualization sensing of ClO.

As we all know, excessive hypochlorite will be transformed into highly toxic substances, while insufficient hypochlorite can not completely kill bacteria and viruses in water. Therefore, it is desirable to develop a new analytical method to detect ClO- in environmental water. Here, a novel and simple fluorescence sensor was constructed for monitoring ClO- by an effective strategy. An Acriflavine@lanthanide metal-organic framework (Acr@Eu(BTEC)) was designed by covalently integrating amino-rich dye (Acr) and carboxyl-rich Eu(BTEC) via post-synthesis method. The created fluorescence sensor has two emission centers originating from Acr and Eu(BTEC), respectively. In the presence of ClO-, the strong green fluorescence derived from Acr was significantly quenched, while the invariant red emission from Eu3+ acted as the reference signal. Thus, Acr@Eu(BTEC) with two emissions was developed as a ratiometric fluorescence sensor for highly sensitive and selective detection of ClO-. The limit of detection (LOD) was as low as 10.75 nM. Moreover, visual detection of ClO- by the naked eyes is feasible with obvious fluorescent color changes from green to orange and then red. This method shows excellent performance in practical application, which suggests that it has great potential in water quality monitoring.

SIRT6 inhibits cholesterol crystal-induced vascular endothelial dysfunction via Nrf2 activation.

Sirtuin 6 (SIRT6), a nicotinamide adenine dinucleotide-dependent deacetylase, participates in various age-related disorders, such as dyslipidemia and cardiovascular diseases. Recent studies have revealed that minute cholesterol crystals (CCs), which are generated after excess free cholesterol accumulation, form not only in mature atherosclerotic plaques but also extremely early in atherosclerosis. Since endothelial dysfunction is an early feature of atherogenesis, this study was designed to investigate the role of SIRT6 in minute CC-induced endothelial dysfunction and the related mechanism. We found that minute CCs could be endocytosed by endothelial cells (ECs), which then decreased nitric oxide (NO) levels and endothelial nitric oxide synthase (eNOS) activity and expression, upregulated the expression of adhesion molecules and enhanced monocyte adhesion to ECs. In addition, minute CCs significantly suppressed SIRT6 expression in ECs. Moreover, the overexpression of SIRT6 could mitigate minute CC-induced endothelial dysfunction. In addition, the expression of Nuclear factor erythroid2-related factor2 (Nrf2) was suppressed after minute CC treatment, whereas SIRT6 overexpression reversed this decrease in Nrf2 expression. More importantly, Nrf2 activation also notably attenuated minute CC-induced endothelial dysfunction. In vivo experiments further indicated that endothelium-specific SIRT6 depletion impaired vascular endothelial function and suppressed Nrf2 expression in hyperlipidemic mice. Taken together, these results indicate that SIRT6 rescues minute CC-induced endothelial dysfunction partly via Nrf2 activation.

Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping.

Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen-deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrometry (CXMS), and disulfide trapping to map interactions within protein complexes. HDXMS measures changes in solvent accessibility and hydrogen bonding upon complex formation; a decrease in HDX rate could account for newly formed intermolecular or intramolecular interactions. To distinguish between inter- and intramolecular interactions, we use a CXMS method to determine the position of direct interface regions by trapping intermolecular residues in close proximity to various cross-linkers (e.g., disuccinimidyl adipate (DSA)) of different lengths and reactive groups. Both MS-based experiments are performed on high-resolution mass spectrometers (e.g., an Orbitrap Elite hybrid mass spectrometer). The physiological relevance of the interactions identified through HDXMS and CXMS is investigated by transiently co-expressing cysteine mutant pairs, one mutant on each protein at the discovered interfaces, in an appropriate cell line, such as HEK293. Disulfide-trapped protein complexes are formed within cells spontaneously or are facilitated by addition of oxidation reagents such as H2O2 or diamide. Western blotting analysis, in the presence and absence of reducing reagents, is used to determine whether the disulfide bonds are formed in the proposed complex interface in physiologically relevant milieus. The procedure described here requires 1-2 months. We demonstrate this approach using the ß2-adrenergic receptor-ß-arrestin1 complex as the model system.

Protective effect of dihydromyricetin on LPS-induced acute lung injury.

Dihydromyricetin (DHM), a bioactive flavonoid component isolated from Ampelopsis grossedentata, is known to have anti-inflammatory effect, but the effect of DHM on acute lung injury (ALI) is largely unknown. Here, we investigated the effect of DHM on ALI and the underlying mechanism by bioinformatic analyses and animal experiments. We found that pretreatment with DHM ameliorated lung pathological changes and suppressed the inflammation response in lung tissues after LPS challenge. The potential targets of DHM were predicted by DDI-CPI and DRAR-CPI tools and analyzed using the STRING server to predict the functionally related signaling pathways, such as MAPK signaling. Molecular docking calculations indicated that DHM could be embedded tightly into the binding pocket of ERK, JNK, and p38. Furthermore, the activation of MAPK signaling induced by LPS was inhibited by DHM. In conclusion, these findings suggest that DHM may exert its protective effect on ALI by inhibiting MAPK signaling. The present study supports a potential clinical application for DHM in treating ALI and provides a novel design that combines in silico methods with in vivo experiments for drug research.

Hydroxytyrosol regulates the autophagy of vascular adventitial fibroblasts through the SIRT1-mediated signaling pathway.

Hydroxytyrosol (HT), a phenolic compound in olive oil, exerts an anti-inflammatory effect in cardiovascular diseases. Recent studies found that autophagy was a therapeutic target of diseases. However, the effect of HT on autophagy in vascular adventitial fibroblasts (VAFs) remains unknown. Thus, in this study, we aimed to determine the effect of HT on cell autophagy and related signaling pathway and whether HT regulates the inflammatory response through autophagy in VAFs. Our results showed that HT promoted cell autophagy by increasing the conversion of LC3 and Beclin1 expression and the autophagic flux in VAFs stimulated with tumor necrosis factor-α (TNF-α). HT also upregulated the expression of the deacetylase sirtuin 1 (SIRT1) protein and mRNA compared with the TNF-α group. The molecular docking studies showed the good compatibility between HT and SIRT1, indicating that HT might act through SIRT1. Further study found that HT regulated autophagy through SIRT1-mediated Akt/mTOR suppression in VAFs. In addition, HT inhibited TNF-α-induced inflammatory response in VAFs through SIRT1. Furthermore, the study showed that HT inhibited the inflammatory response of VAFs through autophagy. These findings indicate that HT regulates the autophagy of VAFs through SIRT1-mediated Akt/mTOR suppression and then inhibits the inflammatory response of VAFs.

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.

SIRT6 inhibits TNF-α-induced inflammation of vascular adventitial fibroblasts through ROS and Akt signaling pathway.

SIRT6, with both deacetylase and ADP-ribosyltransferase activities, is predominantly expressed in the nucleus. It has been revealed that SIRT6 regulates various biological functions including metabolism, aging and stress resistance. This study aims to investigate the role of SIRT6 in vascular inflammation and it molecular mechanism. We found that tumor necrosis factor-α (TNF-α) did not alter the localization of SIRT6 in vascular adventitial fibroblasts (VAFs), vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). The expression of SIRT1, SIRT6 was decreased in TNF-α-treated VAFs. In contrast, TNF-α significantly increased the expression of monocyte chemotactic protein 1 (MCP-1) and interleukin (IL) -6. Knockdown of SIRT1 and SIRT6 by siRNA significantly enhanced TNF-α-induced expression of MCP-1 and IL-6, respectively. Overexpression of SIRT1 and SIRT6 inhibited TNF-α-induced expression of MCP-1 and IL-6 in VAFs. Moreover, we also found SIRT1 positively regulated the expression of SIRT6 in VAFs. In addition, knockdown of SIRT1 and SIRT6 respectively augmented TNF-α-induced generation of reactive oxygen species (ROS) and phosphorylation of protein kinase B (Akt). ROS scavenger N-acetyl-L-cysteine (NAC) and Akt inhibitor MK2206 reduced TNF-α-induced mRNA expression of MCP-1 and IL-6 in VAFs. In vivo studies indicated that the expression of SIRT1, SIRT6 was decreased and the expression of MCP-1, IL-6 and IL-1ß was increased in carotid collar-induced vascular inflammation. Taken together, these findings indicate that SIRT1 and SIRT6 inhibit TNF-α-induced inflammation in VAFs by ROS and Akt pathway.