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.

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.

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.

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.