Marein, an active component of the Coreopsis tinctoria Nutt. plant, is known to improve diabetic nephropathy (DN). However, its anti-diabetic functions in DN and potential mechanisms remain unclear. The aim of this study was to elucidate the effects and mechanisms of Marein in diabetic db/db mice with DN, and in high glucose-treated HK-2 cells. In vivo, treating diabetic db/db mice with Marein for 12 consecutive weeks restored diabetes-induced hyperglycemia and dyslipidemia, and ameliorated renal function deterioration, glomerulosclerosis, and renal ectopic lipid deposition. Marein exerted renoprotective effects by directly inhibiting renal tubule sodium glucose transporter 2 (SGLT2) expression, and then activating the AMP-activated protein kinase (AMPK)/acetyl CoA carboxylase (ACC)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) pathway in db/db mice. Meanwhile, Marein ameliorated fibrosis and inflammation by suppressing the pro-inflammatory factors interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and expression of the extracellular matrix proteins, fibronectin (FN) and collagen 1 (COL1) in diabetic mice. In vitro, MDCK monolayer cells were established to explore the characteristics of Marein transmembrane transport. Marein was found to be absorbed across the membrane at a medium level that involved active transport and this was mediated by SGLTs. In HK-2 cells, Marein decreased uptake of the fluorescent glucose analog, 2-NBDG, by 22 % by inhibiting SGLT2 expression. In high glucose-treated HK-2 cells, Marein decreased SGLT2 expression and increased phosphorylated (p)-AMPK/p-ACC to improve high glucose-induced cellular dysfunction. Furthermore, Marein treatment decreased SGLT2 expression in SGLT2-overexpressing HK-2 cells. In addition, molecular docking and dynamics analysis revealed that SGLT2 was a direct target of Marein. Collectively, our results demonstrated that Marein ameliorates DN by inhibiting renal SGLT2 and activating p-AMPK, suggesting Marein can potentially prevent DN by suppressing renal SGLT2 expression directly.

Coreopsis tinctoria Nutt is an ethnomedicine widely used in Xinjiang, China. It is consumed as a herbal tea by local Uyghur people to treat high blood pressure and diarrhea. Our previous study confirmed that the ethyl acetate extract of Coreopsis tinctoria (AC) had a protective effect on diabetic nephropathy (DN) in an in vivo experiment. Here we aim to elucidate the protective mechanism of AC and marein, the main ingredient in Coreopsis tinctoria on renal fibrosis and inflammation in vitro under high glucose (HG) conditions. A HG-induced barrier dysfunction model in rat mesangial cells (HBZY-1) was established. The cells were exposed to AC and marein and/or HG for 24 h. Then, the renal protective effects of AC and marein via transforming growth factor-β1 (TGF-β1)/Smads, AMP-activated kinase protein (AMPK), and nuclear factor kappa beta (NF-κB) signaling were assessed. Both AC and marein suppressed rat mesangial cell hyperplasia and significantly attenuated the expression of HG-disrupted fibrotic and inflammatory proteins in HBZY-1 cells. It was also confirmed that AC and marein remarkably attenuated HG-induced renal inflammation and fibrosis by regulating the AMPK, TGF-β1/Smads, and NF-κB signaling pathways. These results indicated that AC and marein may delay the progression of DN, at least in part, by suppressing HG-induced renal inflammation and fibrosis. Marein may be one of the bioactive compounds in AC.

Faecal microbiota transplantation (FMT) from Kazak individuals with normal glucose tolerance (KNGT) significantly reduces plasma glycolipid levels in type 2 diabetes mellitus db/db mice. However, t...

The study aims to investigate the effects of the alcohol extract of Coreopsis tinctoria Nutt (AC) on diabetic nephropathy (DN) mice. A total of 30 db/db (DN) mice were divided into 3 groups, which were treated with AC (300 mg/kg/day), metformin (180 mg/kg/day), or saline by gavage for 10 weeks. Ten db/m mice treated with saline were used as normal control (NC group). Body weight (BW) and fasting blood glucose (FBG), HbA1c, 24 h urinary albumin excretion (UAE), and renal pathological fibrosis were analyzed. Expression of miR-192, miR-200b, and proteins in the PTEN/PI3K/AKT pathway was analyzed by qPCR or western blot. The DN mice had significantly higher BW, FBG, and 24 h UAE, as well as more severe pathological fibrosis when compared with NC. Treatment of AC could decrease BW, FBG, and 24 h UAE and alleviated kidney damage. Compared with the NC group, expressions of miR-192 and miR-200b were increased, whereas their target proteins (ZEB2 and PTEN) were reduced in the kidneys of DN mice, which further modulated the expression of their downstream proteins PI3K p85α, P-AKT, P-smad3, and COL4 α1; these proteins were increased in the kidneys of DN mice. In contrast, AC treatment reversed the expression changes of these proteins. These findings demonstrate that AC may protect the kidneys of DN mice by decreasing miR-192 and miR-200b, which could further regulate their target gene expression and modulate the activity of the PTEN/PI3K/AKT pathway to reduce the degree of renal fibrosis.

Flavanomarein (FM) is a major natural compound of Coreopsis tinctoria Nutt with protective effects against diabetic nephropathy (DN). In this study, we investigated the effects of FM on epithelial-mesenchymal transition (EMT) in high glucose (HG)-stimulated human proximal tubular epithelial cells (HK-2) and the underlying mechanisms, including both direct targets and downstream signal-related proteins. The influence of FM on EMT marker proteins was evaluated via western blot. Potential target proteins of FM were searched using Discovery Studio 2017 R2. Gene Ontology (GO) analysis was conducted to enrich the proteins within the protein-protein interaction (PPI) network for biological processes. Specific binding of FM to target proteins was examined via molecular dynamics and surface plasmon resonance analyses (SPR). FM promoted the proliferation of HK-2 cells stimulated with HG and inhibited EMT through the Syk/TGF-β1/Smad signaling pathway. Spleen tyrosine kinase (Syk) was predicted to be the most likely directly interacting protein with FM. Combined therapy with a Syk inhibitor and FM presents significant potential as an effective novel therapeutic strategy for DN.

Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) can protect diabetic kidneys, but the mechanisms are unclear. This work is to investigate the potential mechanisms of C. tinctoria Nutt. in the treatment of diabetic nephropathy based on network pharmacology analysis of its active ingredients. Twelve small molecular compounds of C. tinctoria Nutt. and targets related to diabetic nephropathy were docked by Discovery Studio 3.0. DAVID database was used for GO enrichment and KEGG pathway analysis. Cytoscape 3.6.1 was used to construct active ingredient-target network. Cell viability was detected with MTT. Glucose consumption was analyzed with glucose oxidase method. Protein expression was measured with Western blot and immunofluorescence. Electron microscopy observed autophagosomes. The core active ingredients of C. tinctoria Nutt. included heriguard, flavanomarein, maritimein, and marein. Twenty-one core targets of the 43 potential targets were PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK, and ABL2. These 21 core targets were significantly enriched in 50 signaling pathways. Thirty- four signaling pathways were closely related to diabetic nephropathy, of which the top pathways were PI3K/AKT, insulin, and mTOR, and insulin resistance. The enriched GO terms included biological processes of protein phosphorylation, and the positive regulation of PI3K signaling and cytokine secretion; cellular components of cytosol, extracellular region, and extracellular space; and molecular function of protein kinase activity, ATP binding, and non-membrane spanning protein tyrosine kinase activity. In vitro experiments found that marein increased the expression of phosphorylated AKT/AKT in human renal glomerular endothelial cells of an insulin resistance model induced by high glucose, as well as increased and decreased, respectively, the levels of the microtubule-associated proteins, LC3 and P62. C. tinctoria Nutt. has many active ingredients, with main ingredients of heriguard, flavanomarein, maritimein, and marein, and may exert anti-diabetic nephropathy effect through various signaling pathways and targets.