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TNF-α, IL-6, and IL-8/CXCL1 cytokine levels

BEAS-2B cells were pretreated for 1 hour with 0–200 nM 7(S)-MaR1 or 7(R)-MaR1, followed by 5% HDE for 24 hours. Cell supernatants were collected and IL-6 and IL-8 enzyme-linked immunosorbent assays (ELISAs) were performed as previously described []. Alternatively, mouse lung slices were pre-treated for 1 hour with 0–200 nM MaR1, followed by 5% HDE treatment, or given 5% HDE treatment followed by 0–200 nM 7(S)-MaR1 1 hr after the HDE treatment was given. At 24 hours following HDE treatment, lung slice supernatants were collected and assayed for murine TNF-α, IL-6, and the murine IL-8 cognate CXCL1 using ELISAs.

MaR1 reduces the release of HDE-induced pro-inflammatory cytokines (IL-6 and IL-8) by airway epithelial cells (BEAS-2B cell line)

Previous studies have shown treatment of BECs with 5% HDE for 24 hours leads to significant increases in IL-6 and IL-8 release [,]. To test the effects of MaR1 pretreatment on cytokine release from BECs in response to HDE, BEAS-2B cells were pretreated with 0, 1, 10, 100, or 200 nM MaR1 (in all studies, the 7[S] form of MaR1 was used, unless otherwise stated). After 1 hr, 5% HDE was added to the cultures and allowed to incubate for 24 hours. In cells receiving MaR1 pretreatment, IL-6 and IL-8 cytokine levels were dose-dependently inhibited compared to cells receiving HDE alone, as measured at 24 hours post-HDE exposure (Figure ). In the absence of HDE, MaR1 alone had no effect on IL-6 or IL-8 levels at any concentration tested. These studies were repeated using the 7(R) isoform of MaR1, achieving similar results (data not shown).

Abstract

Background. Inflammation and fibrosis are the important pathophysiologic processes in diabetic nephropathy (DN). Maresin 1 is a potential anti-inflammatory lipid mediator, which has displayed powerful proresolving activities. Aim. We determine whether maresin 1 has protective effect on mouse glomerular mesangial cells (GMCs) induced by high glucose. Methods. We cultured GMCs stimulated by high glucose and categorized as follows: normal glucose group (5.6 mmol/L), high glucose group (30 mmol/L), mannitol group, maresin 1 intervention group (1, 10, and 100 nmol/L), maresin 1 and normal glucose group, and the N-acetylcysteine (NAC) intervention group (10 μmol/L NAC). After 24 h, the expression of ROS, NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β was detected by western-blot, RT-PCR, and immunofluorescence. After 48 h, the expression of TGF-β1 and FN was detected by RT-PCR and ELISA. Results. Compared with normal glucose group, the expression of ROS, NLRP3, caspase-1, IL-1β, TGF-β1, and FN increased in high glucose group (P < 0.05), but it decreased after the treatment of maresin 1 in different concentrations. On the contrary, the expression of procaspase-1 and pro-IL-1β protein was restrained by high glucose and enhanced by maresin 1 in a dose-dependent manner (P < 0.05). Conclusion. Maresin 1 can inhibit NLRP3 inflammasome, TGF-β1, and FN in GMCs; it may have protective effect on DN by mitigating the inflammation and early fibrosis.

2.1. Cell Culture and Stimulation

Mouse glomerular mesangial cells (GMCs) were purchased from Chinese Academy of Sciences and cultured in low glucose Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) with 10% fetal bovine serum (FBS, BOVOGEN, Australia) at 37°C and 5% CO2. GMCs were used between the 2nd and 5th passages for all experiments. To observe the effect of maresin 1, we divided the cells into 8 groups: ① normal control group (NC group): 5.6 mmol/L glucose, ② osmotic pressure control group (OP group): 5.6 mmol/L glucose + 24.4 mmol/L mannitol, ③ high glucose group (HG group): 30 mmol/L glucose, ④ different concentrations of maresin 1 + high glucose group (M + HG group): the GMCs being treated with different concentration of maresin 1 (1, 10, and 100 nmol/L, Cayman Chemical, USA) for 30 min before being exposed to high glucose (30 mmol/L) [, ], 1 nmol/L maresin 1 + 30 mmol/L glucose (M1 + HG group), 10 nmol/L maresin 1 + 30 mmol/L glucose (M2 + HG group), 100 nmol/L maresin 1 + 30 mmol/L glucose (M3 + HG group), ⑤ maresin 1 + normal control group (M + NC group): the most obvious effect of maresin 1 concentration + 5.6 mmol/L glucose, ⑥ N-acetylcysteine (NAC) intervention group as positive control (10 μmol/L NAC + HG group) [, ]. Each test was independently repeated more than 3 times.

3.1.1. The Expression of NLRP3, Caspase-1, Procaspase-1, IL-1β, and Pro-IL-1β in GMCs Induced by 30 mmol/L Glucose for Each Group

Compared with NC group, the mRNA expression of NLRP3, caspase-1, and IL-1β increased significantly after exposing to 30 mmol/L glucose for 6 h, 12 h, 24 h, and 48 h (P < 0.05), and the expression was positively correlated with time changes (Figures and ). Moreover, in M + HG group for 24 h, the protein and mRNA expression of NLRP3, caspase-1, and IL-1β decreased, but procaspase-1 and pro-IL-1β were enhanced obviously as a concentration-dependent manner of maresin 1 compared with HG group (P < 0.05) (Figures , , , and ). And we used immunofluorescence to detect the level of NLRP3, caspase-1, and IL-1β in each group, and we observed the same change (). There were no apparent differences among NC group, OP group, and M + NC group (P > 0.05) (Figures , , , and ).

3.1.2. The ROS Level of GMCs Which Were Treated with NAC and Different Doses of Maresin 1 for 30 min and Were Exposed to High Glucose (30 mmol/L) for 24 h by Using Spectrophotometer and Fluorescence Microscope

As ROS plays an important role in high glucose-induced damage, we explored ROS level in GMCs using DCFH-DA. In spectrophotometer analysis, we observed that high glucose caused a significant ROS generation in GMCs (P < 0.05) compared with NC group. Notably, ROS generation was significantly inhibited by NAC and maresin 1 (1, 10, and 100 nmol/L) (P < 0.05), and the ROS level decreased in a concentration-dependent manner of maresin 1 (). Meanwhile, we got a similar result by using fluorescence microscopy ().

3.2. Antifibrosis Effect of Maresin 1 on GMCs Induced by High Glucose

Compared with NC group, the expression of TGF-β1 and FN upregulated in HG group for different time (P < 0.05), especially for 48 h (Figures , , and ). However, it downregulated significantly in M + HG group by a negative correlation with concentration of maresin 1 (P < 0.05) (Figures , , and ). It seemed that 10 nmol/L maresin 1 was effective enough for counteracting the expression of TGF-β1 and FN enhanced by high glucose. Therefore, we chose 10 nmol/L as a protective concentration in the following experiments. And then, we detected that the mRNA level of TGF-β1 and the expression of FN decreased significantly in M2 + HG group compared with HG group (P < 0.05), and there was no significant difference between NC group and other groups (Figures , , and ).

4. Discussion

TGF-β1 is a key regulator of EMT in pathological processes of glomerular sclerosis in DN, which participate in the renal fibrosis of DN commonly [, ]. Extracellular high glucose can be used as profibrotic factors in GMCs to promote the synthesis and secretion of TGF-β1 and increase the expression of FN that would prompt deposition of extracellular matrix and destruct the complete basement membrane. Finally, EMT and stable mesenchymal phenotype occurred []. This study in vitro found that high glucose could upregulate the expression of TGF-β1 and FN in GMCs in time dependence, suggesting that the high glucose may promote glomerular sclerosis and renal fibrosis in DN. What is more, maresin 1 could restrain the expression of TGF-β1 and FN induced by high glucose in GMCs in a dose-dependent manner, indicating that maresin 1 may inhibit EMT in GMCs and reduce DN glomerular fibrosis. Therefore, we hypothesized that maresin 1 potentially offers a therapeutic option for better treatment of DN through its anti-inflammatory effect and inhibition of renal fibrosis. However, this experiment is not enough; since the limit experimental time, we failed to investigate the specific mechanism of action, which needs the further basis experiments to prove.