Maresin 1 Mitigates High Glucose-Induced Mouse Glomerular Mesangial Cell Injury by Inhibiting Inflammation and Fibrosis

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.

Figure 1

The mRNA expression of NLRP3, caspase-1, and IL-1β in mesangial cells treated with 30 mmol/L glucose for different time. (a) RT-PCR strip chart for different time. NLRP3, caspase-1, and IL-1β mRNA increased significantly after 6 h, 12 h, 24 h, and 48 h of exposure to 30 mmol/L glucose. (b) The corresponding relative gray value statistics graph of the mRNA level. ^∗P < 0.05 versus NC group.

Figure 2

The mRNA and protein level of NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β in each group. (a) RT-PCR detected the expression of NLRP3, caspase-1, and IL-1β mRNA after intervened by high glucose and maresin 1 for 24 h in GMCs, and the mRNA expression of NLRP3, caspase-1, and IL-1β increased in HG group significantly, but compared with HG group, it decreased obviously in M + HG group as a concentration-dependent manner of maresin 1. (b) The corresponding relative gray value statistics graph of the mRNA level. ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group. (c) Western-blot detected the expression NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β protein after intervened by high glucose and maresin 1 for 24 h in GMCs, and the protein expression of NLRP3, caspase-1, and IL-1β increased and procaspase-1 and pro-IL-1β decreased in HG group significantly, but compared with HG group, the protein expression of NLRP3, caspase-1, and IL-1β decreased and procaspase-1 and pro-IL-1β increased obviously in M + HG group as a concentration-dependent manner of maresin 1. (d) The corresponding relative gray value statistics graph of the protein level. ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group.

Figure 3

The level of NLRP3, caspase-1, and IL-1β in each group detected by immunofluorescence. The expression of NLRP3, caspase-1, and IL-1β increased in HG group significantly, but it decreased obviously in M3 + HG group, and there was no difference between NC group and NC + M3 group.

Figure 4

The level of NLRP3, caspase-1, P-caspase-1, IL-1β, and P-IL-1β in each group. (a) RT-PCR detected the expression NLRP3, caspase-1, and IL-1β mRNA after intervened by high glucose and 100 nmol/L maresin 1 in GMCs, and in HG group, the mRNA expression of NLRP3, caspase-1, and IL-1β was higher than that in NC group, but for M3 + HG group, the expression reduced visibly compared to the HG group. (b) The corresponding relative gray value statistics graph of the mRNA level. ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group. (c) Western-blot detected the expression of NLRP3, caspase-1, P-caspase-1, IL-1β, and P-IL-1β protein after intervened by high glucose and 100 nmol/L maresin 1 in GMCs, and the protein expression of NLRP3, caspase-1, and IL-1β was higher than that in NC group, but for M3 + HG group, the expression reduced visibly compared to the HG group. The expression of P-caspase-1 and P-IL-1β was just the reverse. (d) The corresponding relative gray value statistics graph of the protein level. ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group.

Figure 5

(a) DCFH-DA detected the expression of ROS in GMC after the treatment of NAC and maresin 1; ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group. (b) GMCs were stained with DCFH-DA probe and images were captured using fluorescence microscope. Compared with NC group, the ROS generation increased in HG group, but it decreased in NAC + HG and M + HG group in a concentration-dependent manner.

Figure 6

The expression of TGF-β1 and FN in mesangial cells treated with 30 mmol/L glucose for different time. (a) RT-PCR strip chart for different time. Compared with NC group, the expression of TGF-β1 upregulated in HG group for different time, especially for 48 h. (b) The corresponding relative gray value statistics graph of the mRNA level. ^∗P < 0.05 versus NC group. (c) FN ELISA kit detected the level of FN in supernatant of GMCs treated with 30 mmol/L glucose for different time. Compared with NC group, the expression of FN upregulated in HG group for different time, especially for 48 h; ^∗P < 0.05 versus NC group.

Figure 7

The expression of TGF-β1 and FN in each group. (a) Expression of TGF-β1 in GMCs which were treated with different does of maresin 1 for 30 min and were exposed to high glucose (30 mmol/L) for 48 h. Compared with NC group, expression of TGF-β1 was significantly increased in HG group, and compared with HG group, expression of TGF-β1 was significantly lower in M + HG group, and there were effective changes in M2 + HG group. (b) The corresponding relative gray value statistics graph of the mRNA level. ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group. (c) FN ELISA kit detected the level of FN in each supernatant of GMCs; we detected the similar change with TGF-β1; ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group.

Figure 8

The level of TGF-β1 and FN in each group. (a) RT-PCR detected the expression of TGF-β1 mRNA after the treatment of maresin 1 for 48 h; compared with HG group, TGF-β1 mRNA was significantly decreased in M2 + HG group. (b) The corresponding relative gray value statistics graph of the mRNA level. ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group. (c) FN ELISA kit detected the level of FN in supernatant of GMCs after the treatment of maresin 1; ^∗P < 0.05 versus NC group, ^#P < 0.05 versus HG group.