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Published on: November 28, 2015
[Effects of metformin on the polarization and Notch 1 expression of RAW264.7 macrophages]
Miaojiao Chen1, Jingjing Zhang1, Shiping Liu2
1Institute of Metabolism & Endocrinology, Second Xiangya Hospital, Central South University, Diabetes Center of Central South University, Key Laboratory of Diabetes Immunology, Ministry of Education, National Clinical Research Center for Metabolic Diseases, Changsha 410011, China.
Objective:
To explore the possible effects of metformin on regulating the Notch1 expression and the polarization of RAW264.7 macrophages.
Methods:
RAW264.7 macrophages were divided into two groups according to different metformin concentrations (0, 1, 5, 10 mmol/L) and treatment length (0, 12, 24, 48 h). The expressions of interleukin-1β (IL-1β) and interleukin-10 (IL-10) mRNA were detected by real time quantity-polymerase chain reaction (RTQ-PCR). RAW264.7 macrophages were divided into control, metformin-treated, lipopolysaccharide (LPS)-treated and LPS-metformin-treated groups. Then the expressions of IL-1β, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), arginase 1 (Arg1), IL-10, interleukin-4 (IL-4), macrophage galactose-type lectin-1 (Mgl1) and Notch1 mRNA were detected with RTQ-PCR. The expression of Notch1 protein was examined by Western blot.
Results:
At the concentrations of 0-10 mmol/L for 0-48 h, metformin in concentration and time-dependent ways promoted the expression of IL-10 mRNA and inhibited the mRNA expression of IL-1β in RAW264.7 macrophages (both P < 0.05). In metformin-treated cells, the expressions of Arg1 (P = 0.009), IL-10 (P = 0.015) and IL-4 (P = 0.001) mRNA increased while the expressions of IL-1β (P = 0.001) and IL-6 (P = 0.032) mRNA decreased. And it was the same as the levels of Notch1 mRNA (P = 0.004) and protein. The LPS-treated group could up-regulate the expressions of IL-1β (P < 0.001), IL-6 (P = 0.002), TNF-α (P = 0.005) mRNA and Notch1 (P = 0.002) mRNA (protein). And there was a declining trend for the expressions of Arg1, IL-10, IL-4 and Mgl1 mRNA in macrophages (both P>0.05). On the contrary, in LPS-metformin-treated cells, the gene expressions of IL-1β (P < 0.001), IL-6 (P = 0.005), TNF-α (P = 0.029) and Notch1 (P = 0.006) mRNA (along with protein) were down-regulated while Arg1 (P = 0.005), IL-10 (P < 0.001), IL-4 (P < 0.001) and Mgl1 (P = 0.001) mRNA expressions up-regulated.
Conclusions:
Metformin regulates RAW264.7 macrophages with/without LPS toward a M2 phenotype. And Notch1 signaling may play a critical role.
Insights
Metformin promotes M2 macrophage polarization by upregulating IL-10 and downregulating IL-1β, influencing Notch1 expression. This suggests metformin
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Macrophage polarization is crucial in immune responses, with M1 and M2 phenotypes playing distinct roles.
- Notch1 signaling is implicated in various cellular processes, including immune cell differentiation and function.
- Metformin, a widely used antidiabetic drug, has demonstrated immunomodulatory effects in preclinical studies.
Purpose of the Study:
- To investigate the impact of metformin on the polarization of RAW264.7 macrophages.
- To determine the effect of metformin on Notch1 expression in macrophages.
- To explore the role of Notch1 signaling in metformin-induced macrophage polarization.
Main Methods:
- RAW264.7 macrophages were treated with varying concentrations and durations of metformin.
- Gene expression of key polarization markers (IL-1β, IL-10, Arg1, IL-4, IL-6, TNF-α, Mgl1) and Notch1 was quantified using RTQ-PCR.
- Notch1 protein levels were assessed via Western blot analysis.
- Macrophages were also treated with lipopolysaccharide (LPS) with or without metformin to mimic inflammatory conditions.
Main Results:
- Metformin treatment dose- and time-dependently increased IL-10 mRNA and decreased IL-1β mRNA expression.
- Metformin promoted M2-like polarization markers (Arg1, IL-10, IL-4) and downregulated M1 markers (IL-1β, IL-6) in macrophages.
- Metformin upregulated Notch1 expression at both mRNA and protein levels.
- In LPS-stimulated macrophages, metformin reversed the M1-skewing effects of LPS, promoting M2 polarization and downregulating Notch1.
Conclusions:
- Metformin effectively modulates RAW264.7 macrophage polarization towards an M2 phenotype, both in the presence and absence of LPS.
- The study indicates that metformin's immunomodulatory effects on macrophages are associated with alterations in Notch1 signaling.
- Notch1 signaling may play a significant role in mediating the macrophage polarization effects of metformin.
