Alliin, a garlic (Allium sativum) compound, prevents LPS-induced inflammation in 3T3-L1 adipocytes

Abstract

Garlic (Allium sativum L.) has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide), a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24 h with 100 μmol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100 ng/mL LPS for 1 h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile.

Figure 1

Messenger RNA (mRNA) expression levels of proinflammatory genes. Differentiated adipocytes were incubated with 0.1 mM/mL alliin for 24 h and stimulated with 100 ng/mL of lipopolysaccharides (LPS) for 1 h. Values are expressed as arbitrary units (AU) after normalization of expression levels against a control gene. Results are mean ± standard deviations (SD) of three independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 2

Protein expression levels of proinflammatory and anti-inflammatory proteins secreted by 3T3-L1 adipocytes. Cells were incubated with 0.1 mM alliin for 24 h and exposed to 100 ng/mL of lipopolysaccharides (LPS) for 1 h. Cytokine and protein concentration in cell culture supernatants for 30 min, 1, 3, 6, 12, and 24 h after LPS exposure were determined by Luminex technology. Values are expressed in pg/mL of supernatant. Results are mean ± standard deviations (SD) of three independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 3

Levels of phosphoextracellular signal-regulated kinase (ERK1/2 p44/p42) in mouse 3T3-L1 adipocytes. Cells were pretreated for 24 h with alliin 0.1 mM and subsequently exposed to 100 ng/mL of lipopolysaccharides (LPS) for 1 h afterward. (a) Representative Western blot with phospho-ERK1/2 and ERK1/2 antibodies; (b) protein levels of phospho-ERK1/2 and ERK1/2 in total cell extracts. CT control; AU arbitrary units. Data are expressed as mean ± standard deviations (SD) of three independent experiments *P ≤ 0.05; **P ≤ 0.01.

Figure 4

A proposal of how alliin could counteract the inflammatory state promoted by lipopolysaccharides (LPS) in 3T3-L1 adipocytes. (a) Activation of proinflammatory signaling pathway by lipopolysaccharides (LPS). (b) Alliin could reduce the Toll-like receptor-4 (TLR-4) pathway, possibly by diminishing the expression of related genes and proteins such as interleukin-6 (IL-6), monocyte chemostatic protein-1 (MCP-1), and early growth receptor-1 (Egr-1) and therefore regulates extracellular signal-regulated kinase (ERK1/2) activity.