Junbin Chen1, Hidayat Ullah1, Zhongdaixi Zheng1, Xiangfu Gu1, Chuhong Su1, Lingyu Xiao1, Xinglong Wu1, Fei Xiong1, Qing Li2, Longying Zha3. 1. Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1838 Guangzhou Avenue North, Guangzhou, 510515, People's Republic of China. 2. Department of Dietetics, Nanfang Hospital, Southern Medical University, No.1838, Guangzhou, 510515, Guangdong, People's Republic of China. 1620351982@qq.com. 3. Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1838 Guangzhou Avenue North, Guangzhou, 510515, People's Republic of China. lyzha@smu.edu.cn.
Abstract
BACKGROUND: Previous studies indicate that soyasaponins may reduce inflammation via modulating toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling. However, its underlying mechanisms are still not fully understood. METHODS: Lipopolysaccharide (LPS)-challenged inflamed male ICR mice were intervened by intragastrical administration with 10 and 20 μmol/kg·BW of soyasaponin A1, A2 or I for 8 weeks. The serum inflammatory markers were determined by commercial kits and the expression of molecules in TLR4/MyD88 signaling pathway in liver by real-time PCR and western blotting. The recruitments of TLR4 and MyD88 into lipid rafts of live tissue lysates were detected by sucrose gradient ultracentrifugation and western blotting. LPS-stimulated RAW264.7 macrophages were treated with 10, 20 and 40 μmol/L of soyasaponin A1, A2 or I for 2 h. MyD88-overexpressed HEK293T cells were treated with 20 and 40 μmol/L of soyasaponins (A1, A2 or I) or 20 μmol/L of ST2825 (a MyD88 inhibitor) for 6 h. The expression of molecules in TLR4/MyD88 signaling pathway were determined by western blotting. Data were analyzed by using one way analysis of variance or t-test by SPSS 20.0 statistical software. RESULTS: Soyasaponins A1, A2 or I significantly reduced the levels of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and nitric oxide (NO) in serum (p < 0.05), and decreased the mRNA levels of TNFα, IL-6, IL-1β, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) (p < 0.05), the protein levels of myeloid differentiation protein 2 (MD-2), TLR4, MyD88, toll-interleukin1 receptor domain containing adaptor protein (TIRAP), phosphorylated interleukin-1 receptor-associated kinase 4 (p-IRAK-4), phosphorylated interleukin-1 receptor-associated kinase 1 (p-IRAK-1) and TNF receptor associated factor 6 (TRAF6) (p < 0.05), and the recruitments of TLR4 and MyD88 into lipid rafts in liver (p < 0.05). In LPS-stimulated macrophages, soyasaponins A2 or I significantly decreased MyD88 (p < 0.05), soyasaponins A1, A2 or I reduced p-IRAK-4 and p-IRAK-1 (p < 0.05), and soyasaponin I decreased TRAF6 (p < 0.05). In MyD88-overexpressed HEK293T cells, soyasaponins (A1, A2 or I) and ST2825 significantly decreased MyD88 and TRAF6 (p < 0.05). CONCLUSION: Soyasaponins can reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts. This study provides novel understanding about the anti-inflammatory mechanism of soyasaponins.
BACKGROUND: Previous studies indicate that soyasaponins may reduce inflammation via modulating toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling. However, its underlying mechanisms are still not fully understood. METHODS:Lipopolysaccharide (LPS)-challenged inflamed male ICR mice were intervened by intragastrical administration with 10 and 20 μmol/kg·BW of soyasaponin A1, A2 or I for 8 weeks. The serum inflammatory markers were determined by commercial kits and the expression of molecules in TLR4/MyD88 signaling pathway in liver by real-time PCR and western blotting. The recruitments of TLR4 and MyD88 into lipid rafts of live tissue lysates were detected by sucrose gradient ultracentrifugation and western blotting. LPS-stimulated RAW264.7 macrophages were treated with 10, 20 and 40 μmol/L of soyasaponin A1, A2 or I for 2 h. MyD88-overexpressed HEK293T cells were treated with 20 and 40 μmol/L of soyasaponins (A1, A2 or I) or 20 μmol/L of ST2825 (a MyD88 inhibitor) for 6 h. The expression of molecules in TLR4/MyD88 signaling pathway were determined by western blotting. Data were analyzed by using one way analysis of variance or t-test by SPSS 20.0 statistical software. RESULTS:Soyasaponins A1, A2 or I significantly reduced the levels of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and nitric oxide (NO) in serum (p < 0.05), and decreased the mRNA levels of TNFα, IL-6, IL-1β, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) (p < 0.05), the protein levels of myeloid differentiation protein 2 (MD-2), TLR4, MyD88, toll-interleukin1 receptor domain containing adaptor protein (TIRAP), phosphorylated interleukin-1 receptor-associated kinase 4 (p-IRAK-4), phosphorylated interleukin-1 receptor-associated kinase 1 (p-IRAK-1) and TNF receptor associated factor 6 (TRAF6) (p < 0.05), and the recruitments of TLR4 and MyD88 into lipid rafts in liver (p < 0.05). In LPS-stimulated macrophages, soyasaponins A2 or I significantly decreased MyD88 (p < 0.05), soyasaponins A1, A2 or I reduced p-IRAK-4 and p-IRAK-1 (p < 0.05), and soyasaponin I decreased TRAF6 (p < 0.05). In MyD88-overexpressed HEK293T cells, soyasaponins (A1, A2 or I) and ST2825 significantly decreased MyD88 and TRAF6 (p < 0.05). CONCLUSION:Soyasaponins can reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts. This study provides novel understanding about the anti-inflammatory mechanism of soyasaponins.