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The Anti-inflammatory Effects of Lignan Glycosides from Cistanche tubulosa stems on LPS/IFN-γ-induced RAW264.7 Macrophage Cells via PI3K/ AKT Pathway

[ Vol. 22 , Issue. 10 ]

Author(s):

Jinhan Guo, Shuming Tang, Yuyang Miao, Lanlan Ge, Junfa Xu* and Xiaobin Zeng*   Pages 1380 - 1391 ( 12 )

Abstract:


Background: Cistanche tubulosa is a tonic in traditional Chinese medicines and has a broad spectrum of biological activity, including anti-inflammatory. However, the anti-inflammatory major constituents of C. tubulosa and their underlying mechanisms are still unknown.

Objective: The aim of the current study was to explore the separation and structural characterization of lignan glycosides from C. tubulosa (Schenk) Wight., their anti-inflammatory activity and the underlying mechanism.

Materials and Methods: Fractionation and isolation of the 85% EtOH extract of C. tubulosa (Schenk) Wight. were carried out and the primary ingredients lignan glycosides (1-6) were structurally characterized. CCK8 methods were used to evaluate the cytotoxic effect of lignan glycosides (1-6). Effects of lignan glycosides (1-6) on NO production in LPS/IFN-γ-induced RAW264.7 macrophages cells were measured using Griess reagent by reaction with nitrite. The mRNA expression levels of iNOS, COX-2, IL-1β, IL-6, TNF-a, and TGF-β treated RAW264.7 cells with various concentrations (0, 25 and 50 μg/ml) of lignan glycosides (1, 4) in the presence of LPS (10 ng/ml) and IFN-γ (20 ng/ml) for 24 h were analyzed by quantitative RT-PCR. Also, the protein expressions of iNOS, COX-2, PI3K, AKT, p-AKT and β-actin were determined using Western blot analysis. A molecular docking study was performed to investigate the interactions between the lignan glycosides and the PI3K using Autodock vina 1.1.2 package.

Results: Six lignan glycosides (1-6) were isolated from stems of C. tubulosa. Among them, (+)- pinoresinol-4-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (5) and eleutheroside E (6) were firstly isolated from C. tubulosa. Of these lignans, 1 and 4 exhibited pronounced inhibitions on NO production with the values of 33.63 ± 4.78 and 39.28 ± 5.52 % at 50 μg/ml, respectively. Additionally, LPS/IFN-γ-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-a (TNF-a) was significantly suppressed by pre-treatment of 1 and 4 in a dose-dependent manner. While 1 and 4 increased the mRNA levels of anti-inflammatory cytokines (TGF-β). Furthermore, 1 and 4 significantly inhibited the protein levels of PI3K and p-AKT in a dose-dependent manner.

Conclusion: Taken together, these results suggest that 1 and 4 play an important role in the attenuation of LPS/IFN-γ-induced inflammatory responses in RAW264.7 cells and that the mechanisms involve down-regulation of the PI3K/AKT pathway.

Keywords:

Cistanche tubulosa, lignan glycosides, anti-inflammatory activity, structure-activity relationship, PI3K/AKT.

Affiliation:

Ultrasonography Department of Longhua Branch, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, Guangdong, Department of Clinical Laboratory, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University), Shenzhen 518020, Guangdong, Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, Guangdong, Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, Guangdong, Institute of Laboratory Medicine, Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, Guangdong, Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, Guangdong



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