A novel flavonol-polysaccharide from Tamarix chinensis alleviates influenza A virus-induced acute lung injury. Evidences for its mechanism of action

Background: Tamarix chinensis Lour. is a Chinese medicine used for treating inflammation-related diseases and its crude polysaccharides (MBAP90) exhibited significant anticomplement activities in vitro. Purpose: To obtain anticomplement homogenous polysaccharides from MBAP90 and explore its therapeutic effects and potential mechanism on influenza A virus (IAV)-induced acute lung injury (ALI). Methods: Anticomplement activity-guided fractionation of the water-soluble crude polysaccharides from the leaves and twigs of T. chinensis were performed by diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns to yield a homogeneous polysaccharide MBAP-5, which was further characterized using ultra-highperformance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MS) and nuclear magnetic resonance (NMR) analysis. In vitro, the anticomplement activity of MBAP-5 through classical pathway was measured using a hemolytic test. The therapeutic effects of MBAP-5 on ALI were evaluated in H1N1-infected mice. H&E staining, enzyme linked immunosorbent assay (ELISA), immunohistochemistry, and western blot were used to systematically access lung histomorphology, inflammatory cytokines, degree of complement component 3c, 5aR, and 5b-9 (C3c, C5aR, and C5b-9) deposition, and inflammasome signaling pathway protein expressions in lung tissues. Results: MBAP-5 was a novel flavonol-polysaccharide with the molecular weight (Mw) of 153.6 kDa. Its structure was characterized to process a backbone of -> 4)-alpha-D-GlcpA-(1 ->, -> 6)-alpha-D-Glcp-(1 ->, -> 3,4)-alpha-D-Glcp-(1 ->, -> 3,4,6)-alpha-D-Glcp-(1 ->, and -> 4,6)-beta-D-Glcp-(1 ->, as well as branches of alpha-L-Araf-(1 -> and beta-D-Galp-(1 ->. Particularly, O-3 of -> 3,4,6)-alpha-D-Glcp-(1 -> was substituted by quercetin. In vitro assay showed that MBAP-5 had a potent anticomplement activity with a CH50 value of 102 +/- 4 mu g/ml. Oral administration of MBAP-5 (50 and 100 mg/kg) effectively attenuated the H1N1-induced pulmonary injury in vivo by reducing pulmonary edema, virus replication, and inflammatory responses. Mechanistically, MBAP-5 inhibited the striking deposition and contents of complement activation products (C3c, C5aR, and C5b-9) in the lung. Toll-like receptor 4 (TLR4) /transcription factor nuclear factor kappa B (NF-kappa B) signaling pathway was constrained by MBAP-5 treatment. In addition, MBAP-5 could suppress activation of the inflammasome pathways, including Nod-like receptor pyrin domain 3 (NLRP3), cysteinyl aspartate specific proteinase-1/12 (caspase-1/12), apoptosis-associated speck-like protein (ASC), gasdermin D (GSDMD), interleukin (IL)-1 beta, and IL-18 expressions. Conclusions: A novel flavonol-polysaccharide MBAP-5 isolated from T. chinensis demonstrated a therapeutic effect against ALI induced by IAV attack. The mechanism might be associated with inhibition of complement system and inflammasome pathways activation.