Structural Characterization and Anti-inflammatory Activities of Anticomplementary Polysaccharides from Rhododendron principis.

Rhododendron principis leaves have been used as "Dama", a Traditional Tibetan Medicine for treating inflammatory diseases. R. principis crude polysaccharides with anticomplementary activity demonstrated promising anti-inflammatory effects on acute lung injury induced by lipopolysaccharide. R. principis crude polysaccharides significantly decreased the levels of TNF-α and interleukin-6 in both serum and blood and bronchoalveolar lavage fluid in lipopolysaccharide-induced acute lung injury mice by intragastric administration (100 mg/kg). A heteropolysaccharide, ZNDHP, was obtained from R. principis crude polysaccharides with successive anticomplementary activity-guided separation. ZNDHP was characterized as a branched neutral polysaccharide with a backbone composed of → 2)-ß-Glcp-(1→, → 2,6)-α-Glcp-(1→, → 6,3)-ß-Galp-(1→, → 2,6)-α-Galp-(1→, → 6,2)-ß-Glcp-(1→, → 4)-α-Glcp-(1→, → 5)-ß-Araf-(1→, → 3,5)-α-Araf-(1→, and → 4,6)-ß-Manp-(1→, and the backbone structure was further confirmed by partial acid hydrolysis. In addition to anticomplementary and antioxidant activities, ZNDHP exhibited potent anti-inflammatory activity by significantly inhibiting the secretion of nitric oxide, TNF-α, interleukin-6, and interleukin-1ß of lipopolysaccharide-treated RAW 264.7 cells. However, all of these activities decreased greatly after partially hydrolyzing, indicating the importance of the multibranched structure for its bioactivity. Therefore, ZNDHP might be an important component of R. principis for treating inflammation.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.