Inhibition of lipopolysaccharide (LPS)-induced neuroinflammatory response by polysaccharide fractions of Khaya grandifoliola (C.D.C.) stem bark, Cryptolepis sanguinolenta (Lindl.) Schltr and Cymbopogon citratus Stapf leaves in raw 264.7 macrophages and U87 glioblastoma cells

Abstract

Background: Khaya grandifoliola (C.D.C.) stem bark, Cymbopogon citratus (Stapf) and Cryptolepis sanguinolenta (Lindl.) Schltr leaves are used in Cameroonian traditional medicine for the treatment of inflammatory diseases. Several studies have been performed on the biological activities of secondary metabolites extracted from these plants. However, to the best of our knowledge, the anti-neuro inflammatory and protective roles of the polysaccharides of these three plants have not yet been elucidated. This study aimed at investigating potential use of K. grandifoliola, C. sanguinolenta and C. citratus polysaccharides in the prevention of chronic inflammation.

Methods: Firstly, the composition of polysaccharide fractions isolated from K. grandifoliola stem bark (KGF), C. sanguinolenta (CSF) and C. citratus (CCF) leaves was assessed. Secondly, the cytotoxicity was evaluated on Raw 264.7 macrophages and U87-MG glioblastoma cell lines by the MTT assay. This was followed by the in vitro evaluation of the ability of KGF, CSF and CCF to inhibit lipopolysaccharides (LPS) induced overproduction of various pro-inflammatory mediators (NO, ROS and IL1β, TNFα, IL6, NF-kB cytokines). This was done in Raw 264.7 and U87-MG cells. Finally, the in vitro protective effect of KGF, CSF and CCF against LPS-induced toxicity in the U87-MG cells was evaluated.

Results: CCF was shown to mostly contain sugar and no polyphenol while KGP and CSP contained very few amounts of these metabolites (≤ 2%). The three polysaccharide fractions were non-toxic up to 100 μg.mL^{- 1}. All the polysaccharides at 10 μg/mL inhibited NO production, but only KGF and CCF at 12.5 μg/mL down-regulated LPS-induced ROS overproduction. Finally, 100 μg/mL LPS reduced 50% of U87 cell viability, and pre-treatment with the three polysaccharides significantly increased the proliferation.

Conclusion: These results suggest that the polysaccharides of K. grandifoliola, C. citratus and C. sanguinolenta could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.

Keywords: Anti-neuroinflammatory; Cryptolepis sanguinolenta; Cymbopogon citratus; Khaya grandifoliola; Lipopolysaccharides; Plant polysaccharide.

Fig. 1

Cytotoxicity of polysaccharide fractions of Khaya grandifoliola stem bark (KGF), Cryptolepis sanguinolenta (CSF) and Cymbopogon citratus (CCF) leaves on RAW 264.7 macrophages cell lines (a) and U87-MG glioblastoma cell line (b)

Fig. 2

Inhibition of LPS toxicity on U87 cell lines by various polysaccharide fractions and diclofenac potassium; (a) C-control cells; L-LPS (100 μg/mL), KGF, CSF, CCF polysaccharides of Khaya grandifoliola stem bark, Cryptolepis sanguinolenta and Cymbopogon citratus leaves were tested at 100 μg/mL and Diclofenac potassium at 50 μg/mL (DK50). Data are expressed as mean ± SEM, *** denote statistical significance p < 0.001 in comparison to the control group; (b) KGF, CSF, CCF polysaccharides were tested at 12.5, 50 and 100 μg/mL and DK50 #, ##, ### denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the LPS treated group

Fig. 3

Effects of polysaccharides fractions of Khaya grandifoliola stem bark (KGF), Cryptolepis sanguinolenta (CSF) and Cymbopogon citratus (CCF) leaves and Aspirin (Asp) on the secretion of NO by LPS-stimulated RAW 264.7 macrophages cells. C-Non-treated cells; L-LPS (10 μg/mL) treated cells, Asp-Aspirin 1 μM (180 μg/mL) treated cells, Data expressed as mean ± SEM (n = 3). *, **, *** denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the control group. #, ##, ### denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the LPS treated group (Dunnet multiple comparison, graph Pad Prism 6.0)

Fig. 4

Effects of polysaccharides isolate from Khaya grandifoliola stem bark (KGF), Cryptolepis sanguinolenta (CSF) and Cymbopogon citratus (CCF) leaves at 12.5 μg/mL and Diclofenac potassium on the secretion of ROS by LPS-stimulated U87-MG cells. C-control cells; L-LPS (10 μg/ml), DK50-Diclofenac potassium 50 μg/mL. Data expressed as mean ± SEM (n = 3). *, **, *** denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the control group. #, ##, ### denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the LPS treated group (Dunnet multiple comparaison, graph Pad Prism 6.0)

Fig. 5

Normalised gene expression levels of proinflammatory cytokines in LPS induced U87-MG cell lines (n = 3). The mRNA expression values were given as mean ± SEM normalised to GAPDH levels in each sample. Y-axis values represent the number of mRNA copies relative to the number of GAPDH copies. Control-untreated cell line; LPS (100 μg/mL), K. grandifoliola stem bark (FKG) and C. citratus leaves (FCC) were tested at 12.5 μg/mL, DK50-Diclofenac potassium 50 μg/mL treated cells. Data are expressed as mean ± SEM. #, ##, ### denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the LPS treated group. *, **, *** denote statistical significance at p < 0.05, p < 0.01 & p < 0.001 in comparison to the control group