Malva sylvestris Inhibits Inflammatory Response in Oral Human Cells. An In Vitro Infection Model

Abstract

The aim of this study was to investigate the in vitro anti-inflammatory activity of Malva sylvestris extract (MSE) and fractions in a co-culture model of cells infected by Aggregatibacter actinomycetemcomitans. In addition, we evaluated the phytochemical content in the extract and fractions of M. sylvestris and demonstrated that polyphenols were the most frequent group in all samples studied. An in vitro dual-chamber model to mimic the periodontal structure was developed using a monolayer of epithelial keratinocytes (OBA-9) and a subepithelial layer of fibroblasts (HGF-1). The invasive periodontopathogen A. actinomycetemcomitans (D7S-1) was applied to migrate through the cell layers and induce the synthesis of immune factors and cytokines in the host cells. In an attempt to analyze the antimicrobial properties of MSE and fractions, a susceptibility test was carried out. The extract (MIC 175 μg/mL, MBC 500μg/mL) and chloroform fraction (MIC 150 μg/mL, MBC 250 μg/mL) were found to have inhibitory activity. The extract and all fractions were assessed using a cytotoxicity test and results showed that concentrations under 100 μg/mL did not significantly reduce cell viability compared to the control group (p > 0.05, viability > 90%). In order to analyze the inflammatory response, transcriptional factors and cytokines were quantified in the supernatant released from the cells. The chloroform fraction was the most effective in reducing the bacterial colonization (p< 0.05) and controlling inflammatory mediators, and promoted the down-regulation of genes including IL-1beta, IL-6, IL-10, CD14, PTGS, MMP-1 and FOS as well as the reduction of the IL-1beta, IL-6, IL-8 and GM-CSF protein levels (p< 0.05). Malva sylvestris and its chloroform fraction minimized the A. actinomycetemcomitans infection and inflammation processes in oral human cells by a putative pathway that involves important cytokines and receptors. Therefore, this natural product may be considered as a successful dual anti-inflammatory-antimicrobial candidate.

Fig 1. Cytotoxicity test.

Cytotoxic effect of MSE, CLF and AF on fibroblasts HGF-1 cells. Data are expressed as mean ± SEM using one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison tests as compared to non-treated. The level of statistical significance was set at 0.05.

Fig 2. Cytotoxicity effect in the dual chamber model.

Cytotoxic effects of MSE, chloroform and aqueous fraction on fibroblast HGF-1 and keratinocyte OBA-9 cell lines were found after 24 h hours of invasion by A. actinomycetemcomitans. Data are expressed as mean ± SEM using one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison tests as compared to the control group (non-treated). The level of statistical significance was set at 0.05.

Fig 3. Comparison of colony-forming units.

Comparison of colony-forming units (CFU/mL) among groups treated with MSE, CLF and AF after A. actinomycetemcomitans infection. Data are expressed as mean ± SEM using one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison tests as compared to vehicle control. The level of statistical significance was set at 0.05.

Fig 4. Gene expression analysis of lower chamber co-culture invasion assay.

The expression levels of IL-1alfa, IL-1beta, IL-6, IL-8, IL-19, CD14, PTGS, MMP-1, FOS, BCL2 were evaluated and compared to the control (non-infected cells). Quantification of the relative transcript amounts was performed using qPCR with 50 ng of each cDNA. Data quantification was performed using the 2^ΔΔCT method. Statistical analyses included one-way ANOVA followed by Dunnet’s post-hoc tests. A significance level of p < 0.05 (*) indicates differences from the vehicle group.

Fig 5. Cytokine assay.

Quantification of IL-1alpha, IL-1beta, IL-6, IL-8, IL-10 and GM-CSF in the co-culture supernant after 24h of A. actinomycetemcomitans invasion. Cells were treated with 75μg/mL of MSE and fractions and bacteria inocula were established at 2x10⁶ CFU/mL. Data are expressed as mean±SD, n = 6. Symbols indicate statistical differences (p<0.05, Dunnet’s test). # indicates p<0.05 compared to non-treated group; * indicates p<0.05 compared to vehicle group.