Periodontitis is an infectious inflammatory disease resulting in periodontal pocket formation, progressive bone reduction and teeth loss in many industrialized countries [1
]. Common treatment strategies include systemic use of antibiotics and local synthetic antiseptic substances, both leading to undesirable side effects and increased resistance of bacteria [3
]. In consequence, prolonged and/or repeatable treatment is risky, inefficient and fails to stop disease remission and further progression. In fact, as a response to the extensive use of drugs, bacteria have developed a new mechanism to skip and counteract antibiotics activity: resistant polysaccharide envelope, more efficient efflux pumps, intracellular modifications and genetic mutations are some of the pathways exploited by bacteria to withstand drugs effect [4
]. However, it is important to consider that not all body-resident bacteria are pathogens: commensal strain present in the microbiota play a pivotal role in preserving homeostasis in the skin and mucosal physiological systems of the human body [5
]. The use of very strong chemicals such as chlorhexidine [7
] can be exploited only for short periods to prevent severe side effects that can occur after prolonged exposure [8
]. It follows that an ideal new antibacterial compound should be able to affect bacteria metabolism by a different mechanism than those exploited by antibiotics but at the same time would be harmless to the healthy cells and commensal bacteria. In this light, multicomponent plant-derived antibacterial substances like proanthocyanidins (PACN) make a promising alternative and adjunctive therapy candidates for periodontitis treatment because of a lower risk of resistance development and side effects [9
PACN are condensed tannins constructed form flavan-3-ol units [10
]. The compounds possess a range of biological activities including anti-inflammatory and antibacterial [11
]. The capacity of PACN to suppress inflammation is related to both strong antioxidant and metalloproteinase (MMP) inhibiting properties [12
], whereas antibacterial efficiency is achieved due to prevention of bacterial adhesion and biofilm formation [14
]. The chemical nature of PACN in crude extracts varies depending on plant species used. Pelargonium sidoides
DC, a medicinal plant native to South Africa, is one of the most PACN-enriched plants. Medicinal raw materials—roots of the plant—are used in the treatment of infectious and inflammatory disorders, and P. sidoides
root extracts (PSREs) possess the same properties with enhanced efficiency [15
]. PSREs mediate their pharmacological effects via two classes of compounds, namely oxygenated coumarins and prodelphinidins that belong to the PACN group [18
]. The common properties of these compounds isolated from various sources suggest the significant part of the activities of PSREs might be assigned to PACN. Indeed, we have recently shown that namely prodelphinidin fraction from PSRE more efficiently suppress periodontal pathogens Porphyromonas gingivalis
compared to PSRE itself [19
]. Moreover, the activity appeared to be strain selective: reducing the viability of the pathogens while preserving the metabolic activity of the beneficial oral commensal Streptococcus salivarius
Based on these promising results, in the present study we decided to extend the examination of antibacterial efficiency of PACN to other broad-range pathogens and commensals: two commercial drug-resistant Staphylococcus aureus
and Aggregatibacter actinomycetemcomitans
strains, a clinical isolate pathogen Staphylococcus epidermidis
strain and a commensal Escherichia coli
strain. Next, after verifying extract cytocompatibility towards gingival fibroblasts, a “race for the surface” model of bacteria-cells co-culture [20
] was carried out to verify the extract ability to reduce bacteria proliferation while preserving cells viability in the same microenvironment where cells and bacteria compete for the same surface. Finally, we have made an extensive investigation on PACN activity in bacterial lipopolysaccharide (LPS)-mediated inflammation, including measurement of secretion of inflammatory cytokines and other mediators, inflammatory gene expression and viability of gingival fibroblasts, macrophages and blood leukocytes.
2. Materials and Methods
2.1. Pelargonium sidoides Root Extract and Proanthocyanidin Fraction
The P. sidoides
root extract (PSRE) was purchased from Frutarom Switzerland Ltd. Rutiwisstrasse 7 CH-8820 Wadenswil (batch no. 0410100). Proanthocyanidins (PACN) from PSRE were purified as described by Hellström and co-authors [21
] with some modifications [19
]. Briefly, 4 g of PSRE was dissolved in 200 mL of 50% methanol, the solution was centrifuged at 2000× g
for 20 min and filtered through 0.45 µm nylon filters. The solution was purified by gel adsorption over Sephadex LH-20. The proanthocyanidins were released from the gel with 70% aqueous acetone (500 mL) and concentrated under vacuum at 35 °C. The aqueous aliquot was freeze-dried. The freeze dried PACN preparation yielded in 1.37 ± 0.07 g and comprised about 34.25% of the loaded PSRE.
2.2. Bacterial Strains and Growth Conditions
Commercially available strains Staphylococcus aureus (S. aureus, pathogen, ATCC 43300), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans, pathogen, ATCC 33384) and Escherichia coli (E. coli, non-pathogen, ATCC BAA-1427) were purchased from the American Type Culture Collection (ATCC, MA, USA) and cultivated following the manufacturer’s instructions. A clinical isolate of Staphylococcus epidermidis (S. epidermidis, pathogen) was collected at the Clinical Microbiology Unit at the Novara Maggiore Hospital (Novara, Italy). The clinical isolate was obtained after patient’s informed consent in full accordance with the Declaration of Helsinki. Clinical strain was cultivated in Luria-Bertani medium (LB, Sigma-Aldrich, Milan, Italy) at 37 °C. For experiments, a single colony from each strain was collected and inoculated in 9 mL of LB broth at 37 °C overnight (18 h). After incubation, a new fresh LB tube diluted 1:10 was prepared and incubated at 37 °C for 3 h to achieve the logarithmic growth phase. Finally, broth cultures were diluted in LB broth until the optical density was 0.001 at 600 nm, corresponding to a final concentration of 1 × 105 cells/mL.
2.3. Antibacterial Efficiency Evaluation
To test antibacterial activity, PSRE and PACN were used at the following concentrations: 10, 30, 50, 70, 80, 90 and 100 μg/mL. PSRE and PACN powders were mixed and diluted directly into LB medium containing the desired bacteria concentration (described in 2.2. chapter); 1 mL of the obtained mix solutions (LB containing bacteria + PSRE/PACN) was seeded in the wells of a 24 multiwell plate (SPL, BioSigma, Milan, Italy) and incubated 24 h at 37 °C. Bacteria cultivated into pure LB medium were considered as control. After incubation, bacteria viability was evaluated by means of the colorimetric metabolic assay Alamar blue (alamarBlue®, Thermo-Fisher, Waltham, MA, USA) following the manufacturer’s instructions. Briefly, the ready-to-use solution was added to each well in a 1:10 ratio and incubated for 4 h in the dark at 37 °C; then, fluorescence was recorded at 590 nm using a spectrophotometer (Spark, Tecan, Basel, Switzerland).
2.4. Co-Cultures of Human Gingival Fibroblasts and Bacteria
To verify PSRE and PACN ability to preserve cells metabolism in the presence of infection, a race for the surface cells-bacteria co-culture experiment was set up. S. aureus and A. actinomycetemcomitans were selected and used with cells to simulate the oral and mucosal environments, respectively. Human primary gingival fibroblasts (HGF, ATCC PCS-201-018) were used as a cellular model to be tested with bacteria. Cells were seeded at a defined number (1.5 × 104 cells/well) in the wells of a 24 multiwell plate and allowed to adhere overnight. Afterwards, the medium was removed and replaced by 1 mL of solution composed by an antibiotics-free medium (minimal essential medium Eagle alpha-modification, from Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and 100 μg/mL of either PSRE or PACN and 1 × 105 bacteria. The plate was incubated 24 h at 37 °C to allow cells-bacteria direct contact. Then, cells and bacteria were detached from plate wells by a collagenase (1 mg/mL) trypsin-EDTA (0.25%) solution and collected. The number of viable cells was determined by trypan blue and Burker chamber count. Cells cultivated with fresh medium without bacteria were considered as a control.
2.5. Rat Gingival Fibroblast Cell Culture and Treatments
All experimental procedures were performed according to the Law of the Republic of Lithuanian Animal Welfare and Protection (License of the State Food and Veterinary Service for working with laboratory animals No. G2-80). The mice were maintained and handled at Lithuanian University of Health Sciences animal house in agreement with the ARRIVE guidelines. Primary gingival fibroblasts were isolated from gingiva of P5-7 rat pups. After isolation, the cells were grown in 75 cm2 flasks in DMEM with high glucose and Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS and Pen/Strep. At 70%–90% confluence, the cells were detached by 0.025% Trypsin/EGTA and plated in 96 well plates at a density of 2 × 105 cells/well. The treatments were made 24 h after plating. All treatments were made simultaneously, without pre-incubations, and lasted 24 h. LPS was used at concentration of 2 mg/mL (1 mg/mL LPS did not induce significant increase in cell death), filtered PSRE and PACN solutions at 50 and 100 μg/mL (both preparations induced toxicity starting from 200 μg/mL).
2.6. Bone Marrow-Derived Macrophages
For bone marrow-derived macrophages (BMDM) isolation male C57BL6/J inbred mice (18–20 weeks old, Envigo, Netherlands) were used. The experimental procedures were carried out in accordance with the guidelines of the European Community (2010/63/EU), local laws and policies and were approved by the Latvian Animal Protection Ethical Committee, Food and Veterinary Service, Riga, Latvia. Mice were euthanized by decapitation, and bone marrow cells were extracted from femur bones and differentiated for 7 days in RPMI-1640 with Glutamax (Gibco,) supplemented with 10% FBS, 1% antibiotics and 10 ng/mL M-CSF (monocyte-colony stimulating factor, PeproTech, London, UK). Then cells were detached by 0.5% trypsin (Sigma Aldrich), and plated in a 12-well plate (11 × 105 cells/mL) in DMEM-high glucose medium supplemented with 10% FBS, 1% antibiotics. After 1h incubation in 37 °C incubator, cells were stimulated with PSRE and PACN at 100 µg/mL and LPS 10 ng/mL with murine IFNγ (interferon gamma, PeproTech) 100 U/mL for proinflammatory gene expression and macrophage polarization to M1 (pro-inflammatory) phenotype for 2 h and 24 h, respectively.
2.7. Human Peripheral Blood Mononuclear Cells
Human peripheral blood mononuclear cells (PBMCs) were purchased from ATCC (ATCC® PCS-800-011™, Manassas, VA, USA). The cells were cultured at 3.3 × 106 cells/mL (12 well plate) in RPMI medium supplemented with 10% FBS, 1% antibiotics. After 1 h incubation in 37 °C incubator, cells were stimulated with 1 µg/mL LPS (lipopolysaccharide, Sigma-Aldrich) in the presence of 100 µg/mL PSRE or PACN, for 6 h.
2.8. Analysis of Cell Viability by Lactate Dehydrogenase Release, Alamarblue and MTT Assay
PBMCs’ viability was assessed by measuring lactate dehydrogenase (LDH) release in cell culture media. LDH activity was measured using a method based on the reduction of a tetrazolium salt (yellow) to formazan (red) [22
]. The absorbance of kinetic parameters was determined spectrophotometrically at 503 nm on Hidex Sense microplate reader. The reaction mixture contained 30 mM lactate, 150 µM NAD+ (nicotinamide adenine dinucleotide), 0.4 mM 2-(4-iodophenyl)3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) and 3.25 mM N
-methylphenazonium methyl sulphate (PMS) in 100 mM Tris buffer solution (pH 8.0). In addition, PBMCs viability after 24 h incubation with different concentrations of PSRE and PACN was determined with alamarBlue®
, (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s instructions. Briefly, the ready-to-use solution was added to each well in a 1:10 ratio and incubated for 2 h in the dark at 37 °C; then, fluorescence (Ex 544 nm/Em 590 nm) and optical density (570 and 600 nm) using Hidex Sense microplate reader.
BMDM viability after 24 incubation with different concentrations of PSRE and PACN was determined using MTT assay. After incubation, BMDM were incubated with MTT (TCI Europe) solution (1 mg/mL) for 1 h, the formazan crystals formed during incubation were dissolved in isopropanol, and optical density at 570 nm corresponding to the amount of viable cells was measured in a Hidex Sense microplate reader.
2.9. Necrosis Evaluation by Double Nuclear Staining
The level of necrotic cell death was assessed by double nuclear fluorescent staining with Hoechst33342 (10 mg/mL) and propidium iodide (PI, 5 mg/mL), 5 min at 37 °C. PI-positive nuclei indicating lost nuclear membrane integrity were considered necrotic. Cells were visualized under fluorescent microscope OLYMPUS IX71S1F-3 (Olympus Corporation, Tokyo, Japan), counted in fluorescent micrographs and expressed as the percentage of total cell number per image. The data are presented as averages ± standard deviation. LD50 was calculated by SigmaPlot v.13 (Systat Software Inc, London, UK) using the equation proposed by a dynamic curve fitting tool.
2.10. Apoptosis Evaluation by Annexin V staining
The viable cells were analyzed on a BD FACS Melody™ (BD Biosciences, San Jose, CA, USA) flow cytometer using Annexin V-allophycocyanin (BioLegend, San Diego, CA, USA) staining. For analysis of BMDMs apoptosis, treated cells were stained with fluorescent annexin V antibody and, afterwards, the proportion of apoptotic (Annexin V positive) cells was evaluated.
2.11. Caspase Activity Assessment
Caspase-3 activity in cell lysates after treatments was measured by means of a Caspase 3 Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol, by assessing Ac-DEVD-7-amido-4-methylcoumarin cleavage and subsequent increase in 7-amido-4-methylcoumarin (AMC) fluorescence in a fluorometric plate reader Ascent Fluoroskan (Thermo Fisher Scientific, Waltham, MA, USA; λex = 360 nm, λem = 460 nm). The data are presented as averages ± standard deviation of AMC concentration increase rate normalized for mg of cellular protein, averages ± standard deviation.
Caspase-8 in cell lysates after treatments was measured by means of a colorimetric Caspase 8 Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol, by assessing Ac-IETD-p-Nitroaniline cleavage and increase in p-Nitroaniline (pNA) concentration in a spectrophotometric plate reader Multiskan Go 1510 (Thermo Fisher Scientific, Waltham, MA, USA) reading absorbance at λex = 405 nm. The data are presented as averages ± standard deviation of pNA concentration increase rate normalized for mg of cellular protein, averages ± standard deviation.
2.12. Detection of Secreted Inflammatory Mediators
Medium collected after treatments was assayed for cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) production using TNF-α mouse (Millipore), IL-6 human (Sabbiotech), IL-8 rat (Abbexa) and PGE2 rat (Abbexa) kits following the manufacturer’s protocols.
2.13. Bone Marrow-Derived Macrophage Polarisation to M1 Phenotype and Analysis by Flow Cytometry
BMDMs were incubated with PSRE and PACN (100 μg/mL each) and LPS + IFNγ (10 ng/mL/100 U/mL) for 24 h. The cells were washed twice with HBSS and harvested by trypsin (0.5%), then DMEM-high glucose medium with 10% FBS was added and cell suspension was centrifuged at 300× g for 5 min. Then cells were incubated with specific conjugated antibody mixtures (in concentration 1:100 in cell wash buffer) for 30 min on ice in the dark. The mixture contained following monoclonal antibodies purchased from BioLegend (San Diego, CA, USA): FITC-conjugated anti-mouse F4/80, phycoerythrin (PE)-conjugated anti-mouse CD86 and biotin-conjugated anti-mouse CD80. Then cells were washed and stained with streptavidin-APC-Cy7. After 30 min, cells were washed and stained for evaluation for apoptosis (see method above). After staining samples were analyzed by flow cytometry (BD FACS Melody™, BD Biosciences, San Jose, CA, USA).
2.14. mRNA Isolation and Quantitative RT-PCR Analysis
Total RNA from cells was isolated using Ambion PureLink RNA Mini Kit (catalogue No. 12183025) according to the manufacturer’s protocol. The first-strand cDNA synthesis was carried out using a High Capacity cDNA Reverse Transcription Kit (Applied BiosystemsTM, Foster City, CA, USA) following the manufacturer’s instructions. The quantitative RT-PCR analysis of gene expression was performed by mixing SYBR Green Master Mix (Applied BiosystemsTM), synthesized cDNA, forward and reverse primers specific for interleukin-1β (IL-1β), interleukin 10 (IL-10), inducible nitric oxide synthase (iNOS), TNF-α, cyclooxygenase 2 (COX-2) and running the reactions on a Mic Real-Time PCR instrument. The relative expression levels for each gene were calculated with the ∆∆Ct method and normalized to the expression of glucose-6-phosphate isomerase gene.
2.15. Statistical Analysis
The quantitative results are presented as mean ± standard deviation (SD) of 3–7 replicates. The data were processed using Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA) and SPSS 20 (IBM, Armonk, NY, USA) software. For antibacterial activity testing and bacterial-fibroblast co-culture experiments, the statistical data analysis was performed by applying the ANOVA with a Tukey HSD post hoc test. For experiments on rat gingival fibroblasts, ANOVA with Dunn’s test was used and data analyzed by SigmaPlot v.13 (Systat Software Inc., London, UK). In all cases, differences were considered statistically significant when p < 0.05.
Increasing antibiotic resistance makes the search for alternative antimicrobial compounds of a crucial importance for global health [33
]. Failure to defeat fast adapting pathogens without significant damage to host tissues is a key challenge in management of chronic infectious-inflammatory disease including periodontitis [34
]. Progressive bacteria-driven inflammatory response causes continuous damage on periodontal cells making them more sensitive to harmful effects of antibiotics and antimicrobial chemicals [3
]. The damage is further exacerbated by the treatment-caused loss of beneficial commensal bacteria [36
]. This suggests reconsidering the possibilities of alternative treatment strategies including use of specific pathogen-targeting bacterial strains [37
] and plant-derived antibacterials, because such strategies are characterized by lower or no side effects and resistance development risk, as well as complex antiinflammatory and tissue renewal stimulating properties. This study explored antibacterial and antiinflammatory properties of PSRE that is known as potent infection-defeating preparation and PSRE-derived PACN possessing stronger antioxidant and antibacterial properties compared to PSRE [17
Both substances were effective in reducing metabolic activity of the selected strains suggesting a broad range of antibacterial properties. This is in line with previous evidence about various extracts prepared from P. sidoides
roots. A commercial aqueous-ethanolic extract from P. sidoides
) is reported to inhibit growth of Streptococcus pyogenes
, Proteus mirabilis, Staphylococcus aureus
, Escherichia coli
, Streptococcus pneumoniae
, Haemophilus influenza
, Staphylococcus epidermidis
and some other gram-negative and gram-positive bacterial strains (summarized in [38
]). Aqueous-acetone PSRE was efficient in decreasing growth of antibiotic-resistant S. aureus
]. The present study for the first time demonstrated the growth-suppressing efficiency of PSRE and PACN on Aggregatibacter actinomycetemcomitans
, one of the most important gram-negative anaerobic periodontal pathogens [40
]. Similarly as in the previously demonstrated case of P. gingivalis
], PACN demonstrated significantly higher toxicity on A. actinomycetemcomitans
, compared to the effect of PSRE. Fifty μg/mL PACN reduced metabolic activity of A. actinomycetemcomitans
nearly 10 times more if compared to the untreated control value (Figure 1
d). The same concentration had no significant toxicity on other investigated strains. The minimal amount of PACN causing a significant effect on metabolic activity of E. coli
and S. aureus
was 80 μg/mL, and for S. epidermidis
the significant toxicity started from 70 μg/mL PACN. The results indicate that there might be a specific interaction of proanthocyanidins from PSRE with the main pathogenic strains (P. gingivalis
and A. actinomycetemcomitans
) responsible for the development of periodontitis. Strain-specific activity of proanthocyanidins was already noticed by other authors. Lacombe and Wu have reviewed the selective pathogen-suppressing and beneficial strain-promoting activity of proanthocyanidins derived from various berries [41
]. However, despite many publications reporting a selective activity of natural extracts towards pathogen and non-pathogen strains, it is still not completely clear how this selection occurs [42
]. It was shown that cranberry-derived proanthocyanidins are able to interfere with a N
-acylhomoserine lactone-mediated quorum sensing of Pseudomonas aeruginosa
]. Moreover, proanthocyanidins have also been shown to compromise adhesion to host cells by mimicking cell surface signaling [44
]. Some authors have proposed the hypothesis that proanthocyanidins might increase bacterial membrane permeability and cause indirect metabolism decrease due to ATP and other intracellular metabolite loss [42
]. A recent study shows that proanthocyanidins can potentiate antibiotics by acting via bacterial multidrug efflux pumps [46
]. Thus, the disturbance in transmembrane transport indeed might be the cause of bacteriotoxicity. However, more studies definitely are required to clarify the mechanism of action of proanthocyanidins against pathogenic bacterial strains.
On the other hand, PSRE was more efficient than PACN in suppressing both of Staphylococcus
strains that were investigated in this study suggesting that other than proanthocyanidin fraction compounds were acting against these bacteria. Most likely, the distinct antibacterial activity of PSRE can be ascribed to other phenolic compounds such as coumarins, phenolic acids, flavonols and flavan-3-ols [16
Bacterial infection simulation in the co-culture “race for the surface” assay revealed that addition of 100 μg/mL of either PSRE or PACN in the medium was effective in preserving viability of human gingival fibroblasts in the presence of both S. aureus
and A. actinomycetemcomitans
). Similarly, B-type linked proanthocyanidin-coated surfaces are shown to inhibit bacterial spreading and promote survival of mammalian cells [47
]. The mechanism proposed to explain the activity is bacterial attachment and biofilm formation prevention by prodelphinidin-rich proanthocyanidins. In our experimental model, a similar efficiency was achieved by PSRE and PACN solutions, indicating that interaction of soluble compounds with the walls of bacteria also could mediate bacterial adhesion and mammalian cell protection. Accordingly, these results are very promising support to the use of natural extracts as an effective alternative antibacterial compound able to preserve the naïve tissue.
Investigation of gingival tissue protecting properties of PSRE and PACN in the bacterial LPS-mediated inflammation model revealed that both preparations efficiently prevent necrosis and apoptosis of fibroblast cells. Both substances were more efficient in decreasing the executing caspase-3 activity compared with the effect on apoptosis triggering caspase-8. However, PSRE was less efficient in suppressing caspase-8 activity than PACN, indicating that the latter had both upstream and downstream targets in the apoptotic cascade. The antiapoptotic activity of proanthocyanidins from grape seeds including decrease in executing caspases-3 and 9 was reported in a rotenone-induced neurotoxicity model of SH-SY5Y cells [48
]. However, exposure of human colorectal carcinoma cells HCT-116 to proanthocyanidins from the same source significantly upregulated mRNAs encoding caspase-2, caspase-3 and caspase-9 [49
]. Another study reports apoptosis induction in lung cancer cells NCI-H460 via stimulation of caspase-3 and mitochondrial cytochrome c release by gallic acid, one of the important constituents of PSRE [50
]. Such controversial data suggest that the effect of PSRE and proanthocyanidins on apoptotic signaling pathways is cell type-dependent and they might have opposite effects in cancerous and non-cancerous cells as well as in different toxicity models.
Evaluation of pro-inflammatory cytokine secretion and gene expression revealed that PSRE and PACN suppress at least three different inflammatory processes: cytokine secretion (IL-8 from gingival fibroblasts and IL-6 from bone marrow-derived macrophages), inflammatory gene expression (IL-1β, iNOS and COX-2) and macrophage conversion to pro-inflammatory M1 phenotype related to the tissue loss in periodontitis. Downregulation of COX-2 coding mRNA in mononuclear leukocytes and PGE2 release from gingival fibroblasts indicate suppression of the prostaglandin inflammatory pathway. PGE2 is the most prominent in the pathogenesis of periodontitis among prostaglandins [51
]. PGE2 is involved in the stimulation of inflammatory mediators and MMPs, as well as osteoclast formation via receptor activator of nuclear factor-κB ligand (RANKL) [52
]. IL-6 and IL-1β also mediate bone resorption via osteoclasts activation [54
], and increase in iNOS leads to reactive nitrogen species-mediated apoptosis of gingival fibroblasts [55
]. By suppressing these inflammatory pathways, PSRE and PACN are expected to significantly improve condition and survival of periodontal tissues. Similar antiinflammatory activity of PSRE together with Coptis chinensis
root extract was recently shown in LPS-stimulated RAW 264.7 cells [56
]. The extract combination significantly decreased the levels of iNOS, PGE2, TNF-α, IL-1β and IL-6 in RAW 264.7 macrophages, and the results were also confirmed in vivo in a paw oedema rat model. Although the study reported lower levels of TNF-α secretion from LPS-stimulated RAW 264.7 cells, in our study, we did not observe significant changes on TNF-α gene expression in both LPS-stimulated leukocytes and LPS/IFN-γ-stimulated macrophages after PSRE and PACN treatment. Proinflammatory cytokine TNF-α plays a critical role not only in inflammatory cell migration, but also in both innate and adaptive immune responses, by up-regulating antigen presentation and the bactericidal activity of phagocytes [57
]. In periodontitis, TNF-α is one of the key signals initiating several signaling pathways leading to chemotaxis of other inflammatory cells, tissue destruction and osteoclast formation [59
]. The fact that PSRE and PACN had no effect on TNF-α expression level while suppressing several other related genes indicate the targets of the substances are located either downstream of the TNF-α signal or in the TNF-a excluding pathway.
Although antiinflammatory properties of PACN and PSRE revealed in the study were of comparative levels, PACN had stronger efficiency in suppressing caspases and preventing mediator release. Stronger anti-inflammatory activity of PACN might be due to greater amounts of prodelphinidins. These compounds possess higher antioxidant capacity and share certain important structural peculiarities, namely hydroxyl groups in B ring (especially in C4’ position and catechol group), hydroxyl groups in the A ring at the C5 and C7 positions [61