Protein J DOI 10.1007/s10930-017-9727-9
Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 Activity Eleonora Nicolai1 · Federica Sinibaldi1 · Gianpaolo Sannino2 · Giuseppina Laganà2 · Francesco Basoli3 · Silvia Licoccia3 · Paola Cozza2 · Roberto Santucci2 · Maria Cristina Piro1
© Springer Science+Business Media, LLC 2017
Abstract Polyunsaturated fatty acids have been reported to play a protective role in a wide range of diseases characterized by an increased metalloproteinases (MMPs) activity. The recent finding that omega-3 and omega-6 fatty acids exert an anti-inflammatory effect in periodontal diseases has stimulated the present study, designed to determine whether such properties derive from a direct inhibitory action of these compounds on the activity of MMPs. To this issue, we investigated the effect exerted by omega-3 and omega-6 fatty acids on the activity of MMP-2 and MMP-9, two enzymes that actively participate to the destruction of the organic matrix of dentin following demineralization operated by bacteria acids. Data obtained (both in vitro and on ex-vivo teeth) reveal that omega-3 and omega-6 fatty acids inhibit the proteolytic activity of MMP-2 and MMP-9, two enzymes present in dentin. This observation is of interest since it assigns to these compounds a key role as MMPs inhibitors, and stimulates further study to better define their therapeutic potentialities in carious decay.
* Maria Cristina Piro
[email protected] 1
Department of Experimental Medicine and Surgery, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy
2
Department of Clinical Sciences and Translational Medicine, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy
3
Department of Chemical Sciences and Technology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, 00133 Rome, Italy
Keywords Matrix metalloproteases inhibition · omega-3 and omega-6 fatty acids · Dentin degradation · Caries · Field emission-scanning electron microscopy Abbreviations MMP-2 Matrix metalloproteinase 2 MMP-9 Matrix metalloproteinase-9 FE-SEM Field emission scanning electron microscope DHA Docosahexaenoic acid
1 Introduction Human matrix metalloproteinases (MMPs) are a family of (at least) 23 enzymes, whose role consists in degrading the extracellular matrix components [1, 2]. MMPs have been divided into six different groups, mainly based on homology and substrate specificity [3]. Most are multidomain enzymes which play a critical role in a variety of biological processes, such as tissue remodeling and angiogenesis, and in a number of diseases, such as cancer, arthritis and neurodegenerative illnesses [4–10]. This has stimulated studies aimed at identifying classes of compounds able to inhibit the enzymatic action of MMPs; among others, long-chain omega-3 fatty acids were reported to exert an anti-inflammatory activity which reduces the risk of chronic diseases (as arthritis, diabetes, obesity, inflammation, cancer, cardiovascular disease and mental health) [11–16], and the omega-6 linoleic, γ-linolenic, and dihomo-γ-linolenic acids were observed to exert an anti-cancer activity [17] (note that other omega-6 fatty acids, particularly the arachidonic acid, are instead implicated in cancer promotion [18–20]). Omega-3 and omega-6 fatty acids contribute to the structure and function of the phospholipid bilayers in cellular membranes and act as precursors of lipid-mediated
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signaling molecules [21–23]. Omega-6 fatty acids mostly come from plant oils (corn oil, soybean oil, sunflower oil) or from nuts and seeds; omega-3 fatty acids derive from fatty fishes, such as salmon, mackerel, and tuna, or from walnuts and flaxseeds oil [11, 17]. Humans are able to convert omega-3 fatty acids with shorter carbon chain (as the α-linolenic acid) to longer chain forms (as the eicosapentaenoic acid, or EPA, and the docosahexaenoic acid, or DHA); these processes take place competitively with the omega-6 fatty acids, which utilize the same desaturase and elongase proteins to synthesize regulatory proteins [18, 24]. MMP-2 and MMP-9 (also called gelatinases A and B) are present in dentin, a tissue composed of minerals, proteins (approx. 90% type-1 collagen), and water, in an approx. 50:30:20 vol% ratio [25–27]. The observation that MMP-2 and MMP-9 actively contribute to the carious process by degrading the organic matrix of dentin after demineralization induced by bacterial acids [28–33], has promoted studies aimed at identifying compounds able to oppose the activity of the MMPs [34–36]. It was recently shown that omega-3 and omega-6 polyunsaturated fatty acids exert an anti-inflammatory effect in periodontitis, a process characterized by the loss of toothsupporting structures (as the connective tissue attachment) caused by an increased MMPs activity [37–42]. This finding has stimulated the present study, aimed at determining whether these compounds can exert an inhibitory effect on the activity of the MMPs (i.e., MMP-2 and MMP-9) present in dentin. This is an important point to be defined, given the great impact that could have on therapies applied to carious decay [28–33]. Linoleic, α-linolenic, γ-linolenic, and decosahexaenoic (DHA) acids were the omega-3 and omega-6 fatty acids investigated in the present study. Measurements, carried out in vitro and on ex-vivo teeth, show that the fatty acids inhibit the MMP-2 and MMP-9 activity. This assigns to these compounds a role as MMPs inhibitors and stimulates further study to better define their therapeutic potentialities against carious decay.
2 Materials and Methods 2.1 Materials MMP-2 and MMP-9 progelatinases, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, DHA, p-aminophenylmercuric acetate (APMA), TIMP-2 (Tissue Inhibitor of Metalloproteinase-2) and Mca-KPLGL-(Dpa)-AR-NH2 (MMP fluorogenic substrate), were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All reagents were of analytical grade.
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2.2 Activation of Pro‑MMPs MMP-2 and MMP-9 progelatinases were fully activated as previously reported [43], by incubating the proenzymes with 1 mM APMA (p-aminophenylmercuric acetate) for 1 h (MMP-2), or overnight (MMP-9), at 37 °C. The buffer was 50 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 10 mM CaCl2, and 0.05% Brij L23. 2.3 Teeth Surface Preparation Teeth (canines and premolars) were extracted for orthodontic reasons after obtaining patients’ informed consent for the extraction of the teeth as part of an overall treatment plan (University of Rome ‘Tor Vergata’ ethical committee approval, No. 129/16). The exclusion criteria were abrasions, caries, enamel cracks, hypoplasia, previous endodontic and restorative treatment, exposure to bleaching procedures. Soft tissues and calculus were carefully removed with Gracey’s curettes 5–6 (Hu-Friedy, Chicago, IL, USA) by the same operator. Before use, teeth were decontaminated in a 0.5% chloramine T solution and stored in a saline solution (Merck, Darmstadt, Germany). Two indentations were made on both the mesial and the distal surfaces of each specimen using a diamond disk on a low-speed handpiece. The teeth were fractured in a mesiodistal direction with a straight extraction lever, to avoid alteration of the inner dentinal tissue due to cutting instrument passing. Sectioned teeth surfaces were then exposed (4 h) to a solution containing 8 nM MMP-9 proteinase in 50 mM Tris–HCl buffer, pH 7.5, containing 150 mM NaCl and 10 mM CaCl2, previously activated. In the samples containing the inhibitor, MMP-9 was incubated for 1 h with one of the fatty acids (acid concentration: 800 μM), at 20 °C, prior to addition onto the teeth surface. The sectioned surfaces of the teeth were then processed for the Field Emission-Scanning Electron Microscope (EF-SEM) measurements, and kept at 4 °C before use. 2.4 Fluorescence Measurements and Analysis of Data The inhibitory effect of fatty acids versus MMP-2 and MMP-9 was analyzed using the fluorescent quenched substrate Mca-KPLGL-(Dpa)-AR-NH2 (MMP fluorogenic substrate) [43]. Fluorescence signal was monitored with a PC1 spectrofluorometer (ISS Inc, Urbana-Champaign, IL, USA) by exciting the samples with a Xenon arc lamp at: λexc = 325 nm, and collecting the emission signal at λem = 387 nm. Measurements were carried out in 50 mM HEPES buffer, pH 7.5, containing 150 mM NaCl+ 5 mM CaCl2. During measurements the temperature was kept constant at 22 °C by an external bath circulator, and checked in the sample holder by a thermocouple. The metalloproteases
Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix…
(200 pM concentration) were pre-incubated with fatty acids (at concentration ranging between 0 and 80 µM) for 30 min (MMP-2) or 60 min (MMP-9), at 22 °C; 2 µM McaKPLGL-(Dpa)-AR-NH2 were then added to the sample, and the substrate fluorescence signal was collected. To calculate the best estimates of the inhibition constant, Ki, non-linear regression analysis was applied by using the following equation [44]: 𝜈i =1− 𝜈0
(
}1 ) {( )2 2 [E]0 + [I]0 + Ki − [E]0 + [I]0 + Ki − 4[E]0 [I]0 2[E]0
where vi and vo are the rates of substrate hydrolysis in the presence and in the absence of the inhibitor, while [E]0 and [I]0 represent the initial concentrations of the enzyme and the inhibitor, respectively. 2.5 Field Emission‑Scanning Electron Microscope (EF‑SEM) Analysis Samples were prepared as previously described [45]; briefly, the material was fixed in 4% formaldehyde for 4 h, dehydrated (through immersion in ethanol solutions
(1)
Fig. 1 Inhibition by fatty acid of MMP-9. Reaction rate in the presence of linoleic acid (0–70 µM)/reaction rate in its absence (a); α-linolenic acid (0–70 µM)/reaction rate in its absence (b); γ-linolenic acid (0–70 µM)/reaction rate in its absence (c); docosahexaenoic acid (0–70 µM)/reaction rate in its absence (d); the data for each fatty acid concentration is the average of at least three different measurements (black dots). Lines represent the best fit curves
obtained fitting the dataset to Eq. 1. Ordinates: the v i/vo ratio indicates the ratio between initial reaction rate in the presence ( vi) and in the absence (vo) of the inhibitor. The fluorescence signal was monitored by exciting the samples with a Xenon arc lamp at λexc = 325 nm, and collecting the emission signal at λem = 387 nm. Measurements were carried out in 50 mM HEPES buffer, pH 7.5, containing 150 mM NaCl and 5 mM CaCl2. The temperature was 22 °C
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Table 1 Inhibitory effect of omega-3, omega-6 and omega-9 fatty acids on MMP-2 and MMP-9 activity Fatty acid
Ki MMP-2
Oleic Linoleic α-Linolenic
(18:1 cis 9) (ω9) (18:2 cis,cis 9,12) (ω6) (18:3 cic,cis,cis 9,12,15) (ω3) γ-Linolenic (18:3 cic,cis,cis 6,9,12) (ω6) Docosahexaenoic (22:6 cic,cis,cis,cic,cis,cis 4,7,10,13,16,19) (ω3)
MMP-9
7.8 ± 0.4 10.3 ± 0.9 10.7 ± 1.5 13.6 ± 1.7 21.8 ± 2.0 18.8 ± 2.1 25.2 ± 2.3 17.2 ± 2.5 18.5 ± 6.7
9.6 ± 2.2
The equilibrium dissociation constants, Ki, for the two MMPs were calculated by nonlinear regression analysis using the equation (Eq. 1) reported in the “Materials and Methods” section. MMP-2 and MMP-9 were incubated with fatty acids prior addition of the fluorogenic substrate in solution. Data on oleic acid are reported for comparative purposes
of increasing concentration: 30, 50, 70, 80, 95 and 100%) and let to dry overnight in a dehydration jar (Corning Inc., NY, USA). Samples were then mounted on metallic stubs with double-sided carbon tape (Leit Adhesive Carbon Tabs, Agar Scientific Ltd., Essex, UK) and metalized for 1 min at 25 mA, by using a sputter-coated (EMITECH K550X, Quorum Technologies Ltd, West Sussex, UK) with a gold target. A Zeiss Supra 35 Field Emission Scanning Electron Microscope (FE-SEM) (Carl Zeiss SMT, Oberkochen, Germany) was used for SEM analysis. The main operating parameters of the instrument, 5 keV as gun Voltage and a working distance of about 11 μm, were chosen to avoid an excessive charging of the specimens. A Second Electron (SE2) detector was used, as the interest was mainly focused on the topography of the canal structure. Two photomicrographs were obtained from the center of each sample, with magnifications of ×1000 and ×10,000 and then assessed (previously calibrated and blind to the experimental groups) by an examiner. The examiner accomplished three consecutive readings for each micrograph. The predominant score of the three readings was considered representative of the respective sample.
3 Results 3.1 Fluorescence Measurements The inhibition in vitro was evaluated by fluorescence spectroscopy, using the Mca-KPLGL-(Dpa)-AR-NH2 fluorogenic compound as substrate. Incubation with MMP-2 or MMP-9 was performed before addition of the substrate. The omega-3 and omega-6 fatty acids investigated in the
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present study, namely linoleic, α-linolenic, γ-linolenic, and DHA possess a different chain length and degree of unsaturation (see next section for a detailed description). This made it possible to determine the influence of these factors on the inhibitory effect of fatty acids on the MMPs activity. Figure 1 shows the inhibition exerted by the fatty acids on the activity of MMP-9; a very similar effect was observed on MMP-2 (data not shown). Data obtained reveal that the fatty acids (omega-3 and omega-6) inhibit the MMP-2 and MMP-9 activity in a dose-dependent manner. The equilibrium dissociation constant, K i, was determined for each fatty acid. The K i values, determined by nonlinear regression analysis [Eq. (1) in the “Materials and Methods” section], are reported in Table 1. The results obtained provide clear evidence that the fatty acids inhibit the activity of both MMP-2 and MMP-9; this suggests that the number and position of the double bonds in the chain of the acid does not significantly affect the interaction with the MMP. 3.2 EF‑SEM Measurements and Analysis of Data Defined that the MMP-2 and MMP-9 activity is inhibited by omega-3 and omega-6 fatty acids in vitro, we extended the investigation by determining the effect exerted by fatty acids on dentin of ex vivo teeth. Dentin is a tissue containing, among others, type-1 collagen, the natural MMPs substrate [46]. The process was followed on central sections of ex vivo teeth, by using EF-SEM. Samples were prepared as described in the “Materials and Methods” section. The fatty acids all exerted an inhibitory effect on the activity of the two MMPs, reducing their proteolytic action on dentin. This is shown in Fig. 2 which illustrates the effect of the γ-linolenic acid (one of the fatty acids exerting low inhibition, see Table 1), on the activity of MMP-9. The micrograph of panel A illustrates the tubule longitudinal sections of specimens treated with MMP-9 in the absence of the fatty acid. Comparison with to the micrograph of specimen not treated with MMP-9, shown in panel D, provides clear evidence for the degrading action exerted by the MMP. The tubule appears open and the periluminal organic matrix shows an extensive degradation. Most of type-1 collagen fibers were digested, and only few residual collagen filaments on the bottom of the dentin tubules remain visible. The intertubular dentin does not reveal filamentous fibers. The residual organic matrix still observable in the micrograph may be attributed to enzyme instability or ascribed to a partial protection exerted by the inorganic matrix of dentin. Some porosity is observable due to innervations, while fractures of the structure, which are present in some regions of the micrograph, are attributable to dehydration and/or the presence of high vacuum in the chamber of the FE-SEM. The micrographs of panels B and C show dentin tubules located in two different regions of the
Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix…
Fig. 2 Electron micrographs of the dentin surface treated with MMP-9 in the absence and in the presence of γ-linolenic acid. a Micrograph of dentinal tubule longitudinal sections of specimens treated with MMP-9 in the absence of inhibitor. Tubule appears clearly open and the periluminal organic matrix shows extensive degradation after the MMP-9 treatment. Most of type-1 collagen fibers were digested, leaving only a few residual collagen filaments on the bottom of the dentin tubules. The intertubular dentin does not reveal filamentous fibers. The residual organic matrix still observable in the picture may result from a partial protection from the proteolitic activity of MMP-9. Some observable porosity is due to innervations (arrowheads), while fractures of the structure (arrows), present at some points of the micrograph, are due to the dehydration process and to the presence in the chamber of the FE-SEM of a high vacuum. Original magn. ×10,000; bar 1 µm. b, c Micrographs of dentin tubules longitudinally fractured and treated with MMP-9 in presence of γ-linolenic acid as inhibitor. Micrographs were taken
on two different regions of the same sample. The micrographs show an extended area of a three-dimensional crosslinked fibrillar and not fibrillar organic matrix. Collapsed collagen proteins are visible as a dense network of filaments (arrowheads) covering the tubules and the intertubular dentin structure. Major collagen fibrils and minor branching ones are visible. Fibers are coated with a large amount of nonfibrillar organic material visible as small particles measuring from 50 to 500 nm (arrows), probably representing proteoglycans and glycoproteins. Original magn. ×10,000, bar 1 µm. d Micrograph of undigested dentin incubated with buffer only (control), illustrated here for comparable purposes. Original magn. ×10,000, bar 1 µm. As in panel a, fractures of the structure (arrows) are due to the dehydration process and to the presence in the chamber of the FE-SEM of a high vacuum. Arrowheads show collapsed collagen fibers, visible as a dense network of filaments covering the tubules and the intertubular dentin structure
sample, longitudinally fractured and treated with MMP-9 in presence of γ-linolenic acid. An extended area of a threedimensional crosslinked fibrillar and not fibrillar organic matrix is clearly observable; collapsed collagen is visible as a dense network of filaments covering the tubules and the intertubular dentin structure. Major collagen fibrils and minor branching ones are well visible. Furthermore fibers
are coated with a large amount of non-fibrillar organic material, visible as small particles measuring from 50 to 500 nm, probably representing proteoglycans and glycoproteins. Taken as a whole, the reduced dentin degradation observed in the presence of the fatty acids demonstrates that these compounds inhibit the MMP-2 and MMP-9 activity.
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Fig. 3 Structure of fatty acids. With the exception of the oleic acid, which possesses only one double bond in the structure, the other fatty acids are polyunsaturated. The linoleic acid possesses two double bonds, three double bonds are present in the α- and γ-linolenic acids, and six double bonds are present in the structure of the DHA. All fatty acids are cis-double bonds. The blue numbers indicate the position of the double bonds with respect to the carboxylic group (which is considered the beginning of the chain), while the red numbers indicate the distance of the double bonds from the methyl end. (Color figure online)
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ω 1
9 1
9
18
Oleic acid (18:1, ω9)
Linoleic acid (18:2, ω6)
α-linolenic acid (18:3,
γ-linolenic acid (18:3,
3
HO
1
4
7
10
13
16
ω 1
19
Docosahexaenoic acid (22:6, ω3)
4 Discussion The structures of DHA, linoleic, α-linolenic, and γ-linolenic acids (i.e., the omega-3 and omega-6 fatty acids investigated in the present study) are shown in Fig. 3. Linoleic and γ-linolenic acids are two 18-carbonchain polyunsaturated omega-6 fatty acids, the former containing two cis-(at positions 9, 12) double bonds, the latter three cis-(at positions 6, 9, 12) double bonds. In both, the first double bond is located at the sixth carbon from the methyl end (omega-6). α-Linolenic acid and DHA are two polyunsaturated fatty acids, the first containing three cis-double bonds (at positions 3, 6, 9) in a 18-carbon-chain, the second six cis-double bonds (at positions 4, 7, 10, 13, 16, 19) in a 22-carbon-chain. In both, the first double bond is located at the third carbon from the methyl end (omega-3). As reported in Table 1, the fatty acids inhibit the activity of the two gelatinases
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with similar efficacy. These results are in good agreement with a previous study asserting that chain unsaturation and a 18–20 carbon chain length are two factors favoring the inhibition of MMPs activity by fatty acids [43]. In the table, the value of K i relative to the oleic acid is also reported; this compound, which possesses one cis-double bond at position 9 from the chain end (thus, it is classified as a omega-9 fatty acid), is known to possess a strong inhibitory power vs the MMP-2 and MMP-9 activity [43]. Among the fatty acids investigated herein, linoleic acid is the one exerting the strongest inhibition on the MMPs activity; the values of Ki determined for this compound are similar to those previously calculated for the oleic acid. Both fatty acids possess a 18-carbon chain but differ for the number of double bonds (one double bond in the oleic acid, two double bonds in the linoleic acid); this suggests that the additional double bond at position 6 in the linoleic acid is responsible, at least in part, for
Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix…
the differences in the effect shown versus the two MMPs. On the other hand, the number of double bonds present in the acid chain cannot be considered the only factor affecting (and governing) the fatty acid-MMPs interaction; the inhibition exerted by DHA, a compound with six double bonds in the structure, is comparable to that of the α-linolenic and γ-linolenic acids (each possessing three double bonds in the chain). Also, the similar effect shown by the (omega-3) α-linolenic acid and the (omega-6) γ-linolenic acid suggests that the position of the first double bond from the chain end is not relevant, since it does not significantly affect the interaction with the MMP. The inhibitory effect of the fatty acids investigated herein is highlighted by the experimental results, which are supported by a good agreement between EF-SEM and fluorescence measurements. Such agreement can be summarized as follows: (a) the residual type-1 collagen still observed in the presence of fatty acids by EF-SEM is in good agreement with the reduced MMPs activity detected in vitro by fluorescence; (b) the finding that similar FESEM images are obtained (at the same magnification) in the presence of different fatty acids having the same concentration is in good agreement with data in vitro showing that same concentrations of different fatty acids inhibit the MMPs activity of about the same extent. This also indicates that the differences observed for the Ki values are not crucial since they do not affect the behavior of fatty acids in dentin of ex-vivo teeth. Note that, to our knowledge, this is the first report in which the direct inhibition of omega-3 and omega-6 fatty acids on the activity of MMPs is demonstrated. In conclusion, it was reported that the anti-inflammatory effects elicited by fatty acids decrease the risk of a number of chronic diseases such as arthritis, cancer, and cardiovascular diseases [26, 27, 30–34]; data reported herein extend the potential of these compounds in the treatment of the carious decay. Preservation of the collagen matrix integrity is an important issue to improve dentin durability; thus, the finding that omega-3 and omega-6 fatty acids inhibit MMPs activity vs dentin degradation is important not only per se, it opens new perspectives in therapeutic field to limit caries progression in dentin. omega-3 and omega-6 fatty acids appear to be suitable candidates for such issues. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflicts of interest. Ethical Approval Teeth were extracted after obtaining patients’ informed consent, as part of an overall treatment plan (University of Rome ‘Tor Vergata’ ethical committee approval, No. 129/16).
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