**3.1. Results**

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trichrome stain and studied using light microscopy (Dialux 22 Leitz).

the entire paw and digits (Szabo *et al.*, 1998). Arthritic index for each mouse was calculated by adding the four scores of individual paws. Clinical severity was also determined by quantitating the change in the paw volume using plethysmometry (model 7140; Ugo Basile).

*Histological examination -* On day 35, animals were sacrificed while they were under anesthesia (sodium pentobarbital, 45 mg/kg, i.p), and paws and knees were removed and fixed in 10% formalin. The paws were then trimmed, placed in decalcifying solution for 24 h, embedded in paraffin, sectioned at 5 µm, stained with hematoxylin/eosin and Masson's

*Immunohistochemical localization of nitrotyrosine, Poly ADP Ribose (PAR), iNOS, and COX-2 -* On day 35, the joints were trimmed, placed in decalcifying solution for 24 h and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% normal goat serum in phosphate buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. Sections were incubated overnight with 1) anti-rabbit polyclonal antibody directed at iNOS (1:1000 in PBS, v/v) (DBA, Milan, Italy) or 2) anti-COX-2 goat polyclonal antibody (1:500 in PBS, v/v) or 3) anti- nitrotyrosine rabbit polyclonal antibody (1:1000 in PBS, v/v) or 4) with anti-PAR goat polyclonal antibody rat (1:500 in PBS, v/v) or 5). Controls included buffer alone or non-specific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG (for nitrotyrosine and iNOS) or with a biotin-conjugated goat anti-rabbit IgG (for PAR and COX-2) and avidin-biotin peroxidase complex. In order to confirm that the immunoreaction for the nitrotyrosine was specific some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PAR, COX-2 and iNOS, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections indicating that the immunoreaction was positive in all the experiments carried out. Immunocytochemistry photographs (N=5) were assessed by

densitometry by using Optilab Graftek software on a Macintosh personal computer.

Milan, Italy) with a lower detection limit of 10 pg/ml.

*Measurement of cytokines* **-** Tumor Necrosis Factor-α (TNF-α) levels were evaluated in the plasma from CIA and sham mice as previously described (Cuzzocrea *et al.*, 2006). The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-Novabiochem Co.,

*Measurement of chemokines -* Levels of chemokines MIP-1α and MIP-2 were measured in the aqueous joint extracts. Briefly, joint tissues were prepared by first removing the skin and separating the limb below the ankle joint. Joint tissues were homogenized on ice in 3 ml lysis buffer (PBS containing: 2 mM PMSF, and 0,1 mg/ml [final concentration], each of aprotinin, antipain, leupeptin, and pepstatin A) using Polytron (Brinkinarm Instr., Westbury, NY). The homogenized tissues were then centrifuged at 2,000 g for 10 min. Supernatant were sterilized with a millipore filter (0.2 µm) and stored at –80oC until

*Effect of 3,4-DHPEA-EA on joint injury during experimental arthritis -* To imitate the clinical scenario of RA, mice were subjected to CIA. CIA developed rapidly in mice immunized with CII and clinical signs (periarticular erythema and edema) (Fig. 6B) of the disease first appeared in hind paws between 24 and 26 days post-challenge (Fig. 6D) leading to a 100% incidence of

CIA at day 28 (Fig. 6D). Hind paw erythema and swelling increased in frequency and severity in a time-dependent mode with maximum arthritis indices of approximately 10 observed between day 29 to 35 post immunization (Fig. 6D) in CIA-control mice. 3,4-DHPEA-EA treatment demonstrated a significant reduction of joint inflammation, as identified by a significant reduction in the incidence of arthritis (Figure 6C). CIA-3,4-DHPEA-EA mice showed a 40% reduction in the development of arthritis and a significantly lower arthritis index compared to CIA- control mice (Fig. 6D). There was no macroscopic evidence of either hind paw erythema or edema in the sham-control group mice (Fig. 6A and D). The data in Figure 6E demonstrate a time-dependent increase in hind paw volume (each value represents the mean of both hind paws). The CIA-3,4-DHPEA-EA mice showed a significant reduction of paw edema formation when compared to CIA-control mice (Fig. 6E). No increase in hind paw volume over time was observed in the sham-control mice (Fig. 6E). The rate and the absolute gain in body weight were comparable in sham-control and CIA-control mice in the first week (Fig. 6F). From day 25, the CII-challenged mice gained significantly less weight than the shamcontrol mice, and this trend continued through to day 35. 3,4-DHPEA-EA treatment determined a significant increase of the weight gain compared with the vehicle-treatment in CIA-control mice (Fig. 6F). The histological evaluation (on day 35) of the joint from CIAcontrol mice (Fig. 7B) revealed signs of severe arthritis, with inflammatory cell infiltration and bone erosion. The histological alterations of joint were significantly reduced in 3,4-DHPEA-EA-treated mice (Fig. 7C). Moreover Masson's trichrome stain reveals decreased collagen (blue stain) in bone and cartilage of arthritic joint due to bone erosion and cartilage degradation in CIA-control mice (Fig. 7E). The alterations of joint were significantly reduced in 3,4-DHPEA-EA-treated mice (Fig. 7F). There was no evidence of pathology in the sham-control mice (Fig. 7A and D). The histological score (Fig. 7G) was determined by an independent observer.

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mice revealed positive staining for iNOS (Fig. 10, A and A1) and COX-2 (Fig. 9, A and A1), which were primarily localized in inflammatory cells. In contrast, staining for iNOS (Fig. 10B) and COX-2 (Fig. 9B) was markedly reduced in the tibiotarsal joints of CIA-3,4-DHPEA-EA (40 µg/kg) mice. No staining for either iNOS or COX-2 was detected in the tibiotarsal joints obtained from sham control mice (data not shown). Moreover, we also evaluated the

**Figure 6.** Effect of 3,4-DHPEA-EA (Ole aglycone) on the clinical expression of CIA and on body weight. A, no clinical signs were observed in sham mice. CIA developed rapidly in mice immunized with CII and clinical signs such as periarticular erythema and edema (B) were seen with a 100% incidence of CIA at day 28 (D). E, hind paw erythema and swelling increased in frequency and severity in a time dependent mode. CIA-3,4-DHPEA-EA mice demonstrated a significant reduction in the clinical signs of CIA (C), leading to a decrease in the incidence of arthritis in a dose-dependent manner (D). Swelling of hind paws (F) over time was measured at 2-day intervals. G, beginning on day 25, the CII-challenged mice gained significantly less weight and this trend continued through day 35. CIA-3,4-DHPEA-EA mice demonstrated a significant reduced incidence of weight loss (G) as well as less paw edema in a dose dependent manner (F). The figure is representative of all the animals in each group. Values are means ± S.E.M. of 20 animals for each group. \*\*, P < 0.01 versus sham-control; °, P < 0.01 versus CIA.

*Effect of 3,4-DHPEA-EA on cytokines, chemokine expression and neutrophil infiltration -* We initiated studies to assess the effect of 3,4-DHPEA-EA on the expression of chemokines in the aqueous joint extracts during the development of CIA. As shown in Fig. 8, A and B, the expression of MIP-1α and MIP-2, measured by ELISA, was significantly increased in the joint 35 days after CII immunization. MIP-1α and MIP-2 levels in CIA-3,4-DHPEA-EA mice on day 35 were significantly reduced in a dose-dependent manner in comparison with those in vehicle treated CIA-control mice. Assessment of neutrophil infiltration into the inflamed joint tissue was performed by measuring the activity of MPO. It was significantly elevated 35 days after CII immunization in vehicle-treated CIA-control mice (Fig. 8F), whereas in the CIA-3,4-DHPEA-EA group, MPO activity was markedly reduced in a dose-dependent manner (Fig. 8F). To test whether 3,4-DHPEA-EA modulates the inflammatory process through the regulation of cytokine secretion, we analyzed the plasma levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6. A substantial increase in TNF-α, (Fig. 8C), IL-1β (Fig. 8D), and IL-6 (Fig. 8E) production was found in CIA-control mice 35 days after CII immunization. Levels of TNF-α (Fig. 8C), IL-1β (Fig. 8D), and IL-6 (Fig. 8E) were significantly reduced in a dose-dependent manner in CIA-3,4-DHPEA-EA mice in comparison to CIA-control mice.

*Effect of 3,4-DHPEA-EA treatment on iNOS, COX-2, PGE2, nitrotyrosine, and PAR formation -* Immunohistochemical analysis of the tibiotarsal joint sections obtained from CIA-control mice revealed positive staining for iNOS (Fig. 10, A and A1) and COX-2 (Fig. 9, A and A1), which were primarily localized in inflammatory cells. In contrast, staining for iNOS (Fig. 10B) and COX-2 (Fig. 9B) was markedly reduced in the tibiotarsal joints of CIA-3,4-DHPEA-EA (40 µg/kg) mice. No staining for either iNOS or COX-2 was detected in the tibiotarsal joints obtained from sham control mice (data not shown). Moreover, we also evaluated the

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CIA at day 28 (Fig. 6D). Hind paw erythema and swelling increased in frequency and severity in a time-dependent mode with maximum arthritis indices of approximately 10 observed between day 29 to 35 post immunization (Fig. 6D) in CIA-control mice. 3,4-DHPEA-EA treatment demonstrated a significant reduction of joint inflammation, as identified by a significant reduction in the incidence of arthritis (Figure 6C). CIA-3,4-DHPEA-EA mice showed a 40% reduction in the development of arthritis and a significantly lower arthritis index compared to CIA- control mice (Fig. 6D). There was no macroscopic evidence of either hind paw erythema or edema in the sham-control group mice (Fig. 6A and D). The data in Figure 6E demonstrate a time-dependent increase in hind paw volume (each value represents the mean of both hind paws). The CIA-3,4-DHPEA-EA mice showed a significant reduction of paw edema formation when compared to CIA-control mice (Fig. 6E). No increase in hind paw volume over time was observed in the sham-control mice (Fig. 6E). The rate and the absolute gain in body weight were comparable in sham-control and CIA-control mice in the first week (Fig. 6F). From day 25, the CII-challenged mice gained significantly less weight than the shamcontrol mice, and this trend continued through to day 35. 3,4-DHPEA-EA treatment determined a significant increase of the weight gain compared with the vehicle-treatment in CIA-control mice (Fig. 6F). The histological evaluation (on day 35) of the joint from CIAcontrol mice (Fig. 7B) revealed signs of severe arthritis, with inflammatory cell infiltration and bone erosion. The histological alterations of joint were significantly reduced in 3,4-DHPEA-EA-treated mice (Fig. 7C). Moreover Masson's trichrome stain reveals decreased collagen (blue stain) in bone and cartilage of arthritic joint due to bone erosion and cartilage degradation in CIA-control mice (Fig. 7E). The alterations of joint were significantly reduced in 3,4-DHPEA-EA-treated mice (Fig. 7F). There was no evidence of pathology in the sham-control mice (Fig. 7A and D). The histological score (Fig. 7G) was determined by an independent observer.

*Effect of 3,4-DHPEA-EA on cytokines, chemokine expression and neutrophil infiltration -* We initiated studies to assess the effect of 3,4-DHPEA-EA on the expression of chemokines in the aqueous joint extracts during the development of CIA. As shown in Fig. 8, A and B, the expression of MIP-1α and MIP-2, measured by ELISA, was significantly increased in the joint 35 days after CII immunization. MIP-1α and MIP-2 levels in CIA-3,4-DHPEA-EA mice on day 35 were significantly reduced in a dose-dependent manner in comparison with those in vehicle treated CIA-control mice. Assessment of neutrophil infiltration into the inflamed joint tissue was performed by measuring the activity of MPO. It was significantly elevated 35 days after CII immunization in vehicle-treated CIA-control mice (Fig. 8F), whereas in the CIA-3,4-DHPEA-EA group, MPO activity was markedly reduced in a dose-dependent manner (Fig. 8F). To test whether 3,4-DHPEA-EA modulates the inflammatory process through the regulation of cytokine secretion, we analyzed the plasma levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6. A substantial increase in TNF-α, (Fig. 8C), IL-1β (Fig. 8D), and IL-6 (Fig. 8E) production was found in CIA-control mice 35 days after CII immunization. Levels of TNF-α (Fig. 8C), IL-1β (Fig. 8D), and IL-6 (Fig. 8E) were significantly reduced in a dose-dependent

*Effect of 3,4-DHPEA-EA treatment on iNOS, COX-2, PGE2, nitrotyrosine, and PAR formation -* Immunohistochemical analysis of the tibiotarsal joint sections obtained from CIA-control

manner in CIA-3,4-DHPEA-EA mice in comparison to CIA-control mice.

**Figure 6.** Effect of 3,4-DHPEA-EA (Ole aglycone) on the clinical expression of CIA and on body weight. A, no clinical signs were observed in sham mice. CIA developed rapidly in mice immunized with CII and clinical signs such as periarticular erythema and edema (B) were seen with a 100% incidence of CIA at day 28 (D). E, hind paw erythema and swelling increased in frequency and severity in a time dependent mode. CIA-3,4-DHPEA-EA mice demonstrated a significant reduction in the clinical signs of CIA (C), leading to a decrease in the incidence of arthritis in a dose-dependent manner (D). Swelling of hind paws (F) over time was measured at 2-day intervals. G, beginning on day 25, the CII-challenged mice gained significantly less weight and this trend continued through day 35. CIA-3,4-DHPEA-EA mice demonstrated a significant reduced incidence of weight loss (G) as well as less paw edema in a dose dependent manner (F). The figure is representative of all the animals in each group. Values are means ± S.E.M. of 20 animals for each group. \*\*, P < 0.01 versus sham-control; °, P < 0.01 versus CIA.

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positive staining for PAR (Fig. 12, A and A1). In contrast, no positive PAR was found in the tibiotarsal joints of CII-challenged mice treated with 3,4-DHPEA-EA (40 µg/kg) (Fig. 12B). There was no staining for either nitrotyrosine or PAR in the tibiotarsal joints obtained from

**Figure 8.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on cytokine and chemokine expression and neutrophil infiltration. A substantial increase in the expression of MIP-1 (A), MIP-2 (B), MPO activity (F), plasma TNF-α (C), IL-1β (D), and IL-6 levels (E) was found in CIA-control mice 35 days after CII immunization. CIA-3,4-DHPEA-EA mice demonstrated a significant reduction in the expression of MIP-1 (A), MIP-2 (B), MPO activity (F), plasma TNF-α (C), IL-1β (D), and IL-6 levels in a dose

dependent manner (E). Values are means ± S.E.M. of 20 animals for each group. \*\*, P < 0.01 versus sham

control; °, P < 0.01 versus CIA-control. ND, not detectable.

sham-control mice (data not shown).

**Figure 7.** Morphological changes of CIA. Representative hematoxylin and eosin-stained section of joint was examined by light microscopy. The histological evaluation (on day 35) of joint from CIA-control mice (B and G) revealed signs of severe arthritis, with inflammatory cell infiltration and bone erosion. The histological alterations of the joint were significantly reduced in the tissues from CIA-3,4-DHPEA-EA (40 µg/kg)-treated mice (C and G). Masson's trichrome stain reveals decreased collagen in bone and cartilage of arthritic joint due to bone erosion and cartilage degradation in CIA-control mice (E and G). The alterations of joint were significantly reduced in 3,4-DHPEA-EA (40 µg/kg)-treated mice (F and G). There was no evidence of pathology in the sham-control mice (A, D, and G). The histological score (G) was made by an independent observer. The figure is representative of at least three experiments performed on different experimental days. Values are means ± S.E.M. of 20 animals for each group. \*\*, P < 0.01 versus sham-control; °, P < 0.01 versus CIA.

levels of PGE2, the metabolite of COX-2, in the serum during the development of CIA. A substantial increase in PGE2 production was found in CIA-control mice 35 days after CII immunization (Fig. 9E). Levels of PGE-2 were significantly reduced in CIA-3,4-DHPEA-EA mice in a dose dependent manner compared with those in CIA-control mice (Fig. 9E). The release of free radicals and oxidant molecules during chronic inflammation has been suggested to contribute significantly to the tissue injury (Cuzzocrea *et al.*, 2001). On day 35, positive staining for nitrotyrosine, a marker of nitrosative injury, was found in the tibiotarsal joints of vehicle-treated CIA-control mice (Fig. 11, A and A1). 3,4-DHPEA-EA (40 µg/kg) treatment significantly reduced the formation of nitrotyrosine (Fig. 11B). Immunohistochemical analysis of joint sections obtained from CII-challenged mice revealed positive staining for PAR (Fig. 12, A and A1). In contrast, no positive PAR was found in the tibiotarsal joints of CII-challenged mice treated with 3,4-DHPEA-EA (40 µg/kg) (Fig. 12B). There was no staining for either nitrotyrosine or PAR in the tibiotarsal joints obtained from sham-control mice (data not shown).

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**Figure 7.** Morphological changes of CIA. Representative hematoxylin and eosin-stained section of joint was examined by light microscopy. The histological evaluation (on day 35) of joint from CIA-control mice (B and G) revealed signs of severe arthritis, with inflammatory cell infiltration and bone erosion. The histological alterations of the joint were significantly reduced in the tissues from CIA-3,4-DHPEA-EA (40 µg/kg)-treated mice (C and G). Masson's trichrome stain reveals decreased collagen in bone and cartilage of arthritic joint due to bone erosion and cartilage degradation in CIA-control mice (E and G). The alterations of joint were significantly reduced in 3,4-DHPEA-EA (40 µg/kg)-treated mice (F and G). There was no evidence of pathology in the sham-control mice (A, D, and G). The histological score (G) was made by an independent observer. The figure is representative of at least three experiments performed on different experimental days. Values are means ± S.E.M. of 20 animals for each group. \*\*, P

levels of PGE2, the metabolite of COX-2, in the serum during the development of CIA. A substantial increase in PGE2 production was found in CIA-control mice 35 days after CII immunization (Fig. 9E). Levels of PGE-2 were significantly reduced in CIA-3,4-DHPEA-EA mice in a dose dependent manner compared with those in CIA-control mice (Fig. 9E). The release of free radicals and oxidant molecules during chronic inflammation has been suggested to contribute significantly to the tissue injury (Cuzzocrea *et al.*, 2001). On day 35, positive staining for nitrotyrosine, a marker of nitrosative injury, was found in the tibiotarsal joints of vehicle-treated CIA-control mice (Fig. 11, A and A1). 3,4-DHPEA-EA (40 µg/kg) treatment significantly reduced the formation of nitrotyrosine (Fig. 11B). Immunohistochemical analysis of joint sections obtained from CII-challenged mice revealed

< 0.01 versus sham-control; °, P < 0.01 versus CIA.

**Figure 8.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on cytokine and chemokine expression and neutrophil infiltration. A substantial increase in the expression of MIP-1 (A), MIP-2 (B), MPO activity (F), plasma TNF-α (C), IL-1β (D), and IL-6 levels (E) was found in CIA-control mice 35 days after CII immunization. CIA-3,4-DHPEA-EA mice demonstrated a significant reduction in the expression of MIP-1 (A), MIP-2 (B), MPO activity (F), plasma TNF-α (C), IL-1β (D), and IL-6 levels in a dose dependent manner (E). Values are means ± S.E.M. of 20 animals for each group. \*\*, P < 0.01 versus sham control; °, P < 0.01 versus CIA-control. ND, not detectable.

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**Figure 10.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on iNOS immunostaining. A marked increase in iNOS (A and in particular A1) staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for iNOS (B) in the paw of CIA-3,4-DHPEA-EA (40 µg/kg) mice. To verify the binding specificity for iNOS, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for iNOS was found in the sections, indicating that the immunoreaction was positive (see negative control C). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for iNOS from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3- 266). Data are expressed as a percentage of total tissue area. \*\*, P < 0.01 versus sham-control; °, P < 0.01

versus CIA. ND, not detectable.

**Figure 9.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on COX-2 immunostaining and on serum PGE2 levels. A marked increase in COX-2 (A and in particular A1) staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for COX-2 (B) in the paw of CIA-3,4- DHPEA-EA (40 µg/kg) mice. To verify the binding specificity for COX-2, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for COX-2 was found in the sections indicating that the immunoreaction was positive (see negative control C). In addition, a marked increase of PGE2 levels was found in the serum of CIA control mice 35 days after CII immunization (E). The treatment with 3,4-DHPEA-EA also caused a significant reduction in a dose-dependent manner of the serum levels of the metabolite of COX-2 (E). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for COX-2 from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total tissue area. \*\*, P < 0.01 versus sham control; °, P < 0.01 versus CIA. ND, not detectable.

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**Figure 9.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on COX-2 immunostaining and on serum PGE2 levels. A marked increase in COX-2 (A and in particular A1) staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for COX-2 (B) in the paw of CIA-3,4- DHPEA-EA (40 µg/kg) mice. To verify the binding specificity for COX-2, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for COX-2 was found in the sections indicating that the immunoreaction was positive (see negative control C). In addition, a marked increase of PGE2 levels was found in the serum of CIA control mice 35 days after CII immunization (E). The treatment with 3,4-DHPEA-EA also caused a significant reduction in a dose-dependent manner of the serum levels of the metabolite of COX-2 (E). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for COX-2 from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total tissue area. \*\*, P <

0.01 versus sham control; °, P < 0.01 versus CIA. ND, not detectable.

**Figure 10.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on iNOS immunostaining. A marked increase in iNOS (A and in particular A1) staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for iNOS (B) in the paw of CIA-3,4-DHPEA-EA (40 µg/kg) mice. To verify the binding specificity for iNOS, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for iNOS was found in the sections, indicating that the immunoreaction was positive (see negative control C). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for iNOS from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3- 266). Data are expressed as a percentage of total tissue area. \*\*, P < 0.01 versus sham-control; °, P < 0.01 versus CIA. ND, not detectable.

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**Figure 12.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on PARP immunostaining. A marked increase in PARP (A and in particular A1), staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for PARP (B) in the paw of CIA-3,4-

control; °, P<0.01 versus CIA. ND, not detectable.

DHPEA-EA (40 g/kg)-treated mice. To verify the binding specificity for PARP, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for PARP was found in the sections, indicating that the immunoreaction was positive (see negative control C). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for PARP from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total tissue area. \*\*, P <0.01 versus sham-

**Figure 11.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on nitrotyrosine immunostaining. A marked increase in nitrotyrosine (A and in particular A1) staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for nitrotyrosine (B) in the paw of CIA-3,4-DHPEA-EA (40 µg/kg)-treated mice. To verify the binding specificity for nitrotyrosine, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for nitrotyrosine was found in the sections, indicating that the immunoreaction was positive (see negative control C). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for nitrotyrosine from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total tissue area. \*\*, P < 0.01 versus sham control; °, P < 0.01 versus CIA. ND, not detectable.

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**Figure 11.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on nitrotyrosine immunostaining. A marked increase in nitrotyrosine (A and in particular A1) staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for nitrotyrosine (B) in the paw of CIA-3,4-DHPEA-EA (40 µg/kg)-treated mice. To verify the binding specificity for nitrotyrosine, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for nitrotyrosine was found in the sections, indicating that the immunoreaction was positive (see negative control C). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for nitrotyrosine from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total

tissue area. \*\*, P < 0.01 versus sham control; °, P < 0.01 versus CIA. ND, not detectable.

**Figure 12.** Effect of 3,4-DHPEA-EA (Ole aglycone) treatment on PARP immunostaining. A marked increase in PARP (A and in particular A1), staining was evident in the paw 35 days after initiation of CIA. There was a marked reduction in the immunostaining for PARP (B) in the paw of CIA-3,4- DHPEA-EA (40 g/kg)-treated mice. To verify the binding specificity for PARP, some sections were also incubated with only the secondary antibody (no primary antibody). No positive staining for PARP was found in the sections, indicating that the immunoreaction was positive (see negative control C). The figure is representative of at least three experiments performed on different experimental days. Densitometry analysis of immunocytochemistry photographs (n = 5) for PARP from paw section was assessed (D). The assay was performed by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as a percentage of total tissue area. \*\*, P <0.01 versus shamcontrol; °, P<0.01 versus CIA. ND, not detectable.

**3,4-DHPEA-EA Inhibits the Progression of Established Arthritis -** To confirm that 3,4- DHPEA-EA exerts beneficial effects in the experimental model of collagen-induced arthritis, we have also evaluated its effect in a therapeutic regimen of post-treatment (40 µg/kg), starting the treatment at day 28. CIA-3,4-DHPEA-EA post-treatment mice also showed a reduction in the development of arthritis and a significantly lower arthritis score compared with those in CIA-control mice as shown in Fig. 13A. 3,4-DHPEA-EA post-treatment also significantly reduced paw edema formation (Fig. 13B). In addition, 3,4-DHPEA-EA posttreated mice showed significantly reduced histological alterations of the tibiotarsal joint as shown in the histological score (Fig. 13C) and increased body weight (Fig. 13D).

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tissue with the potential for bone erosion and cartilage degradation known as pannus (Filippin *et al.*, 2008). Therefore, it is necessary to establish and characterize experimental animal models to assess cellular and molecular events that contribute to the pathogenesis of joint inflammation. Of interest, CII-induced arthritis in the mouse has proven to be a useful model, because it possesses many of the cellular and humoral immune events found in human rheumatoid arthritis. Oxidative stress describes an imbalance between ROS synthesis and antioxidants. Many studies have demonstrated a role of ROS in the pathogenesis of inflammatory chronic arthropathies, such as rheumatoid arthritis (Filippin *et al.*, 2008). In this regard, we investigate here the effects of 3,4-DHPEA-EA, a hydrolysis product of oleuropein, in a mouse model of CII-induced arthritis. Although T-cell and antibody responses against CII are a crucial event for the initiation of CIA (Holmdahl *et al.*, 1989), it has been demonstrated that several cytokines also appear to direct cell-to-cell communication in a cascade fashion during the progression of CIA such as IL-1 (Hom *et al.*, 1992), TNF-α (Dong *et al.*, 2010), and IL-6 (Ferraccioli *et al.*, 2010). In addition, it has been demonstrated that monocyte chemotactic protein-1, MIP-α, MIP-1β, and regulated on activation normal T cell expressed and secreted are differentially chemotactic for lymphocyte subsets and are expressed in tissue from the inflamed joints of patients with rheumatoid arthritis (Koch *et al.*, 1994). In this study, we have confirmed that the cytokines (TNF-α, IL-1β, and IL-6) as well as the chemokines (MIP-1α and MIP-2) are expressed at sites of inflamed joints and probably contribute in different capacities to the progression of chronic joint inflammation. Several cytokines, including TNF-α and IL-1β, are known initiators of the nuclear factor (NF- κB) activation cascade (Filippin *et al.*, 2008) and are under its transcriptional control, constituting a positive feedback loop. Recent studies have observed that the acute consumption of olive oil decreased the activation of the NF- κB system on mononuclear cells from healthy men (Perez-Martinez *et al.*, 2007) and that 3,4- DHPEA-EA, *trans*-resveratrol, and hydroxytyrosol incubated with human umbilical vein endothelial cells inhibit lipopolysaccharide-triggered NF- κB and activator protein-1 activation (Carluccio *et al.*, 2003). Of interest, using 3,4-DHPEA-EA, we have demonstrated an inhibition of the release of proinflammatory cytokines and chemokines and a reduction of leukocyte infiltration measured by MPO activity. Several studies also showed that the potential cardioprotective activity of oleuropein in acute cardiotoxicity induced by doxorubicin treatment was determined in vivo in rats (Andreadou *et al.*, 2007) by inhibiting lipid peroxidation products, decreasing oxidative stress, and reducing iNOS in cardiomyocytes and that the olive oil polyphenols are capable of down-regulating COX-2 expression in colonic cancer cells by a mechanism involving the early inhibition of p38 mitogen-activated protein kinase and downstream inhibition of the transcription factor cAMP response element-binding protein (Corona *et al.*, 2007). We show here that 3,4- DHPEA-EA decreased iNOS and COX-2 expression by immunohistochemical staining and also reduced the levels of the metabolite of COX-2, PGE2, in the serum of 3,4-DHPEA-EA treated mice. Reactive nitrogen species, such as the peroxynitrite radical (ONOO\_) generated by the reaction between O2 . and nitric oxide, can also cause oxidative damage (Soneja *et al.*, 2005). The addition of ONOO\_ to body cells, tissues, and fluids leads to fast protonation, which may result in the depletion of -SH groups and other antioxidants, oxidation and

**Figure 13.** Effect of 3,4-DHPEA-EA (Ole aglycone) post-treatment on joint inflammation. Starting the treatment at day 28, we have also demonstrated that 3,4-DHPEA-EA post-treatment (40 µg/kg) caused a significantly lower arthritis score (A) and a reduction of foot increase (B) compared with the CIAcontrol. In addition, we have also shown a reduction in the histological damage (C) and increased body weight (D) in 3,4-DHPEA-EA -treated mice. Data are expressed as a percentage of total tissue area. \*\*, *P<* 0.01 versus sham-control; °, *P<* 0.01 versus CIA.

#### **3.2. Discussion**

Rheumatoid arthritis is an inflammatory disease characterized by chronic inflammation of the synovial joints associated with proliferation of synovial cells and infiltration of activated immunoinflammatory cells, including memory T cells, macrophages, and plasma cells, leading to progressive destruction of cartilage and bone (Hitchon *et al.*, 2004). Another central feature of RA synovitis is the transformation of fibroblast-like synovial cells into autonomously proliferating cells with a tissue-infiltrating nature, forming hyperplastic

**3,4-DHPEA-EA Inhibits the Progression of Established Arthritis -** To confirm that 3,4- DHPEA-EA exerts beneficial effects in the experimental model of collagen-induced arthritis, we have also evaluated its effect in a therapeutic regimen of post-treatment (40 µg/kg), starting the treatment at day 28. CIA-3,4-DHPEA-EA post-treatment mice also showed a reduction in the development of arthritis and a significantly lower arthritis score compared with those in CIA-control mice as shown in Fig. 13A. 3,4-DHPEA-EA post-treatment also significantly reduced paw edema formation (Fig. 13B). In addition, 3,4-DHPEA-EA posttreated mice showed significantly reduced histological alterations of the tibiotarsal joint as

shown in the histological score (Fig. 13C) and increased body weight (Fig. 13D).

**Figure 13.** Effect of 3,4-DHPEA-EA (Ole aglycone) post-treatment on joint inflammation. Starting the treatment at day 28, we have also demonstrated that 3,4-DHPEA-EA post-treatment (40 µg/kg) caused a significantly lower arthritis score (A) and a reduction of foot increase (B) compared with the CIAcontrol. In addition, we have also shown a reduction in the histological damage (C) and increased body weight (D) in 3,4-DHPEA-EA -treated mice. Data are expressed as a percentage of total tissue area. \*\*,

Rheumatoid arthritis is an inflammatory disease characterized by chronic inflammation of the synovial joints associated with proliferation of synovial cells and infiltration of activated immunoinflammatory cells, including memory T cells, macrophages, and plasma cells, leading to progressive destruction of cartilage and bone (Hitchon *et al.*, 2004). Another central feature of RA synovitis is the transformation of fibroblast-like synovial cells into autonomously proliferating cells with a tissue-infiltrating nature, forming hyperplastic

*P<* 0.01 versus sham-control; °, *P<* 0.01 versus CIA.

**3.2. Discussion** 

tissue with the potential for bone erosion and cartilage degradation known as pannus (Filippin *et al.*, 2008). Therefore, it is necessary to establish and characterize experimental animal models to assess cellular and molecular events that contribute to the pathogenesis of joint inflammation. Of interest, CII-induced arthritis in the mouse has proven to be a useful model, because it possesses many of the cellular and humoral immune events found in human rheumatoid arthritis. Oxidative stress describes an imbalance between ROS synthesis and antioxidants. Many studies have demonstrated a role of ROS in the pathogenesis of inflammatory chronic arthropathies, such as rheumatoid arthritis (Filippin *et al.*, 2008). In this regard, we investigate here the effects of 3,4-DHPEA-EA, a hydrolysis product of oleuropein, in a mouse model of CII-induced arthritis. Although T-cell and antibody responses against CII are a crucial event for the initiation of CIA (Holmdahl *et al.*, 1989), it has been demonstrated that several cytokines also appear to direct cell-to-cell communication in a cascade fashion during the progression of CIA such as IL-1 (Hom *et al.*, 1992), TNF-α (Dong *et al.*, 2010), and IL-6 (Ferraccioli *et al.*, 2010). In addition, it has been demonstrated that monocyte chemotactic protein-1, MIP-α, MIP-1β, and regulated on activation normal T cell expressed and secreted are differentially chemotactic for lymphocyte subsets and are expressed in tissue from the inflamed joints of patients with rheumatoid arthritis (Koch *et al.*, 1994). In this study, we have confirmed that the cytokines (TNF-α, IL-1β, and IL-6) as well as the chemokines (MIP-1α and MIP-2) are expressed at sites of inflamed joints and probably contribute in different capacities to the progression of chronic joint inflammation. Several cytokines, including TNF-α and IL-1β, are known initiators of the nuclear factor (NF- κB) activation cascade (Filippin *et al.*, 2008) and are under its transcriptional control, constituting a positive feedback loop. Recent studies have observed that the acute consumption of olive oil decreased the activation of the NF- κB system on mononuclear cells from healthy men (Perez-Martinez *et al.*, 2007) and that 3,4- DHPEA-EA, *trans*-resveratrol, and hydroxytyrosol incubated with human umbilical vein endothelial cells inhibit lipopolysaccharide-triggered NF- κB and activator protein-1 activation (Carluccio *et al.*, 2003). Of interest, using 3,4-DHPEA-EA, we have demonstrated an inhibition of the release of proinflammatory cytokines and chemokines and a reduction of leukocyte infiltration measured by MPO activity. Several studies also showed that the potential cardioprotective activity of oleuropein in acute cardiotoxicity induced by doxorubicin treatment was determined in vivo in rats (Andreadou *et al.*, 2007) by inhibiting lipid peroxidation products, decreasing oxidative stress, and reducing iNOS in cardiomyocytes and that the olive oil polyphenols are capable of down-regulating COX-2 expression in colonic cancer cells by a mechanism involving the early inhibition of p38 mitogen-activated protein kinase and downstream inhibition of the transcription factor cAMP response element-binding protein (Corona *et al.*, 2007). We show here that 3,4- DHPEA-EA decreased iNOS and COX-2 expression by immunohistochemical staining and also reduced the levels of the metabolite of COX-2, PGE2, in the serum of 3,4-DHPEA-EA treated mice. Reactive nitrogen species, such as the peroxynitrite radical (ONOO\_) generated by the reaction between O2 . and nitric oxide, can also cause oxidative damage (Soneja *et al.*, 2005). The addition of ONOO\_ to body cells, tissues, and fluids leads to fast protonation, which may result in the depletion of -SH groups and other antioxidants, oxidation and

nitration of lipids, DNA disruption, and nitration and deamination of DNA bases (Filippin *et al.*, 2008). In this report, an intense immunostaining of nitrotyrosine formation also suggested that a structural alteration of joint had occurred, most probably due to the formation of highly reactive nitrogen derivatives ROS produce strand breaks in DNA, which triggers energy-consuming DNA repair mechanisms and activates the nuclear enzyme poly(ADP-ribosyl) polymerase (PARP). There is various evidence that the activation of PARP may also play an important role in inflammation (Genovese *et al.*, 2005). Continuous or excessive activation of PARP produces extended chains of ADP-ribose (PAR) on nuclear proteins and results in a substantial depletion of intracellular NAD and subsequently, ATP, leading to cellular dysfunction and, ultimately, cell death (Chiarugi, 2002). We demonstrate here that 3,4-DHPEA-EA treatment reduced the activation of PARP with a decrease in PAR expression in the joint during CIA. In this regard, several studies demonstrated that hydroxytyrosol, a hydrolysis product of 3,4-DHPEA-EA, also exerts an inhibitory effect on peroxynitrite-dependent DNA base modifications and tyrosine nitration (Deiana *et al.*, 1999). Likewise, Salvini *et al.* (2006) showed a 30% reduction of oxidative DNA damage in peripheral blood lymphocytes during intervention in postmenopausal women with virgin olive oil containing high amounts of phenols. Thus, 3,4-DHPEA-EA, given at the onset of the disease, reduced paw swelling, clinical score, and the histological severity of the disease when injected after the onset of clinical arthritis. Amelioration of joint disease was associated with near to full inhibition of cytokines as well as inhibition of neutrophil infiltration, which is a key player in RA. Therefore, 3,4-DHPEA-EA was also administered from day 28 after collagen immunization, targeting this early initiation phase of CIA. Then, with treatment starting at day 28, 3,4-DHPEA-EA post-treatment caused a significant reduction of inflamed joints collected at day 35. In conclusion, RA is a complex chronic inflammatory disease dependent on multiple interacting environmental and genetic factors, making it difficult to understand its pathogenesis and thereby to find effective therapies. Taken together, the results of the present study enhance our understanding of the role of ROS generation in the pathophysiology of CII-induced arthritis, implying that olive oil compounds such as 3,4-DHPEA-EA may be useful in the therapy of inflammation.

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 313

**Author details** 

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