Avaliação do dano celular e da modulação das citocinas TNF-α, IL-6 e IL-10 em macrófagos expostos à terapia fotodinâmica mediada pela PpIX

Pouco se sabe se a morte celular induzida pela terapia fotodinâmica (PDT) compromete os macrófagos (MΦ), envolvidos nas respostas imunes induzidas pela PDT. Neste estudo, foram avaliados parâmetros de citotoxicidade dos MΦ mediada pela PDT e a produção de citocinas, frente à protoporfirina IX (PpIX). MΦ peritoneais de camundongos BALB/c foram estimulados in vitro com PDT, luz, PpIX ou lipopolissacarídeo (LPS). Após isto, a viabilidade celular (VC), a lipoperoxidação, os níveis de óxido nítrico (NO), de DNA degradado, de TNF-α, IL-6 e IL-10 foram avaliados. A exposição curta à PDT reduziu a VC em 10-30%. Os níveis de NO e de DNA degradado duplicaram, sem aumento da lipoperoxidação. Houve aumento de TNF-α e IL-10, sendo maior na presença de LPS. Já a produção de IL-6 reduziu em dez vezes. A PDT induz estresse celular, gera radicais NO e causa dano ao DNA, tornando o microambiente citotóxico. Ainda, modula citocinas pró e anti-inflamatórias em MΦ.

Palavras-chave:
fotoquimioterapia; macrófago; dano ao DNA; citocinas; estresse oxidativo

Autoria

R. D. R. Tiveron

Núcleo de Biotérios – NUBIO, Biotério Central, Universidade Federal do Triângulo Mineiro – UFTM, Avenida Tutunas, 600, CEP 38061-500, Uberaba, MG, BrasilUniversidade Federal do Triângulo MineiroBrasilUberaba, MG, BrasilNúcleo de Biotérios – NUBIO, Biotério Central, Universidade Federal do Triângulo Mineiro – UFTM, Avenida Tutunas, 600, CEP 38061-500, Uberaba, MG, Brasil

http://orcid.org/0000-0002-9424-5636

D. A. Costa

Universidade de Uberaba – UNIUBE, Avenida Nenê Sabino, 1801, CEP 38055-500, Uberaba, MG, BrasilUniversidade de Uberaba BrasilUberaba, MG, BrasilUniversidade de Uberaba – UNIUBE, Avenida Nenê Sabino, 1801, CEP 38055-500, Uberaba, MG, Brasil

http://orcid.org/0000-0002-0263-6550

M. D. I. Leite

Universidade de Uberaba – UNIUBE, Programa de Mestrado em Odontologia, Avenida Nenê Sabino, 1801, CEP 38055-500, Uberaba, MG, BrasilUniversidade de UberabaBrasilUberaba, MG, BrasilUniversidade de Uberaba – UNIUBE, Programa de Mestrado em Odontologia, Avenida Nenê Sabino, 1801, CEP 38055-500, Uberaba, MG, Brasil

http://orcid.org/0000-0002-1612-3449

C. B. S. Vaz

http://orcid.org/0000-0003-4451-3628

M. Sousa

http://orcid.org/0000-0002-5716-6410

S. M. C. F. Carlos

Laboratório Multidisciplinar do Centro de Educação Profissional – CEFORES, Universidade Federal do Triângulo Mineiro – UFTM, Praça Manoel Terra, 330, CEP 38025-180, Uberaba, MG, BrasilUniversidade Federal do Triângulo MineiroBrasilUberaba, MG, BrasilLaboratório Multidisciplinar do Centro de Educação Profissional – CEFORES, Universidade Federal do Triângulo Mineiro – UFTM, Praça Manoel Terra, 330, CEP 38025-180, Uberaba, MG, Brasil

http://orcid.org/0000-0002-3502-3910

C. J. F. Oliveira

http://orcid.org/0000-0003-2211-7333

R. R. Machado

Faculdade de Farmácia – FAFAR, Universidade Federal de Minas Gerais – UFMG, Avenida Presidente Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, BrasilUniversidade Federal de Minas GeraisBrasilBelo Horizonte, MG, BrasilFaculdade de Farmácia – FAFAR, Universidade Federal de Minas Gerais – UFMG, Avenida Presidente Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brasil

http://orcid.org/0000-0002-1284-1195

T. P. Paulino ^**e-mail: tony.paulino@uftm.edu.br

http://orcid.org/0000-0001-8856-4684

*e-mail: tony.paulino@uftm.edu.br

SCIMAGO INSTITUTIONS RANKINGS

Figuras

Figuras (6)

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Figure 1
Evaluation of cell viability exposing MΦ to LED light only (20 J cm^-2), to PpIX only (60 µg mL^-1 for 2 h), and to PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), as well as cell viability in LPS (20 µg mL^-1 for 1 h with or without PDT). X-axis indicates presence (+) or absence (-) of light, PpIX and LPS. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to the negative control group (p < 0.05).

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Figure 2
Evaluation of malondialdehyde induced by lipid peroxidation in MΦ exposed to LED light only (20 J cm^-2), to PpIX only (60 µg mL^-1 for 2 h), and to PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), as well as malondialdehyde in the H₂O₂ positive control group (50% H₂O₂ + 2 mM FeSO₄ for 1 h). X-axis indicates presence (+) or absence (-) of light, PpIX and H₂O₂. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test).*Represents statistically significant results compared to the negative control group (p < 0.05).

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Figure 3
Quantification of NO produced after 1 h of LED light only (20 J cm^-2), PpIX only (60 µg mL^-1 for 2 h), and PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), and also oxi-reduction of 10 µM NO₂ + 10 µM NO₃ on medium for 1 h (positive control). X-axis indicates presence (+) or absence (-) of light, PpIX and NO₂ with NO₃. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to the negative control group (p < 0.05).

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Figure 4
Quantification (%) of DNA degradation in comet tails when MΦ were exposed to LED light only (20 J cm^-2), to PpIX only (60 µg mL^-1 for 2 h) and to PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), as well as DNA degradation in comet tails in the H₂O₂ positive control group (50% H₂O₂ + 2 mM FeSO₄ for 1 h). X-axis indicates presence (+) or absence (-) of light, PpIX and H₂O₂. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to the negative control group (p < 0.05).

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Figure 5
Representative microscopy images of comets found in the negative control group (A), showing preserved genetic material, and in the PDT group (B), showing genetic material degradation that forms a comet tail due to the lower weight of DNA fragments.

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Figure 6
Quantification of cytokines released 21 h after application of PpIX alone (60 µg mL^-1 for 2h), PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), LPS alone (20 µg mL^-1 for 1 h) and PDT + LPS (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h + LPS for 1 h). (A) TNF-α data are presented as median + standard deviation (Kruskal–Wallis; 2×2 comparisons by Mann-Whitney test). X-axis indicates presence (+) or absence (-) of light, PpIX and LPS. Data of IL-10 (B) and IL-6 (C) are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to negative control group (p < 0.05). Letter “a” represents statistically significant results in comparison with groups represented by letter “b”.

Figure 1 Evaluation of cell viability exposing MΦ to LED light only (20 J cm^-2), to PpIX only (60 µg mL^-1 for 2 h), and to PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), as well as cell viability in LPS (20 µg mL^-1 for 1 h with or without PDT). X-axis indicates presence (+) or absence (-) of light, PpIX and LPS. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to the negative control group (p < 0.05).

Figure 2 Evaluation of malondialdehyde induced by lipid peroxidation in MΦ exposed to LED light only (20 J cm^-2), to PpIX only (60 µg mL^-1 for 2 h), and to PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), as well as malondialdehyde in the H₂O₂ positive control group (50% H₂O₂ + 2 mM FeSO₄ for 1 h). X-axis indicates presence (+) or absence (-) of light, PpIX and H₂O₂. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test).*Represents statistically significant results compared to the negative control group (p < 0.05).

Figure 3 Quantification of NO produced after 1 h of LED light only (20 J cm^-2), PpIX only (60 µg mL^-1 for 2 h), and PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), and also oxi-reduction of 10 µM NO₂ + 10 µM NO₃ on medium for 1 h (positive control). X-axis indicates presence (+) or absence (-) of light, PpIX and NO₂ with NO₃. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to the negative control group (p < 0.05).

Figure 4 Quantification (%) of DNA degradation in comet tails when MΦ were exposed to LED light only (20 J cm^-2), to PpIX only (60 µg mL^-1 for 2 h) and to PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), as well as DNA degradation in comet tails in the H₂O₂ positive control group (50% H₂O₂ + 2 mM FeSO₄ for 1 h). X-axis indicates presence (+) or absence (-) of light, PpIX and H₂O₂. Data are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to the negative control group (p < 0.05).

Figure 5 Representative microscopy images of comets found in the negative control group (A), showing preserved genetic material, and in the PDT group (B), showing genetic material degradation that forms a comet tail due to the lower weight of DNA fragments.

Figure 6 Quantification of cytokines released 21 h after application of PpIX alone (60 µg mL^-1 for 2h), PDT (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h), LPS alone (20 µg mL^-1 for 1 h) and PDT + LPS (20 J cm^-2 + 60 µg mL^-1 PpIX for 2 h + LPS for 1 h). (A) TNF-α data are presented as median + standard deviation (Kruskal–Wallis; 2×2 comparisons by Mann-Whitney test). X-axis indicates presence (+) or absence (-) of light, PpIX and LPS. Data of IL-10 (B) and IL-6 (C) are presented as mean + standard deviation (ANOVA, Tukey’s post-hoc test). * represents statistically significant results compared to negative control group (p < 0.05). Letter “a” represents statistically significant results in comparison with groups represented by letter “b”.

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Avaliação do dano celular e da modulação das citocinas TNF-α, IL-6 e IL-10 em macrófagos expostos à terapia fotodinâmica mediada pela PpIX

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[1] *e-mail: tony.paulino@uftm.edu.br