Učinak azitromicina na ekspresiju TLR-4, TNFalfa i TGFbeta u crijevnoj sluznici miševa s eksperimentalnim kolitisom [The effect of azithromycin on TLR-4, TNF alpha and TGF beta expression in bowel mucosa in a murine model of experimental colitis]

Pleško, Sanja (2011) Učinak azitromicina na ekspresiju TLR-4, TNFalfa i TGFbeta u crijevnoj sluznici miševa s eksperimentalnim kolitisom [The effect of azithromycin on TLR-4, TNF alpha and TGF beta expression in bowel mucosa in a murine model of experimental colitis]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Changes in expression of pro- and anti-inflammatory cytokines and membrane receptors are documented patients with IBD. The aim of this study was to investigate the effect of azithromycin on expression of TLR-4, TNFalpha i TGFbeta in bowel mucosa in murine model of experimental colitis induced by enema 2, 4 –dinitrofluorobenzen. Immunohistochemistry was performed from paraffin embedded murine intestinal tissue for TLR-4, TNFalpha i TGFbeta. Experimental groups of animals were treated with azithromycin, metronidazole or methylprednisolone for five days, respectively and the control group was the group of healthy mice and group of mice with experimental colitis. Results of this study showed the changes in expression of pro- and anti-inflammatory cytokines (TLR-4, TNFalpha i TGFbeta) in manipulation of experimental colitis induced by DNFB. The expected differences in expression markers: significantly increased expression of TNFα i TLR4, and significantlly decreased expression of TGFβ in bowel mucosa in a murine model of experimental colitis compared to bowel mucosa of healthy mice. The results of this study documented the differences in expression of observed markers; smaller expression of TNFα i TLR4 in bowel mucosa treated with methilprednizolone and azithromycin and larger expression of TGFβ in bowel mucosa of mice with experimental colitis treted with methilprednizolone, azithromycin and metronidazol. In murine bowel mucosa treated with metronidazol no differences were noted compared with group with experimental colitis. Metronidazol showed weak antiinflammatory effect compared to methilprednizolone and azithromycin. The effect of treatment on expression of markers documented that the effect of metronidazole on the reduction of TNF and TLR4 expression on mononuclear cells of submucosa was significantly weaker than the effect of methylprednisolone and azithromycin. There was no significant difference between methylprednisolone and the effect of azithromycin on the reduction of TNF and TLR4 expression. There was no significant difference between the effect of methylprednisolone, azithromycin and metronidazole on expression of TGFβ. The antiinflammatory effect of azithromycin was not significantly different from the antiinflammatory effect of methylprednisolone. According to the results azithromycin shows the corresponding anti-inflammatory effect which manifested itself in a significant reduction of expression of TNFα and TLR4 with potential clinical implications, regarding the activity patterns of inflammation perpetuation and the period of the biological course of inflammatory bowel disease in humans.

Abstract in Croatian

Promjene ravnoteže proupalnih i protuupalnih citokina te membranskih biljega imaju važnu ulogu u etiopatogenezi UBC (upalne bolesti crijeva). Cilj ovog istraživanja bio je pokazati učinak azitromicina na izraženost TLR-4, TNFalfa i TGFbeta u sluznici debelog crijeva u modelu eksperimentalnog kolitisa izazvanog intrarektalnom primjenom 2,4- dinitrofluorobenzena. Učinjena je imunohistokemijska analiza ekspresije TLR4, TNFalfa i TGFbeta iz parafinskih kocki u kojima je bilo pohranjeno tkivo crijeva miševa. Eksperimentalne skupine miševa tretirane su primjenom azitromicina ili metronidazola ili metilprednizolona tijekom pet dana uz kontrolne skupine koje su činile grupa zdravih miševa i grupa miševa s eksperimentalnim kolitisom. Rezultati ovog istraživanja pokazuju promjene proupalnih i protuupalnih patogenetskih markera (TNFα, TGFβ i TLR4) u manipulaciji izvorno opisanog modela eksperimentalnog kollitisa izazvanog primjenom DNFB. Dokumentirane su očekivane razlike izraženosti biljega u sluznici miševa s eksperimentalnim kolitisom i sluznice zdravih miševa: značajno veća ekspresija TNFα i TLR4 te značajno manja ekspresija TGFβu sluznici miševa s eksperimentalnim kolitisom, u odnosu na sluznicu zdravih životinja. Rezultati istraživanja razlike izraženosti promatranih biljega između tretiranih i netretiraniih miševa s eksperimentalnim kolitisom pokazuju: manju ekspresiju TNFα i TLR4 u sluznici miševa tretiranih metilprednizolonom i azitromicinom i veću ekspresiju TGFβ u sluznici miševa s eksperimentalnim kolitisom koji su tretirani metilprednizolonom, azitromicinom i metronidazolom. U sluznici životinja tretiranih metronidazolom nije zabilježena razlika ekspresije TNFα i TLR4, u odnosu na sluznicu miševa s eksperimentalnim kolitisom. U svjetlu izraženosti biljega, metronidazol pokazuje slabiji protuupalni učinak od učinka metilprednizolona i azitromicina. Razlike učinka pojedinog tretmana na izraženost biljega pokazuju da je učinak metronidazola na smanjenje izraženost TNFα i TLR4 na mononuklearnim stanicama submukoze bio značajno manji od učinka metilprednizolona i azitromicina. Nije zabilježena značajna razlika učinka metilprednizolona i azitromicina na smanjenje izraženosti TNFα i TLR4. U odnosu na izraženost TGFβ nije zabilježena razlika učinka metilprednizolna, azitromicina i metronidazola. Protuupalni učinak azitromicina nije se značajno razlikovao od protuupalnog učinka metilprednizolona. Prema rezultatima istraživanja azitromicin pokazuje odgovarajući protuupalni učinak koji se očitovao u značajnom smanjenju izraženosti TNFα i TLR4 s potencijalnim kliničkim implikacijama, uvažavajući aktivnost obrazaca podržavanja upale i razdoblju biološkog tijeka upalne bolesti crijeva u ljudi.

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