The Cytokines And Matrix Metalloproteinases Expression Induced By Neuwiedase, A PI Metaloproteinase Isolated from Bothrops neuwiedi pauloensis Snake Venom

¹Baldo, C; ^{1Dias-Oliveira}, J. D.; ¹Izidoro, L. F. M.; ¹Goulart, L. R.; ¹Hamaguchi, A.; ¹Homsi-Brandeburgo, M. I and ¹Rodrigues, V. M.

1 Instituto de Genética e Bioquímica, UFU, Uberlândia-MG.

This study evaluated the mRNA expression of IL-6 and matrix metalloproteinases (MMP2 and MMP9) as consequence of i.m. injections of neuwiedase, a PI metalloproteinase isolated from Bothrops neuwiedi pauloensis snake venom. The mRNA expression was performed by a RT-PCR after i.m. injection of 200 mg of neuwiedase or 50 mL of PBS in the gastrocnemius muscle of C57BL/6 mice. After 1, 3, 6 and 24 h the gastrocnemius muscle was collected and the total RNA was extracted by the Tri-Reagent (Sigma) method following the manufacturer’s instructions. The RT-PCR was carried out by using “Ready-to-Go RT-PCR beads” (Amersham Biosciences). The RT-PCR reactions was achieved  with 1mg RNA and the reaction conditions were established for each primer sequence. The sequence of primers used in the experiments of RT-PCR was designed by Primer Designer 2.0 Software. The RT-PCR showed that neuwiedase can induce the mRNA expression for IL-6, MMP2 and MMP9 in gastrocnemius muscle at 3, 6 and 24 hours after the treatment, respectively. Zymogramy analysis was also carried out to detect gelatinase activity in muscle homogenates obtained from animals injected with neuwiedase. Samples of muscle homogenates obtained from controls mice showed two main proteolytic bands when analyzed by zymography. A conspicuous increment in the intensity these bands was observed in samples obtained 3 and 6 h after neuwiedase injections. According of the RT-PCR reactions, these bands are probably MMP2 and MMP9. The activation of endogenous matrix metalloproteases may contribute to further tissue damage by promoting extracellular matrix degradation. In adddition, neuwiedase can induce an inflammatory response by up-regulation of IL6 gene.

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