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bioRxiv · 10.1101/2023.02.10.527989

Proteins with anti-apoptotic action in the hemolymph of caterpillars of the Megalopygidae family acts by maintaining the structure of the cellular cytoskeleton

Carvalho, N. D.·Rofatto, H. K.·Villar, K. S.·Magnelli, R. F.·Mendonca, R. Z.
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Abstract

Brazil has a very large biological variety, which is an almost inexhaustible source of substances of pharmacological and biotechnological interest. Several studies have demonstrated the presence of bioactive peptides in insect hemolymph and their potential use as therapeutic agents. However, few data are available regarding molecules extracted from insects with anti-apoptotic action. The objective of this work was to identify and isolate proteins from the hemolymph of caterpillars of the Megalopygidae family with pharmacological and biotechnological interest. Two species of this family were studied, Podalia sp and Megalopyge albicolis. Cytotoxicity tests on Vero and Sf-9 cells revealed that the hemolymph of both caterpillars was cytotoxic only at concentrations greater than 5%v/v. In the anti-apoptotic activity assays, it was verified that the supplementation of cell cultures with only 1% of hemolymph v/v is sufficient to inhibit cell death by apoptosis induced by different inducers such as terbutyl, actinomycin D, hydrogen peroxide, or even by nutrient depletion. For this study, cells were stained with trypan blue, crystal violet, and fluorescent markers to cytoskeleton (actin and tubulin), mitochondria membrane electric potential (JC-1), and apoptosis marker (acridine orange and ethidium). The protein responsible for anti-apoptotic action was isolated through gel filtration chromatography, using an AKTA purifier high-resolution liquid chromatography system. The hemolymph was fractionated into 3 pools for Podalia sp and 6 pools for M. abicolis. In the antiapoptotic tests, semi-purified hemolymph from both caterpillars showed anti-apoptotic effect in VERO and SF-9 cells, pre-treated with only 1% v/v of hemolymph and induced to death by different and apoptotic inductors. Was observed that the molecule with anti-apoptotic effect is present in pool 3 in both hemolymphs. This protector effect blocked and attenuated the disruption of the cytoskeleton (actin filaments), being that the protective effect also was observed on the integrity of the mitochondrial membrane of SF-9 cells pre-treated with both hemolymphs and treated with the apoptosis inducer Terbutil at concentrations of 25 to 100M. By acting on the mitochondrial pathway of death by apoptosis, a pathway that can cause disorders and diseases neurodegenerative such as Parkinsons and Alzheimers diseases, substances present in the hemolymph of these and others caterpillars could be good candidates in studies for the treatment of these diseases.

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Carvalho, N. D., Rofatto, H. K., Villar, K. S., Magnelli, R. F., Mendonca, R. Z.. 2023-02-11. Proteins with anti-apoptotic action in the hemolymph of caterpillars of the Megalopygidae family acts by maintaining the structure of the cellular cytoskeleton. https://doi.org/10.1101/2023.02.10.527989

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