P–801 Effects of Lonomia obliqua venom components on human endometrial stromal cells: A potential source for new cytoprotective biomolecules against recurrent pregnancy loss
Authors: Schneider R , Berger M , Terraciano P B , Zanin. Gotardi D H , Niad. Crispim M , SIlva M. D , Zanon P , Seren. Montenegro I , Santi L , Orland. Bey. d. Silva W , Almeid. Guimarães J , Pandolf. Passos E 
Published: August 2021

Abstract

Study question
Could new molecules like Lonomia obliqua lipocalins and hemolins have cytoprotective effects on endometrial stem cells (hESC)?

Summary answer
Lonomia obliqua venom-induced hESC viability, proliferation and migration occurred mainly by protection against oxidative damage and ERK-dependent pathway activation

What is known already
Recurrent pregnancy loss (RPL) is associated with severe physical and psychological morbidity, for which there is no treatment options. The pathophysiology involves deficiency in proliferation and migration capacities of endometrial stromal cells (hESCs) impairing embryo implantation and development. Animal venoms are rich sources of bioactive molecules and despite its known toxic effects, they also have protective components such as pro-proliferative molecules, growth factor-like, anti-apoptotic and anti-oxidant.

Study design, size, duration
This study was an experimental in vitro with endometrial stem cells. Treatment duration was 8–72h. Every assay had control cells exposed to phosphate buffered saline (PBS).

Participants/materials, setting, methods
hESCs were isolated from fresh human endometrial biopsies and characterized according standard protocols. Then the effects of L. obliqua venomous secretions on cell viability, proliferation and migration were determined using MTT, wound-healing assay, sulforhodamine B (SRB) assay and measuring the immunocontent of Ki67. Venom components involved in cell enhancing effects were also identified by classical chromatographic methods and proteomic analysis. Assays were conducted in triplicate.

Main results and the role of chance
The hESCs in culture showed adhesiveness properties, presented a fusiform fibroblastoid morphology and ability to in vitro differentiate into adipocytes, osteocytes and chondrocytes. The expression of cell surface markers was also characterized by flow cytometry. hESCs were positive for mesenchymal markers (CD105, CD90 and CD73) and negative for hematopoietic markers (CD45 and CD11), indicating that isolated cells have potential for multilineage differentiation. L. obliqua bristle extract (LOBE) increased dose-dependently hESCs viability in a concentration range varying from 0.001 to 0.1 µg/mL, independently of the cell isolation bath. For some cell isolates (patient ID 1, 3, 4, 6 and 7) it was observed a slightly reduction in hESC viability at highest LOBE concentrations (10 µg/mL). Treatment increased hESC viability in the presence of low concentrations of fetal bovine serum (1% FBS) and even in its complete absence. This effect was long lasting, being significant up to 72 h of incubation with LOBE in serum deprivation conditions. r to identify the potential molecules involved in the cytoprotective action, a mass spectrometry-based proteomic analysis was performed. It was identified a total number of 430 proteins in LOBE and 312 proteins in L. obliqua hemolymph.

Limitations, reasons for caution
This study was only conducted in vitro.
Wider implications of the findings: In this work we reported the identification of at least six protein classes with cytoprotective properties through proteomic analysis and isolated one fraction enriched in this cytoprotective factors. L. obliqua secretions induced increase in hESCs viability, proliferation and migration mainly by the protection against oxidative damage and ERK-dependent pathway activation.

Trial registration number
Not applicable