Efeito protetor de Punica granatum L. (romã) em modelo murino de sepse letal induzida por ligadura e perfuração cecal

Abstract

Sepsis is defined as a syndrome in which there is an imbalance between anti and pro-inflammatory responses leading to systemic effects. With the aim of discovering efficient alternatives to contain this picture, studies with natural products are increasingly present in the literature. In this context, we have the plant species Punica granatum L. known as pomegranate and that has anti-inflammatory, antimicrobial and antioxidant action. In this way, the objective of this work was to evaluate the possible anti-inflammatory effect that the Hidroaloolic Crude Extract of Punica granatum (HCEPg) presents against the lethal polymicrobial sepsis induced in mice. To investigate the effect of EBPg (5mg/kg), 25 animals were used in 5 groups: CLP, ATB (treated with Imipenem), CLP treated 6 hours before induction of sepsis, treated with concomitant sepsis and PLC treated 6 hours after the induction of sepsis. The survival of these animals was monitored for 5 days every 12 hours. It was verified that the extract had therapeutic effect and that the animals with treatment concomitant to the induction of sepsis had approximately 65% of survival. To evaluate the cellular and immunological parameters, 15 animals were distributed in 3 groups: SHAM, CLP and CLP+EBPg. After 12h of sepsis induction the animals were euthanized. The percentage of leukocytes in the peripheral blood was initially evaluated, with an increase in the number of lymphocytes and neutrophils in treated septic animals, compared to untreated animals. The effect of HCEPg on the major primary (bone marrow) and secondary lymphoid organs (lymph node and spleen) was evaluated by means of total cell count, as well as in the infectious (peritoneum) focus by means of total and differential count and in the lung, a of the main organs affected in sepsis, by means of differential counting of Broncho Alveolar Lavage (BAL) cells. It was observed that there was no difference in the amount of cells present in the bone marrow, lymph node and BAL between the treated and untreated groups. However, there were fewer cells in the spleens of treated animals compared to untreated animals and increased cells in the peritoneal cavity. Among the cell types found in the peritoneum of septic animals treated or not, neutrophils had a higher percentage. However, the extract promoted the increase of both macrophages and lymphocytes in the infectious focus. In order to investigate the efficiency of the cells in containing the proliferation of microorganisms in the infectious focus the production of hydrogen peroxide was evaluated. The extract promoted the reduction in MFI of the treated and stimulated group with PMA in relation to the CLP group and the percentage of H2O2 of the treated group in relation to sham and CLP+HCEPg, demonstrating that the HCEPg maintains H2O2 production, but not of exacerbated manner. To verify which cell phenotypes were present in the secondary lymphoid organs (lymph node and spleen) and the infectious focus, the immunophenotyping technique was performed. In the lymph node there was an increase in the percentage of activated lymphocytes and dendritic cells, and a decrease in activated lymphocytes activated. In the spleen there was an increase in the adduction of host cells. In cells of the peritoneum there was an increase in activated helper T lymphocytes and a decrease in cytotoxic T lymphocytes. In addition, the major cytokines released in the sera from the animals were found. HCEPg promoted increased clearance of all cytokines (IL-6, IL-10, MCP-1, INF-γ, TNF) with a special highlight for IL-10. Thus, we can infer that the HCEPg has immunomodulatory potential, acting in the control of inflammatory parameters such as increased release of regulatory cytokine, activation of immunological cells involved in regulatory activity and presentation of antigens, control of hydrogen peroxide production and indirect control of infection, by recruiting cells to the infectious focus. Thus, it presents itself as an important therapeutic alternative in cases of sepsi.