**1.5 Evaluation of the expression of transcripts related to inflammation**

The placental explants were evaluated for the expression of the genes encoding IL-1β, TNF-α, LC3-II, beclin-1, and p62 proteins at the transcriptional level. In addition, the gene expression of the inflammasome was evaluated through the NLRP3 and caspase-1 genes. Total RNA was extracted from the placentas using the Total RNA Purification Kit (Norgen Biotek Corp., Thorold, Canada) according to the manufacturer's protocol, and the Reverse Transcription-coupled polymerase chain reaction (RT-qPCR) was performed as described previously [64]. Briefly, isolated RNA was DNAse I Amp Grade (Invitrogen) treated. Subsequently, the synthesis of complementary DNA (cDNA) was conducted using ImProm-IITM Reverse Transcription System,


#### **Table 1.**

*Primers for inflammasome and autophagy proteins, cytokines, and GAPDH. Source: Gen Bank (https://www. ncbi.nlm.nih.gov/genbank).*

according to manufacturer's protocol. The RT-qPCR was made using RT GoTaq-qPCR Master Mix (Promega, Madison, WI, USA), and the primer sequences used in this study are listed in **Table 1**. Each reaction was set in duplicate and the conditions for the RT-qPCR were as follows: initial denaturation at 96°C for 2 min and then 40 cycles at 95°C for 15 s and 60°C for 60 s, followed by a melting curve. Expression values of the analyzed transcripts were normalized to that of the enzyme-encoding glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH).

The calculation of the differential expression of selected genes was carried out by the data processing method compared with a standard curve [65]. To analyze relative gene expression, we standardized the RNA expression levels in all samples to that of a single RNA sample, which was set to a value of 100.

#### **1.6 Cytokine determination**

Cytokine concentrations in culture supernatants of placental explants were determined by enzyme-linked immunosorbent assay (ELISA), using Quantikine

**87**

provided the original work is properly cited.

and Maria Terezinha Serrão Peraçoli

Sao Paulo State University—UNESP, Botucatu, Brazil

\*Address all correspondence to: priscilarezeck@gmail.com

*Autophagy in Preeclampsia*

**1.7 Statistical Analysis**

IL-1β.

**Key points**

responses.

**Author details**

intracellular materials.

*DOI: http://dx.doi.org/10.5772/intechopen.85592*

than 0.05 was considered to be statistically significant.

in various inflammatory and infectious diseases.

preeclampsia and fetal growth restriction.

ELISA kits (R&D Systems) for TNF-α and IL-1β according to the manufacturer's instructions. Assay sensitivity limits were 1.6 pg./mL for TNF-α and 3.9 pg./mL for

All of the data were analyzed using one-way ANOVA, followed by post-hoc Tukey test using the statistical program PRISM (Graph Prism for Windows, version 6.01, GraphPad, EUA) to compare the difference among the groups. A p value less

1.Autophagy is an intracellular degradation system characterized as a natural defense mechanism capable to reduce damages related to inflammatory

2.The main role of autophagy is to maintain cellular homeostasis by recycling

3.Activation of inflammasome and autophagy are essential elements of the innate immune system, and disorders in these processes have been implicated

4.Oxidative stress may also contribute to placental tissue senescence and to the pathophysiology of some placental-related disorders of pregnancy, such as

5.The use of products with antioxidant and anti-inflammatory effects still need to be evaluated in order to reduce oxidative stress, induce autophagy, and

decrease the activation of inflammasome in placental tissue.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Priscila Rezeck Nunes\*, Leandro Gustavo de Oliveira, Mariana Romão Veiga

ELISA kits (R&D Systems) for TNF-α and IL-1β according to the manufacturer's instructions. Assay sensitivity limits were 1.6 pg./mL for TNF-α and 3.9 pg./mL for IL-1β.
