Approval was obtained from the Ethics Committee of Selcuk University Medical Faculty for the study (2018/329). Our study was supported by the Selcuk University Scientific Research Projects Coordination Unit (Project number: 19401029). After obtaining informed consent from the parents of the patients to be included in the study, we conducted our study by the ethical standards stated in the 1964 Helsinki Declaration. Human tonsil samples used in this study were obtained from tonsillectomy patients in Selcuk University, Medicine Faculty Hospital Department of Ear Nose Throat (ENT) between the dates of November 2018 and August 2019.
Study population, sample collection, and protocol
Our study was conducted on 46 patients (25 males, 21 females) aged between 2 and 16 years (mean age 6.2), who were clinically diagnosed with chronic tonsillitis and tonsillar hypertrophy by the ENT clinician. Patients with a history of immunodeficiency or systemic disease were excluded from the study. The tonsillectomy procedure was performed under general anesthesia using the classical dissection method. After the palatal pharyngeal archus mucosa was cut with low-heat plasma and the tonsil capsule was opened, a cryogenic plasma knife was used to remove the tonsil tissue and stop bleeding. Extracted tonsil tissue was classified as 1, 2, 3, and 4 using the Friedman staging system according to their size .
Chronic tonsillitis group (n = 23); Patients with tonsil sizes grade 1 and 2, and tonsillectomy indications included frequently recurrent tonsillary infection, sore throat, and malodorous mouth problems were accepted as the chronic tonsillitis group.
Tonsillar hypertrophy group (n = 23); Patients with tonsil size grades 3 and 4 and tonsillectomy indications included obstructive symptoms such as snoring, open mouth breathing, difficulty in breathing, and swallowing problems were accepted as the tonsillar hypertrophy group.
Extracted tonsil tissue samples were sent to the medical genetics department in TRIzol tubes to investigate the ER stress and apoptosis. Two consultants (NK, FD) from the medical genetics department who blinded to the study groups and the clinical characteristics of the patients, evaluated tonsil tissue samples for ER stress and apoptosis using Real-time PCR and Western blot methods.
Tonsillar hypertrophy and chronic tonsillitis groups were compared in terms of expression levels of genes in the ER stress pathway (ATF4, ATF6, EDEM1, CHOP, GRP78, EIF2AK3, ERN1, GRP94) and apoptosis pathway (BAX, BCL-2).
Real-time PCR analysis
Total RNA extraction from tissue samples obtained from patients was performed using TRIzol® reagent (Invitrogen, Waltham, USA) according to the protocol previously described in the literature . Tissue pieces were dissected by freezing in liquid nitrogen before extraction. cDNA synthesis was performed using the Transcriptor High-Fidelity cDNA Synthesis kit (Roche, Basel, Switzerland) using oligo (dT) and random primers, following the manufacturer's instructions. Oligonucleotide primers were designed using the IDT DNA primer request tool and are manufactured by Biomers Inc. (Ulm, Germany). Primer sequences are given below. All PCR reactions were performed using LightCycler® 480 Instrument II (Roche, Penzberg, Germany) Real-time PCR via SYBR Green Master Mix (Bio-Rad Hercules, California, USA). Before proceeding with the qPCR process of the genes of interest, optimization processes of the primers used were carried out. Housekeeping genes such as 18S rRNA (18S ribosomal RNA), 28S rRNA (28S ribosomal RNA), ACTB (β-actin), β2M (β2-microglobulin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were examined for normalization. ACTB levels of these were found to be more stable, and studies continued with them. ΔCT results were normalized by ACTB. Expression results were calculated by the 2ΔΔct method. Primer sequences were shown in Table 1.
Western blot analysis
Antibodies and Western blot ATF-6 (D4B8) rabbit anti-human monoclonal (# 65880), ATF-4 (D4B8) rabbit anti-human monoclone (# 11815), GRP78 (C50B12) rabbit anti-human monoclonal mAb (# 3177), CHOP (D46F1) ) rabbit anti-human monoclonal antibody (# 5554), PERK (C33E10) Rabbit antihuman mAb (#3192), ERN1 (IRE1α) (14C10) Rabbit antihuman mAb (#3294), Grp94 (D6X2Q) XP® Rabbit antihuman mAb ( #20292), Bax (D2E11) Rabbit antihuman mAb ( #5023), Bcl-2 (D55G8) Rabbit Antihuman mAb (#4223) and β-Actin (13E5) rabbit anti-human monoclonal antibody (# 4970) were obtained from cell signaling technology company (cell signaling tec., Leiden, Netherlands). Western blot analyzes were performed according to protocols previously described in the literature . ACTB levels were used for normalization in Western blot analysis. For each group normalization, their own ACTB values were taken into account in this calculation. Changes in protein levels were determined after this normalization.
PCR primers were designed using IDT PrimerQuest software. Image-based data were analyzed by ImageJ software. The statistical significance was investigated using GraphPad Prism V6 software (GraphPad software Inc., La Jolla, USA). Expression results were calculated by the 2ΔΔct method. CT results were normalized by ACTB. All the experiments were performed as minimum triplicate (N ≥ 3). The SPSS version 22.0 (IBM Statistics) was used to analyze the data of the study. First of all, the conditions of the normal distribution of the data were examined with skewness and kurtosis values (values between 0.846 and 0.924) and Q_Q plot graphics, and it was observed that the data showed a normal distribution. Based on these results, an independent group t-test was used to compare the ER stress and apoptosis gene levels of chronic tonsillitis and tonsillar hypertrophy groups. The level of significance was set to p < 0.05.