Cell culture

The Neuro-2a (N2a) cells, derived from mouse neuroblastoma, were purchased from the American Type Culture Collection (Manassas, VA, USA). N2a cells exhibit properties of neuronal stem cells and can differentiate into neuronal cells when treated with 20M retinoic acid (RA)1,15. The cells were incubated in culture dishes into Dulbeccos modified Eagles medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Serum Source International, Charlotte, NC, USA) and 1% penicillin/streptomycin (Gibco, Rockville, MD, USA) in a humidified 5% CO2 atmosphere at 37C. When the cells reached 7080% of confluency, the medium was replaced as a differentiation medium, which contained 2% FBS and 20M RA in DMEM for four days. Differentiated N2a cells were maintained in a humidified atmosphere of 5% CO2 at 37C, and the differentiation medium was changed every two days.

The following procedures were adapted to establish an in vitro I/R injury model from previous studies1,16,17,18. Differentiated N2a cells were washed three times with phosphate-buffered saline (PBS), and the medium was replaced with deoxygenated, glucose-free balanced salt solution (Gibco) in hypoxic condition (O2 tension 1%) for 3h. Following OGD condition, injured cells were replaced onto the growth medium. PBS and 50 or 100g/ml PDRN (Placentex Integro, Mastelli Srl, Italy) was added to the growth medium of injured cells for 24h according to the following experimental groups. Cells treated with OGD and PBS were classified as the OGD group, and cells treated with OGD and PDRN were classified as the OGD+PDRN group. Differentiated N2a cells without OGD were classified as the non-OGD group (Fig.1A).

Effect of PDRN on an in vitro I/R injury model. (A) Experimental design of PDRN treatment on an in vitro I/R injury model. (B) Effect of PDRN on the cell viability of in vitro I/R injury model. (C) Bar graphs show the number of differentially expressed genes in OGD group compared with Non-OGD group and in OGD+PDRN group compared with OGD group. Values are presented as meansstandard error of the mean (SEM). Statistically significant differences are shown as **p<0.01, ***p<0.001.

To analyze the proliferation of the in vitro I/R injury model, the number of cells in the non-OGD, OGD, and OGD+PDRN groups were calculated using an advanced detection and accurate measurement (ADAM) automatic cell counter (NanoEnTek Inc., Seoul, South Korea).

After the cellular experiments, total RNA was isolated from the cells of all the groups with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for RNA isolation19 according to the manufacturers instructions. A NanoDrop spectrophotometer (Thermo Fisher Scientific) was used to confirm RNA quantity and purity.

RNA-seq transcriptome array of the non-OGD, OGD, and OGD+PDRN groups was performed at Macrogen Inc. (Seoul, Korea) with the HiSequation 2000 platform (Illumina, San Diego, CA, USA) according to methods detailed in our previous study20.

In this study, fold change (FC) criteria (FC|1.7|) were used to identify the differentially expressed genes (DEGs)from the results of RNA-seq transcriptome array. To identify their roles, three different pairs of DEGs were submitted to the Database for Annotation, Visualization and Integrated Discovery (DAVID) v.6.8 annotation tool21.

RT-qPCR was performed to validate the transcriptome analysis results. ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) was used to prepare the cDNA from total RNA, according to the manufacturers instructions. RT-qPCR was performed to measure the mRNA levels of the genes of interest using qPCRBIO SyGreen Mix Hi-ROX (PCR BIOSYSTEMS, London, UK) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The 2CT method was used for data analysis22. Supplementary Table S1 lists the primers used for RT-qPCR.

The proteins were extracted from the cell pellets. Proteins were boiled for 10min and loaded onto 412% bis Tris gels (Invitrogen, Waltham, MA, USA) for separation. The separated proteins were then blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen) with 20% (v/v) methanol in NuPage Transfer Buffer (Invitrogen) at 15V for 4h at 4C. Tris-buffered saline containing 0.01% Tween 20 (TBST) in 5% skim milk (Difco, BD Biosciences, Oxford, UK) was used to block the membranes for 1h. The blots were washed three times with TBST for 10min and then incubated overnight at 4C with primary antibodies specific to the following target proteins: phosphorylated JAK1, phosphorylated JAK2, phosphorylated STAT1 (1:1000; Cell Signaling Technology, Cambridge, UK), CSF1, IL-6, PTPN6, RAC2, TNF, IL-1, IL-1, phosphorylated STAT3, STAT3 ADORA2A, JAK1, JAK2, STAT1, SOCS3, Bax, Bcl-2, and -actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation, the blots were washed thrice with TBST and incubated for 1h with a horse-radish peroxidaseconjugated secondary antibody (1:3000; Santa Cruz) at 25C. Finally, the blots were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).

To analyze neuronal cell death, N2a cells were seeded on a 96-well cell culture plate (SPL Life Sciences, Gyeonggi-do, Korea) and OGD and PDRN treatments were performed as previously described above. The death of the in vitro I/R injury model was evaluated using a cytotoxicity lactate dehydrogenase (LDH) assay kit (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. Briefly, 10l of lysis buffer was added to each well and the cells were cultured at 37C in CO2 for 30min. A total of 100 L of the working solution was then added to each well, and the samples were cultured at room temperature in the dark. Stop solution (50l) was then added to each well, and LDH levels in the culture supernatant were analyzed immediately by measuring the absorbance at 490nm using a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA).

To analyze apoptosis, N2a cells were seeded on a cell culture slide (SPL Life Sciences, Gyeonggi-do, Korea) and OGD and PDRN treatments were performed as previously described above. The DeadEnd Fluorometric TUNEL System (Promega Madison WI USA) was used to assess apoptosis according to the manufacturers protocol. Briefly, the samples were mounted on glass slides with a fluorescent mounting medium with DAPI for imaging using an LSM 700 fluorescence microscope (Carl Zeiss, Gottingen, Germany). The number of positively stained cells over the total number of cells per specimen field was measured, and the percentage of positive cells was calculated. Four individual specimens were analyzed per group.

All data are expressed as the meanstandard error of the mean (SEM), and all experiments were repeated at least four times with four technical replicates in each group. The Statistical Package for Social Sciences v.25.0 (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, v.25.0. Armonk, NY, USA) was used for the statistical analyses. The significance of intergroup differences was estimated using Students paired t-test or one-way analysis of variance (ANOVA). Statistical significance was set at p<0.05.

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