@2024 Afarand., IRAN

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ISSN: 2008-2630    Iranian Journal of War & Public Health 2015;7(1):35-42

Background
… [1-7] Different respiratory diseases such as chronic obstructive pulmonary disease (COPD), respiratory bronchiolitis, obstructive bronchiolitis, emphysema, bronchiectasis, fibrosis, bronchiolitis obliterans, and irritability of the airways, have been shown in the studies about the delayed pulmonary complications due to mustard gas [6, 9-12]. The effective factors on COPD, idiopathic pulmonary fibrosis, and chronic bronchiolitis pathology might play roles in the delayed complications of the lung in the veterans with chemical injuries [9]. … [13] Several complications are due to inflammatory cells in the lung [9, 14]. … [15-21] In the damaged lung tissue in COPD, the lung epithelial cells and alveolar macrophages lead to recruitment of inflammatory cells to the injured site and create an inflammatory focus through secretion of chemokines like CXCL10 [22-24]. In chronic bronchiolitis, alongside the recruitment of the activated T cells, CXCL10, via connecting to CXCR3 on the surface of the epithelial cells of the airways, leads to excessive proliferation of the cells and airways obstruction [24]. CXCL10 plays an important role in fibrosis [25, 26]. … [27-31]
Previous Studies
Serum levels of CCL2/MCP1 and macrophages recruitment chemokine in the chemically-injured veterans of Sardasht, Iran, have increased, while CCL5, IL8/CXCL8, and RANTES have decreased [32]. Inflammatory cytokines including IL-1β, IL-1α, IL-1Ra, and TNF, have decreased in the chemically-injured veterans of Sardasht, Iran. In addition, serum levels of soluble P and L selectins of the veterans have decreased, while serum level of soluble E selectin has increased [33, 34].
Aim(s)
The aim of this study was to investigate CXCL10 Gene Expression in the lung tissues of chemically-injured veterans.
Research type
This is a case-control study.
Research society, place and time
Lung paraffin blocks of veterans with lung complications exposed to mustard gas, sampled via bronchoscopy method for diagnosis purposes were studied at 2013-14. (The blocks were accessible at the pathology archives of Iran hospitals.)
Sampling method and number
16 blocks were selected. The samples of non-exposed persons (control group) were taken from archives of the hospitals. 7 paraffin blocks were selected from non-exposed persons, sampled to diagnose lung diseases. Age and sex of the persons were consistent with the exposure group.
Used devices&materials
Based on the final pathological diagnosis, 3 groups were selected including bronchiolitis obliterans, respiratory bronchiolitis, and antheracosis groups. On the basis, the exposures were divided into 2 groups. 9 samples were included in bronchiolitis obliterans group. 7 samples were included in respiratory bronchiolitis group. On the basis, the non-exposure group was divided into 3 groups including bronchiolitis obliterans (3 samples), respiratory bronchiolitis (1 sample), and antheracosis groups (3 samples). The inclusion criteria were certain exposure to mustard gas (based on the medical records of Martyrs and Veterans Foundation), lung complication (based on the medical records), male, and aged between 30 and 60years. The exclusion criteria were sample loss, insufficient tissues, and cancer cells in the tissue. RNA was extracted from paraffin samples using “RNA Extraction from Paraffin Tissue” kit (Qiagen, Cat: 73504; Germany). At first two 18-micron slices were prepared from the block and then, they were deparaffinized using xylene, at 56°C and for 30min. Absolute alcohol was used to remove xylene. After incubation, one deparaffinized semi-sample with protease K at 45°C was precipitated after passing the RNA column. Finally, the precipitated RNA was dissolved in water free of Rnase/Dnase. The extracted RNA was measured using Nano Drop device (Thermo; USA). To ensure the absence of genomic DNA, some samples were selected randomly and PCR reaction with the extracted RNA product was performed directly and before converting to cDNA with beta-actin primer with the ability to bind to genomic DNA (Table 1). Due to seeing the product band on gel with PCR on the RNA product, treatment was done with DNase I (Fermentas, Cat: EN0521; USA) according to protocol. 1µg RNA product, 2µl MgCl2 with 10Xµl buffer, and 1µl DNase I/RNase-free were added to each reaction and it was reached to 10µl final volume, adding DEPC-treated Water. The samples were incubated for 45min at 37°C and then, 50mmol EDTA was added to hinder Dnase I reaction and it was incubated for 10 more minutes at 56°C. The RNA product, brought at the Dnase I side with the mentioned primer, was converted into PCR for the second time. There was no product due to the PCR and the absence of genomic DNA was confirmed. Then, cDNA was synthesized from the RNA product. The extracted RNA was converted to cDNA, using cDNA Synthesis kit (TAKARA; Japan). To synthesize cDNA, 2µl master mix 2X (TAKARA; Japan) was incubated with 0.5µl oligo primer and 0.5µl random primer with 0.5µl reverse transcriptase enzyme and 500ng of the RNA product in 10µl final volume for 15min and at 37°C (and 5seconds at 85°C to stop the reaction). The primers were designed using NCBI database (Table 1). The favorable conditions for reaction were formed, changing the primer and sample amounts and primer binding temperature. GAPDH gene was considered as reference gene in the analysis of Real-Time PCR results using Livak method (Fig. 1). The functions of the primers were evaluated with positive samples before performing desired stages on Real-Time PCR. The reaction of Real-Time PCR was done using Master Mix containing SYBR Green I (TAKARA, Cat No.: RR820L; Japan) and Real-Time (Applied Biosystem; USA). 20µl reactions were done with 2.5µm cDNA production, 1µl step out primer and 1µl step back primer with 5pmol for CXCL10 and with 10pmol concentration in PCR Real-Time reaction. For GAPDH, 9.6µlit Master Mix and 0.4µlit Rox were done with 1 cycle with 95°C heat for 30seconds, 50 cycles with 95°C heat for 5seconds, and then 50 cycles with 60°C heat for 30seconds and after PCR stages had ended, the melting curve program was performed in the device. Only one pick was observed on the melting curve and the absence of non-specific products was confirmed. Delta CT method was used to investigate relative gene expression. Standard curve was used to compute the efficiency. And 1 to 5 concentrations of control cDNA were used to produce the standard curve. The differences in PCR reaction efficiency of target and standard genes were close to each other [35]. Data was analyzed using SPSS 20 software and Mann-Whitney Test.
Findings by Text
At the time of sampling, the mean ages of exposed and non-exposed groups were 41.95±11.13years and 51.02±10.07years, respectively. Delta CT mean values of the veterans and control samples were 7.37±3.58 and 5.81±3.29. There was no significant difference between the groups and between exposed and non-exposed sub-groups. CXCL10 expression patterns in exposure and non-exposed groups with similar pulmonary pathologic disorders were the same (Fig. 1).
Main comparison to the similar studies
Findings by Text: At the time of sampling, the mean ages of exposed and non-exposed groups were 41.95±11.13years and 51.02±10.07years, respectively. Delta CT mean values of the veterans and control samples were 7.37±3.58 and 5.81±3.29. There was no significant difference between the groups and between exposed and non-exposed sub-groups. CXCL10 expression patterns in exposure and non-exposed groups with similar pulmonary pathologic disorders were the same (Fig. 1).
Suggestions
The research ought to be repeated with more samples. CXCL10 in samples such as phlegm and bronchoalveolar lavage, which can show lung tissue situation, ought to be investigated. More studies ought to be done to investigate the correlation between CXCL10 and the severity of the delayed effects. In addition, the level of the chemokine in different injured tissues of the veterans ought to be measured including skin and eyes.
Limitations
Difficult access to proper samples from archives of the hospitals was one of the limitations for the present study. It was impossible to remove affecting factors, such as consumed medications by the patient, from the study, which might affect the results.
Conclusions
The level and pattern of CXCL10 expression are not different in lung tissues of two exposed and non-exposed groups.
Acknowledgments
The researcher fells grateful to Immunoregulation Research Center.
Conflict of interest
Non-declared
Ethical Permissions
All procedures were approved by Ethics Committee of Shahed University.
Funding Sources
The study was funded by Immunoregulation Research Center.

TABLES and CHARTS

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