Effect of Ionizing Radiation Exposure on NOX4 Expression in-Vitro and in-Vivo Studies: A Systematic Review and Meta-analysis

Effect of Ionizing Radiation Exposure on NOX4 Expression in-Vitro and in-Vivo Studies: A Systematic Review and Meta-analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of Ionizing Radiation Exposure on NOX4 Expression in-Vitro and in-Vivo Studies: A Systematic Review and Meta-analysis Pooya Hajimirzaei, Reza Paydar, Maryam Razmgir, Fatemeh Rajabinasab, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4854221/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Patients exposed to ionizing radiation (IR) from various sources experience several side effects. Understanding the mechanisms through which IR induces these effects could minimize their consequences. This study investigates the activation pathway of NADPH oxidase 4 (NOX4) after exposure to IR. Methods: The analysis incorporated studies that assessed NOX4 expression as an outcome variable. The study involved searches across various databases. A total of 58 articles were included in the meta-analysis, and data extracted from these studies were analyzed using Comprehensive Meta-Analysis Software. Results: Analysis of the impact of IR on NOX4 expression, demonstrated a notable increase in protein expression in animals (SMD=3.452; p<0.001), in normal cells in vitro (SMD=2.689; p<0.001), and in cancer cells (SMD=2.159; p<0.05). Furthermore, there was a significant increase in NOX4 mRNA expression in animals (SMD=5.070; p<0.001), in normal cells in vitro (SMD=3.563; p<0.001), and in cancer cells (SMD=3.280; p<0.001). Subgroup analysis was conducted based on the tests utilized to measure NOX4 expression, various organs, IR parameters, and follow-up time after IR. Conclusion: NOX4 plays a crucial role in mediating radiation-induced damage in many organs. The upregulation of NOX4 expression in these organs is influenced by factors such as the radiation dose and source. Additionally, there was a further increase in NOX4 protein expression over time, highlighting its potential role in the progression of radiation-induced damage in vital organs. Ionizing Radiation NOX4 Radiotherapy Meta-analysis Systematic Review Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Gamma-ray induces higher expression of NOX4 compared to X-ray. The peak in NOX4 mRNA upregulation was observed 1-2 weeks post-radiation exposure. The bone marrow exhibits heightened sensitivity to NOX4 upregulation following radiation. The impact of whole-body irradiation on NOX4 elevation is equivalent to that of local radiation. Introduction Exposure to ionizing radiation (IR) can manifest through various pathways, including but not limited to solar radiation, X-ray devices, and radioactive isotopes emitting gamma rays (γ-rays). The impact of IR on tissue and organ functionality is well-documented, as the potential for negative health outcomes escalates with higher dosage levels, particularly evident in the pediatric and adolescent populations due to their heightened susceptibility. 12 studies have presented comprehensive evaluations of the correlation between exposure to IR from radon and radon decay products and the associated risk of developing lung cancer among miners. ( 1 , 2 ). The utilization of radiation in medical contexts, including diagnostic radiology and radiotherapy, is prevalent, underscoring the significance of comprehending and alleviating radiation-induced harm ( 3 , 4 ). Radio sensitivity, radio susceptibility, and radio degeneration are the three categories into which IR side effects can be subdivided. Humans react to IR in different ways. Radiosusceptibility to radiation is concerned with cancers generated by radiation, radio-degeneration is with non-cancerous consequences typically ascribed to processes other than cell death, and radio-sensitivity is with the early or late unfavorable tissue effects following IR exposure to normal tissues ( 5 , 6 ). Numerous research investigations have documented correlations between insulin resistance (IR) and occurrences of hepatocellular carcinoma and esophageal cancer, as well as, to a lesser degree, multiple myeloma and non-Hodgkin’s lymphoma; however, the findings exhibit variability. Additionally, prolonged exposure to low levels of radiation may result in impairments in T-cell-mediated immune responses ( 7 – 9 ). Exposure to radiation in anatomical regions, such as the thorax, can result in radiation-induced damage, including reversible pneumonitis and irreversible pulmonary fibrosis, which may ultimately result in fatality ( 10 , 11 ). On the contrary, cell types like progenitor cells and bone marrow stem cells, which exhibit a rapid rate of division, appear to display increased sensitivity to radiation ( 12 ). In addition, these cells express pro-apoptotic genes. This makes them vulnerable to radiation and rapidly induces apoptosis ( 13 ). A reduction in the number of circulating blood cells, including red blood cells, white blood cells and platelets, is one of the important side effects of IR ( 14 ). Regardless of whether the organs are directly in the IR field, close by or far away, they can still suffer side effects. This is because radiation can cause both local and systemic reactions (bystander and abscopal effects) ( 15 ). Some examples of radiation-related normal tissue complications include damage to the optic and auditory nerves ( 16 ), bone necrosis ( 17 ), damage to the endothelium of blood vessels, which can lead to cardiovascular and respiratory complications ( 18 ), decreased secretion of exocrine glands ( 19 ), and decreased renal filtration rate (GFR) ( 20 ). A comprehensive understanding of the mechanisms of IR-induced damage is crucial for the management of its adverse effects and the development of radiation protection approaches. Knowledge of the effective radiation doses to induce organ damage and the molecular pathways involved is essential for the design of preventive measures. In addition, the use of IR for therapeutic purposes, such as cancer treatment, underlines the need for effective preventive and protective strategies. Therefore, a comprehensive understanding of the health effects of IR and the development of targeted protective measures are essential to minimize the risks associated with radiation exposure. IR exerts its effects on the living biological environment in two main ways. The first and probably most important pathway is the production of reactive free radicals based on oxygen or nitrogen species ( 21 ). Such chemically reactive molecules interact with macromolecules such as DNA and proteins to produce their damaging effects ( 22 , 23 ). The lifetime of reactive oxygen species (ROS) is no more than a fraction of a second, but the adverse effects persist for much longer ( 24 ). Nuclear factor-kappa beta (NF-κB) is a transcription factor that controls the expression of a number of genes associated with IR ( 25 ), including an increase in ROS production via the mitochondrial pathway ( 26 ). NF-κβ induces a chronic increase in intracellular ROS by increasing the expression of NADPH oxidase (NOX) ( 27 ). NOX is known to be one of the major pro-oxidant enzymes that generate superoxide species via NADPH ( 28 , 29 ). The most important member of the NOX family is NOX4, which acts as a major producer of intracellular ROS, particularly H 2 O 2 ( 30 ). The expression of NOX4 increases the production of H 2 O 2 , and the H 2 O 2 produced in turn leads to the activation of NF-κβ, and this cycle continues so that the damaging effects of radiation are amplified each time ( 10 ). Therefore, the production of ROS, in particular through the activation of NOX4, is a major factor in the damage to healthy tissues following radiation exposure ( 31 ). Considering the major role of NOX4 among all the possible harmful mediators of IR, the level of NOX4 could be a crucial marker in assessing the adverse effects of IR (Fig. 1 ). In this systematic review and meta-analysis, the effects of IR on the expression of NOX4 (protein and mRNA) in various organs of animals and different cell lines, as well as the parameters of radiation (dose, source, and method), follow-up time, and evaluation tests, were investigated. Methods Five databases were searched to identify relevant studies including PubMed, Scopus, Web of Science, Embase, and Google Scholar up to December 20, 2023. No language restrictions were applied. Given that the same search strategy does not work for different databases, a separate strategy was written for each database. In addition to the systematic search, a manual search was performed to obtain additional articles. For instance, a search in Google Scholar was done based on keywords related to the subject. A combination of the following search words was used for the relevant literature: (“NADPH oxidase 4”, “NOX4 protein”, “Lenox NAD(P)H Oxidase “, “renal NAD(P)H Oxidase”, “NOX4”, “radiotherapy”, “radiation therapy”, “radiotherapies”, “rradiation treatment”, “targeted radiotherapy”). The detailed search strategy for PubMed is provided in the Appendix. Inclusion Criteria The inclusion criteria were applied: peer-reviewed studies based on NOX4 measurement after IR, peer-reviewed in-vitro studies that evaluated NOX4 expression after IR, studies that used a healthy or control group in addition to the IR group, and IR exposure carried out by a standard device. Exclusion Criteria The following articles were excluded; review articles, articles not discussing NOX4 expression after IR, studies that did not use appropriate NOX4 evaluation methods, studies without a control group, case reports, letters to the editor, short reports, and congress abstracts. Methods of Article Assessment Information gathering was performed based on a web checklist in conformance with PRISMA rules (32). At first, two co-authors independently screened the articles based on the title and abstract, and then on the full text of the article. Where there was any disagreement a discussion took place, and then a third researcher was recruited to resolve the disagreement. Finally, an Excel spreadsheet was designed to enter the required information, including the c, outcome, and possible biases. Data such as Mean, SEM, and SD were extracted by plot digitizer software and added to the Excel table (33-35). Risk of Bias Assessment The SYRCLE Risk of Bias tool was employed for the quality assessment of preclinical animal studies and evaluated by two independent researchers (36). This scale comprises 10 items covering 5 categories (Table 1). The scale was modified to change the quantitative risk of bias into a subjective appraisal: <50% (poor), 50%-69% (reasonable), 70%-79% (good), and 80%-100% (exceptionally good). The CONSORT statement was updated for in-vitro studies (37) (Table 2). Analysis The data were presented as mean and standard deviation (SD). Comprehensive meta-analysis Software (3.3.070; USA) was used for data analysis. The effect size with a 95% confidence interval (95% CI) was calculated. The random-effect model was applied, and if the heterogeneity was less than 50%, the fixed-effect model was used. The presence of publication bias was examined by Egger's precision-weighted linear regression method and the results were presented as a funnel plot. Leave-one out sensitivity analyses: Sensitivity analyses were conducted by omitting one study at a time to assess the influence of potential bias on the results of the meta-analysis and to gauge the strength of the findings in light of significant heterogeneity, guaranteeing the dependability of the meta-analysis outcomes. The I² statistic was utilized to assess heterogeneity, while subgroup analyses were carried out to probe the sources of variation among the studies included. In both animal and in-vitro studies, the quantification of the NOX4 expression (both mRNA and protein), was analyzed after and without IR exposure. Subgroup analysis in animal studies was done based on evaluation test, organ, radiation dose, radiation source, irradiation method, and follow-up time. Subgroup analysis in in-vitro normal cell studies were done based on cell type, radiation dose, radiation source, and follow-up time. The findings were reported as standardized mean difference and 95% CI, and in all analyses, p < 0.05 was considered as a significant level. In the analysis of NOX4 expression in different articles, it was found that differing follow-up times were used to assess NOX4. To avoid double counting and ensure accurate analysis, the data from different time points were averaged. However, for subgroup analysis based on follow-up time, the data from each time point were classified into different groups, so averaging was not necessary. This approach was taken to ensure that the data were accurately analyzed and that the results were not skewed by double counting. Results 519 articles were extracted based on our search strategy, of which 266 were from PubMed, 170 from Embase, 52 from Scopus, and 31 from Web of Science. After removing duplicate articles by Endnote software, 217 studies remained and were selected for initial screening. Finally, 43 in-vivo (animal) and 15 in-vitro studies were included in the qualitative and quantitative analysis. The results of the different stages of screening are shown in the PRISM flowchart in Figure 2. Characteristics of Articles The characteristics of the articles included in the study are listed in Table 3, divided into in-vivo animal and in vitro sections. Table 3-A includes 43 in-vivo (animal) studies, including 28 studies measuring NOX4 protein and 16 studies measuring NOX4 mRNA. Protein measurement was conducted using Western blot (WB) tests in 15 studies, immunohistochemistry (IHC) in 8 studies, flow cytometry (FC) in 6 studies, and enzyme-linked immunosorbent assay (ELISA) in one study. In addition, mRNA measurement was conducted using real-time PCR (qRT PCR) in 16 studies. NOX4 expression was measured in various organs, including the lungs in 14 studies, bone marrow in 11 studies, cardiovascular system in 7 studies, kidney in 3 studies, salivary gland in 2 studies, and 8 other organs each in one study, comprising the brain, liver, small intestine, spleen, thymus, prostate, parotid gland, and ovarian. The studies employed administered doses of IR that varied from 1 to 75 Gy. Out of the total, 18 studies focused on whole-body irradiation, while 21 studies investigated localized radiation treatment. The radiation sources utilized were: proton beam in one study, electron beam in one study, X-ray (KV) in 12 studies, X-ray (MV) in 9 studies, γ-ray (60Co) in 5 studies, and γ-ray (137Cs) in 15 studies. The time intervals post-irradiation employed for evaluating NOX4 expression varied from 24 hours to 22 months. Table 3-B includes 15 in-vitro studies, with 10 measuring NOX4 protein and 10 measuring NOX4 mRNA, with some studies measuring both. The measurement of protein used WB tests in 8 studies and FC in 2 studies. For mRNA assessment, qRT-PCR was the method used in all 10 studies. NOX4 expression was evaluated in various cell types, including human cells in 11 studies, rat cells in 3 studies, and mouse cells in 2 studies. The doses of IR used in these studies ranged from 3 cGy to 16 Gy, with X-ray used in 10 studies and γ-ray in 5 studies. The follow-up period for NOX4 analysis post-irradiation varied from 15 minutes to 5 days. Of these, 13 studies utilized normal cells, while 2 studies employed cancer cells. The following studies were excluded from the meta-analysis; an in vivo study from Jiyoung Park 2022 (38), and in vitro studies from Jiwon Choi 2022 (39), Urbain Weyemi 2015 (40), Yingchun Zhang 2012 (92), Yingchun Zhou 2022 (93), Sung Hyo Park 2020 (48), Sarah Park-2010 (96), Eun Joo Chung 2019 (97), and XiaoHong Yang 2017 (98) because they did not contain complete analysis, the results were not clearly stated, or were not in line with the inclusion criteria. Based on the search results, there were no clinical studies specifically investigating the effect of IR on NOX4 expression in humans. Meta-analysis (Animal studies) Funnel plot The results of the funnel plot analysis and Egger’s test on the animal studies are reported in Figure 2, indicating possible publication bias for the outcome measures including mRNA expression (Egger's p < 0.05) (Figure 3-A), and protein expression (Egger's p < 0.05) (Figure 3-B). Overall Analysis It was found that 16 studies (containing 19 separate experiments) reported a noteworthy increase in NOX4 mRNA expression in the irradiated animals, with a SMD of 5.070 and CI95% ranging from 3.374 to 6.766;(p < 0.001; I 2 =91.8%). Furthermore, 28 studies (containing separate 38 experiments) reported a significant increase in NOX4 protein expression post-irradiation, with an SMD of 3.452, CI95% of 2.807 to 4.097, (p < 0.001, and; I 2 =74.5%) (Figure 4). Sensitivity Analysis The leave-one-out sensitivity analysis indicated that no single study had a significant effect on the overall findings of NOX4 mRNA and protein expression after IR, suggesting that the results were robust and not unduly influenced by any individual study. Subgroup Analysis Subgroup analysis was conducted based on various factors including the type of tests used to measure NOX4 expression, specific organs studied, dose of IR administered, source of radiation, and the follow-up time after exposure to IR. Subgroup analysis of tests measuring NOX4 expression post-irradiation show varying results. The use of WB (SMD=2.886; P < 0.001), IHC (SMD=6.165; P < 0.001), FC (SMD=2.862; P < 0.001), and qRT-PCR (SMD=5.070; P < 0.001) all provided significant results, while ELISA was only utilized in a single study (Table 5-a). Eight organs (brain, liver, ovaries, parotid gland, prostate, small intestine, spleen, and thymus) were limited to a single study (Table 5-b). Subgroup analysis focusing on different organs where NOX4 expression was assessed showed notable increases following IR exposure in specific organs. Increased NOX4 protein expression was observed in the lungs (SMD=4.391; P < 0.001), bone marrow (SMD=2.862; P < 0.001), cardiovascular system (SMD=2.206; P < 0.01), salivary gland (SMD=3.714; P < 0.001), and kidney (SMD=1.502; P < 0.01) post-irradiation. Moreover, increased NOX4 mRNA expression was observed in the bone marrow (SMD=9.371; P < 0.01) and cardiovascular system (SMD=2.716; P < 0.01) post-irradiation. Subgroup analysis based on the dose of IR showed a substantial increase in NOX4 protein expression across different dose levels: 3-6 Gy (SMD=2.631; P < 0.001); 6-12 Gy (SMD=4.236; P < 0.001); 12-24 Gy (SMD=2.941; P < 0.001); and 75 Gy (SMD=8.303; P < 0.05) (Table 5c). Subgroup analysis based on the dose of IR showed also a substantial increase in NOX4 mRNA expression across different dose levels: 1-3 Gy (SMD=13.904; P < 0.05); 3-6 Gy (SMD=11.505; P < 0.05); 6-12 Gy (SMD=2.706; P < 0.01); and 12-24 Gy (SMD=4.405; P < 0.01) (Table 5-c). Moreover, subgroup analysis based on the irradiation method revealed that both whole body irradiation (SMD=3.907; P < 0.001) as well as local irradiation (SMD=3.581; P < 0.001) had a significant effect on increasing NOX4 protein expression (Table 5d). The same results were observed for NOX4 mRNA expression: whole body irradiation (SMD=6.784; P < 0.001) and local irradiation (SMD=2.736; P < 0.01) (Table 5-d). Subgroup analysis based on the radiation source, showed that various radiation sources, including X-ray (KV) (SMD=2.725; P value < 0.001); X-ray (MV) (SMD=3.162; P value < 0.001); γ-ray (137Cs) (SMD=3.724; P value < 0.001); γ-ray (60Co) (SMD=16.507; P value < 0.05); and electron beam (SMD=1.997; P value < 0.05) all had a significant effect on increasing NOX4 protein expression (Table 5-e). Similarly, for NOX4 mRNA expression similar results were observed: X-ray (KV) (SMD=1.288; P value < 0.01); X-ray (MV) (SMD=17.133; P value < 0.05); γ-ray (137Cs) (SMD=4.823; P value < 0.001); γ-ray (60Co) (SMD=9.211; P value < 0.05); and proton beam (SMD=15.014; P value < 0.01) (Table 5e). Proton beam and electron beam were each only used in one study. Finally, subgroup analysis based on the follow-up time after IR exposure showed a consistent increase in NOX4 protein expression across all follow-up times. This included times below one week (SMD=2.455; P < 0.001), between one and two weeks (SMD=3.588; P < 0.001), between two weeks and one month (SMD=3.169; P < 0.001), and more than one month (SMD=4.653; P < 0.001) (Table 5f). Similarly, for NOX4 mRNA expression similar results were observed: below one week (SMD=8.110; P < 0.001), between one and two weeks (SMD=9.347; P < 0.01), between two weeks and one month (SMD=5.861; P < 0.01), and more than one month (SMD=3.916; P < 0.001) (Table 5-f). Meta-analysis (in-vitro studies) Funnel plot The results of the funnel plot analysis and Egger’s test of the in-vitro studies are reported in (Figure 5), indicating possible publication bias for the outcome measure of mRNA expression in normal cells (Egger's p < 0.05) (Figure 5-A) and protein expression in normal cells (Egger's p 0.05) (Figure 5-C). Analysis of normal and cancer cells General analysis of the normal cell studies revealed that 9 studies (10 independent experiments) had a significant increase in NOX4 (mRNA) expression in the irradiated cells (SMD = 3.563; CI95% 2.297 to 4.829; P < 0.001; I 2 =69 %). Additionally, 8 studies reported a significant increase in NOX4 protein expression after IR (SMD = 2.689; CI95% 1.314 to 4.064; P < 0.001; I 2 =79.5 %) (Figure 6-A). These findings are in agreement with the meta-analysis of the animal studies. In addition, the studies included in Table 3B utilized various methods to assess the effects of IR on NOX4 expression, with the majority of studies showing a significant increase in NOX4 expression in normal cells after irradiation. The general analysis of the cancer cell studies revealed that 1 study observed a significant increase in NOX4 (mRNA) expression in irradiated cells (SMD = 3.280; CI 95% 1.865 to 4.694; P < 0.001). In addition, 2 studies (3 independent experiments) reported a significant increase in NOX4 (protein) expression after IR (SMD = 2.159; CI 95% 0.202 to 4.116; P < 0.05; I 2 =81.5 %) (Figure 6-B). Sensitivity Analysis The leave-one-out sensitivity analysis indicated that no single study had a significant effect on the results of NOX4 mRNA and protein expression in normal cells after IR, suggesting that the results were robust and not heavily influenced by any individual study. The estimated pooled SMD was significant in the original analysis of NOX4 protein expression in cancer cells (P < 0.05). However, the leave-one-out sensitivity analysis revealed that the statistical significance was changed by the removal of the experiments included in Jiyoung Park 2022-3 study (38): 4T1-Luc mouse breast cancer cells independent experiment (SMD = 2.064; CI95% (-1.638 to 5.766); P > 0.05) or MDA-MB-231 human breast cancer cells independent experiment (SMD = 1.319; CI95% (-0.786 to 3.424); P > 0.05). Subgroup Analysis of Normal Cells Subgroup analysis of normal cells was performed based on the tests that measured NOX4 expression after radiation, including WB (SMD=1.945; P value < 0.01), FC (SMD=4.193; P < 0.001), and qRT-PCR (SMD=3.563; P < 0.001) (Table 6-a). Following the initial analysis, further investigation was carried out focusing on the source of the normal cells in which NOX4 expression was assessed. The findings indicated a significant rise in NOX4 protein expression levels after IR in human cells (SMD=2.491; P < 0.01) (Table 6-b). Also, the findings indicated a significant rise in NOX4 mRNA expression levels after IR in human cells (SMD=3.412; P < 0.01), rat cells (SMD=5.652; P < 0.001), and mouse cells (SMD=2.878; P < 0.01) (Table 6-b). Additionally, subgroup analysis was undertaken based on the radiation dose, radiation source, and duration of follow-up. The subgroup analysis concerning the dose of IR revealed a noteworthy impact on the upregulation of NOX4 protein expression in normal cells at two dose levels; 3-50 cGy (SMD=4.193; P < 0.001) and 8-16 Gy (SMD=2.516; P < 0.05) (Table 6c). NOX4 mRNA expression in normal cells was upregulated at all dose levels; 3-50 cGy (SMD=4.149; P < 0.001), 2-6 Gy (SMD=3.173; P < 0.001), and 8-16 Gy (SMD=2.987; P < 0.05) (Table 6-c). Moreover, the subgroup analysis, focusing on the radiation source, showed that both X-ray (SMD=2.601; P < 0.01) and γ-ray (SMD=3.531; P < 0.01) sources had a significant impact on increasing NOX4 protein expression in normal cells (Table 6d). Similarly, for NOX4 mRNA expression in normal cells, the same results were observed: X-ray (SMD=3.020; P < 0.001) and γ-ray (SMD=4.224; P < 0.001) (Table 6-d). Lastly, the subgroup analysis based on the follow-up time post-IR exposure indicated an increase in NOX4 protein expression in normal cells at all follow-up time points: 1 hour (SMD=3.601; P < 0.05), 2-24 hours (SMD=2.590; P < 0.01), and 2-5 days (SMD=2.306; P < 0.001) (Table 6-e). Similarly, for NOX4 mRNA expression in normal cells, the same results were observed: 1 hour (SMD=4.112; P < 0.001), 2-24 hours (SMD=3.843; P < 0.001), and 2-5 days (SMD=2.612; P < 0.01) (Table 6-e). Discussion All animal studies were conducted in normal animals, while a significant number of in vitro studies were conducted in normal cells, with only a limited number of studies focusing on cancer cells. In both animal and in vitro studies, both mRNA and protein levels of NOX4 were found to be increased after IR. In addition, the results of in vitro and in vivo analyses indicated that the effect of IR on NOX4 mRNA was more pronounced than its effect on NOX4 protein levels at various times after exposure. Furthermore, based on the in vitro results, it was found that NOX4 mRNA protein and mRNA expression increased in both normal and cancer cells, with a more pronounced increase observed in normal cells. However, this finding must be interpreted with caution due to the limited number of studies using cancer cells. The subgroup analysis suggested that the lungs, cardiovascular system, salivary glands and bone marrow were highly sensitive to radiation exposure. The lungs showed the highest sensitivity to increases in NOX4 protein, followed by the salivary glands, bone marrow and cardiovascular system. Conversely, the kidney showed the lowest increase in NOX expression levels. In addition, the highest mRNA up-regulation was found in the bone marrow. As mentioned above, the kidneys showed the lowest increase in NOX4 protein expression after radiation exposure. However, it is important to note that only three studies were carried out on the kidney, highlighting the need for further research on this organ. However, this finding may be due to the high levels of NOX4 expression found in the distal tubular cells of the normal human kidney ( 95 ). Conversely, studies have suggested that NOX enzymes are involved in facilitating radiation-induced damage in various organs, such as the kidney. In particular, NOX4 has been identified as a contributor to the development of cisplatin-induced acute kidney injury through the induction of ROS-mediated programmed cell death and inflammation ( 96 , 97 ). Therefore, further research into the role of NOXs in the kidney and the potential for NOX-specific drugs to reduce radiation-induced kidney injury is warranted. In vitro analysis of normal cells showed that increasing dose within the study range (3 cGy to 16 Gy) led to a reduction in the up-regulation of NOX4 mRNA expression levels. Notably, the highest dose group (6–16 Gy) showed no significant changes in NOX4 mRNA expression. Similarly, analysis of NOX4 protein levels showed that increasing the dose from 3 cGy to 6 Gy resulted in a decrease in the upregulation of NOX4 protein expression, with the 2–6 Gy dose group showing no significant changes in NOX4 protein expression. However, when the dose was further increased to 6–16 Gy, NOX4 protein expression was increased, although to a lesser extent than in the cGy group. Interestingly, both NOX4 mRNA and protein analyses in the in vitro normal cell group suggested that the cGy doses were more effective than the Gy range doses in modulating NOX4 expression levels. On the other hand, the in vivo results showed that NOX4 mRNA changes were most pronounced at the lowest IR dose (1 to 3 Gy). Within the dose range of 1 to 12 Gy, dose escalation initially resulted in a reduced effect; however, after 12 Gy, there was a resurgence of the effect on mRNA expression. In essence, dose escalation resulted in an initial decrease in effect, followed by a subsequent increase in effect at even higher doses. The subgroup analysis of NOX4 protein showed that a lower dose of radiation resulted in higher NOX4 expression, but 75 Gy, which was the highest dose, had the greatest effect in increasing NOX4. These findings are supported by various studies, such as the induction of NOX4 expression following a dose-dependent pattern and a significantly higher expression observed after 4 Gy of heavy ion irradiation ( 98 ). In addition, research has shown that both low (< 100 mGy) and high doses (10 Gy) of IR can lead to the up-regulation of NADPH oxidase, contributing to the increased expression of NOX4 ( 99 ). These collective findings highlight the need for a comprehensive understanding of the effect of radiation dose on NOX4 expression and the implications for radiation-induced damage. Although the exact mechanism for the observed increase in NOX4 expression at lower radiation doses is not explicitly explained in the search results, it is evident that NOX4 expression is closely linked to IR dose and the resulting production of ROS. Further research may be required to fully elucidate the specific mechanisms underlying the dose-dependent effects of radiation on NOX4 expression. Based on the results of the in vitro normal cells, the expression levels of both NOX4 protein and mRNA decreased over time after irradiation. Therefore, the highest levels of protein and mRNA were recorded during the first hour after irradiation. While the expression of mRNA decreased with time after IR exposure, the expression of NOX4 protein increased in in vivo studies. After mRNA is transcribed from DNA, it serves as a template for protein synthesis, so mRNA levels can decrease over time as the mRNA molecules are translated into protein. As proteins are synthesised and fulfil their functions, the mRNA molecules may be degraded or have a short half-life compared to proteins. This can lead to a decrease in mRNA levels over time as protein levels increase. This illustrates the complex relationship between mRNA and protein levels, which is influenced by factors such as protein turnover rates and post-translational regulatory mechanisms ( 100 – 102 ). While a slight decrease was observed at follow-ups between two weeks and one month, it returned to a higher level than at the one-week follow-up at one month. This pattern may be an important reason for the spread of damage following radiation exposure. Over time, NOX4 expression may lead to the sustained production of excessive ROS, resulting in oxidative stress and damage to various cellular components such as nucleic acids, proteins and lipids. This could lead to various pathological conditions such as hyperglycemia, inflammation, cancer and vascular abnormalities ( 103 – 104 ). Whole-body irradiation results in a higher production of NOX4 protein and mRNA compared to local irradiation, as suggested by the results. Studies have shown that whole body irradiation causes residual bone marrow injury by inducing excessive NOX activity, which leads to increased ROS production. This underlines the importance of immediate regulation or inhibition of NOX4 activity in individuals who are accidentally exposed to radiation, such as in nuclear accidents, mishandling of radioactive sources, cosmic and solar radiation, terrestrial IR emitted from the earth or from building materials ( 105 – 107 ). The changes in NOX4 expression were influenced by the type of radiation source used. Specifically, X-rays (MV) and γ-rays (60Co) showed the most pronounced effects on mRNA expression, while X-rays (KV) had the least effect on mRNA expression. On the other hand, γ-rays (60Co) had the greatest effect on NOX4 protein expression. The observed significant effect of X-rays (MV) on mRNA should be interpreted with caution due to the high heterogeneity and limited number of articles included in the analysis. In in vitro studies, due to the limited number of articles available, a subgroup analysis was performed based only on the radiation source and not on the energy level. In in vitro normal cell studies, γ-rays showed a more pronounced effect on increasing NOX4 protein and mRNA expression compared to X-rays. These results, which link the effect of radiation sources on NOX4 expression to clinical outcomes and treatment strategies, may provide valuable insights to improve the practice of personalised medicine in radiation oncology. By optimising treatment approaches and minimising side effects, these findings have the potential to improve patient outcomes. NOX4 expression may also serve as an indicator for assessing treatment efficacy, predicting poorer outcomes or determining increased risk of metastasis. In addition, regulating NOX4 activity may be a promising way to enhance treatment efficacy or sensitize cancer cells to radiotherapy. In the in-vitro studies, NOX4 levels were increased in all three types of animal cells. However, the rise in NOX4 protein levels in rat cells was not statistically significant, likely due to limited data and notable heterogeneity within the samples. Some reports have suggested that the increase in NOX4 after radiotherapy in glioblastoma may play an important role in the radioresistance produced by the tumor microenvironment ( 108 ). In pelvic cancer, NOX4 causes radiation nephrotoxicity ( 109 ). However, one study suggested that increased expression of NOX4 may lead to less radiation-induced damage to the hematopoietic system ( 110 ). Conclusion NOX4 plays a role in mediating radiation-induced damage in various organs, including the kidneys, lungs, and heart . The increase in NOX4 expression in various organs depends on the radiation dose radiation, source, and time after exposure, NOX4 protein expression increases over time, in line with the spread of damage. Additionally, the potential role of NOX4 as a therapeutic target for enhancing the sensitivity of cancer cells to ionizing radiation warrants further exploration. The implications of NOX4 expression in the context of biodosimetry and the development of radiation resistance need to be elucidated to advance our understanding of the biological effects of ionizing radiation exposure. Overall, ionizing radiation exposure triggers NOX4 expression, which contributes to cellular senescence, DNA damage, tissue inflammation, oxidative stress and fibrotic responses, highlighting the significance of NOX4 in the biological effects of radiation exposure. Further research is needed to fully elucidate the mechanisms underlying the expression of NOX4 over time in various organs. Clinical impact These findings suggest the ongoing development of NOX4 inhibitors for clinical radiotherapy applications should be continued, particularly in the context of protecting against vascular, lung, bone marrow, and other radiation-induced injuries. Declarations Declaration of Interest The authors declare no competing interests that may have influenced the research, results, or conclusions presented in this manuscript. Ethical Approval and consent to participate The proposal with ethical code (IR.IUMS.REC.1401.705) was approved by the IACUC of the Iran University of Medical Science, Tehran, Iran. Consent to Publication All authors consent to the publication of the article in the journal “BMC Molecular and Cell Biology”. Data Availability statement Data sets generated during the current study are available from the corresponding author on reasonable request. Funding This research was supported by [IR.IUMS.REC.1401.705]. The funding sources had no involvement in the study design, data collection, analysis, interpretation, or writing of the manuscript. Acknowledgment The authors extend their appreciation to the Radiation Biology Research Center (RBRC), Physiology Research Center, and Systematic Review Network under the Vice-Chancellor for Research and Technology at Iran University of Medical Sciences for their assistance and support. References Laurier D, Marsh J, Rage E, Tomasek LJAotI. Miner studies and radiological protection against radon. 2020;49(1_suppl):57-67. Timins JKJHp. Communication of benefits and risks of medical radiation: a historical perspective. 2011;101(5):562-5. Hendee WR, O’Connor MKJR. Radiation risks of medical imaging: separating fact from fantasy. 2012;264(2):312-21. 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Mitigation of radiation‐induced hematopoietic system injury by melatonin. 2020;35(8):815-21. Tables Tables 1 to 6 are available in the Supplementary Files section Appendix Appendix is not available with this version Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tiff Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4854221","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345341682,"identity":"eaccd6d0-889a-4aa3-a1b8-80386c31ba82","order_by":0,"name":"Pooya Hajimirzaei","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Pooya","middleName":"","lastName":"Hajimirzaei","suffix":""},{"id":345341684,"identity":"13c588ec-c541-4f5d-9032-22f1b1989cd3","order_by":1,"name":"Reza Paydar","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"","lastName":"Paydar","suffix":""},{"id":345341685,"identity":"1c8a667a-6368-41fd-a02f-31085cde4593","order_by":2,"name":"Maryam Razmgir","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Razmgir","suffix":""},{"id":345341686,"identity":"08418987-1588-48b3-b148-0db3049b0684","order_by":3,"name":"Fatemeh Rajabinasab","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Rajabinasab","suffix":""},{"id":345341687,"identity":"324d0852-a94f-4099-a1a7-0c21b6230a28","order_by":4,"name":"Faeze AhmadiTabatabaei","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Faeze","middleName":"","lastName":"AhmadiTabatabaei","suffix":""},{"id":345341688,"identity":"f905e461-db11-42c4-8ed7-2a0a4996fda5","order_by":5,"name":"Michael R Hamblin","email":"","orcid":"","institution":"University of Johannesburg","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"R","lastName":"Hamblin","suffix":""},{"id":345341690,"identity":"a79063fc-9315-4b33-8bf4-71dc0ed02060","order_by":6,"name":"Atousa Janzadeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACxgYQycaQAGI/ABI8fCRoYWY2AGlhI84uiBY2CQibAGBuP3v4w48ymzwG9vPHKr/m2MmwMTA/fHQDn8N68hIMe86lFTPwJLPdlt2WDHQYm7FxDl6/5Bgk8LYdTmxgAGqR3MYM1MLDJo1XS/8bg4N/2/4nNvA/ZiuW3FZPhJYZOYbNvG0HEhskktkYP247TIyWN8bMMueSE9skHhtLM247zsPGTMAvhv05xh/flNkl9vMnPvz4c1u1PT9788PHeLU0QBmg6GDmAbGY8SgHAXkUV/4goHoUjIJRMApGJgAAh4BDacSXcQAAAAAASUVORK5CYII=","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Atousa","middleName":"","lastName":"Janzadeh","suffix":""},{"id":345341692,"identity":"34685ecb-1187-48f9-9f40-ab27b65bb210","order_by":7,"name":"Soroush Taherkhani","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Soroush","middleName":"","lastName":"Taherkhani","suffix":""}],"badges":[],"createdAt":"2024-08-03 16:41:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4854221/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4854221/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64306345,"identity":"e9718d02-0dae-4e1f-b304-79bcf8475f87","added_by":"auto","created_at":"2024-09-11 12:54:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":444137,"visible":true,"origin":"","legend":"\u003cp\u003eMechanisms of cell damage caused by exposure to ionizing radiation. When ionizing radiation reaches the cell surface, due to its ionizing nature, it causes the formation of ROS. Further, the increase of ROS levels, causes the destruction of the intracellular structure, including: mitochondrial dysfunction, protein and lipid structures, DNA damage which eventually causes apoptosis. The NOX4 enzyme with its oxidizing function generates more ROS, which further activates the NF-κβ signaling pathway and causes the transcription factor to phosphorylate and enter the nucleus, which subsequently leads to the expression of genes related to inflammatory processes, oxidative stress and cancer.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/3ca974e9c40c2df6e7c28db0.png"},{"id":64307793,"identity":"5432e71a-a803-4a8d-9c4c-6123136c8378","added_by":"auto","created_at":"2024-09-11 13:02:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171390,"visible":true,"origin":"","legend":"\u003cp\u003ePRISMAFlow chart showing the identification and selection process of studies on the effect of IR on NOX4 expression.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/88968d8349534208cc9e4d68.png"},{"id":64307800,"identity":"0c7d5dc6-77ac-455e-81b0-88ff68dd9ad7","added_by":"auto","created_at":"2024-09-11 13:02:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21504,"visible":true,"origin":"","legend":"\u003cp\u003eFunnel plot for the evaluation of publication bias in studies that examined the effect of exposure to IR on NOX4 mRNA (A), and NOX4 protein (B) expression in animal studies.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/1e1bcb3b907162134ee3bd91.png"},{"id":64306351,"identity":"05d61ad8-33ba-487a-8a62-e6e2017e364b","added_by":"auto","created_at":"2024-09-11 12:54:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":515750,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot of evaluation expression of NOX4 after exposure to IR in animal studies that demonstrated a significant increase in expression.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/3be98185b8936f1bf8ab8b72.png"},{"id":64307802,"identity":"2066c5d9-ff45-4982-8ab8-a5e77654c24b","added_by":"auto","created_at":"2024-09-11 13:02:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26655,"visible":true,"origin":"","legend":"\u003cp\u003eFunnel plot for the evaluation of publication bias in studies that examined the effect of exposure to IR in-vitro on NOX4 mRNA (A) and NOX4 protein (B) expression in normal cells; and NOX4 protein expression in cancer cells (C).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/b3f52c53d6ae3645d80096d3.png"},{"id":64306349,"identity":"8683a649-b04f-4f26-9259-e46ff3d54507","added_by":"auto","created_at":"2024-09-11 12:54:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":180963,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot evaluating the expression of NOX4 (mRNA and protein) in normal cells (A) and cancer cells (B) after exposure to IR in in-vitro studies.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/0060cad27658c218f3e70d20.png"},{"id":73759595,"identity":"ec0e38e6-4101-4665-b9ec-93587d4e0e9b","added_by":"auto","created_at":"2025-01-14 11:16:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1995451,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/d17b0e10-1e6e-4399-8e82-0be26700a340.pdf"},{"id":64306347,"identity":"95e5c001-ed2a-49c0-b25c-61079a37c1cf","added_by":"auto","created_at":"2024-09-11 12:54:41","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":460840,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/32a990041dc9fb825efdca02.tiff"},{"id":64306344,"identity":"16cb717d-480f-48c1-886f-b1fabb1d172a","added_by":"auto","created_at":"2024-09-11 12:54:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":96135,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4854221/v1/5eb55193c78edad9e73e6831.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Ionizing Radiation Exposure on NOX4 Expression in-Vitro and in-Vivo Studies: A Systematic Review and Meta-analysis","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eGamma-ray induces higher expression of\u0026nbsp;NOX4\u0026nbsp;compared to X-ray.\u003c/li\u003e\n \u003cli\u003eThe peak in\u0026nbsp;NOX4\u0026nbsp;mRNA upregulation was observed 1-2 weeks post-radiation exposure.\u003c/li\u003e\n \u003cli\u003eThe bone marrow exhibits heightened sensitivity to\u0026nbsp;NOX4\u0026nbsp;upregulation following radiation.\u003c/li\u003e\n \u003cli\u003eThe impact of whole-body irradiation on\u0026nbsp;NOX4\u0026nbsp;elevation is equivalent to that of local radiation.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eExposure to ionizing radiation (IR) can manifest through various pathways, including but not limited to solar radiation, X-ray devices, and radioactive isotopes emitting gamma rays (γ-rays). The impact of IR on tissue and organ functionality is well-documented, as the potential for negative health outcomes escalates with higher dosage levels, particularly evident in the pediatric and adolescent populations due to their heightened susceptibility. 12 studies have presented comprehensive evaluations of the correlation between exposure to IR from radon and radon decay products and the associated risk of developing lung cancer among miners. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The utilization of radiation in medical contexts, including diagnostic radiology and radiotherapy, is prevalent, underscoring the significance of comprehending and alleviating radiation-induced harm (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRadio sensitivity, radio susceptibility, and radio degeneration are the three categories into which IR side effects can be subdivided. Humans react to IR in different ways. Radiosusceptibility to radiation is concerned with cancers generated by radiation, radio-degeneration is with non-cancerous consequences typically ascribed to processes other than cell death, and radio-sensitivity is with the early or late unfavorable tissue effects following IR exposure to normal tissues (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Numerous research investigations have documented correlations between insulin resistance (IR) and occurrences of hepatocellular carcinoma and esophageal cancer, as well as, to a lesser degree, multiple myeloma and non-Hodgkin\u0026rsquo;s lymphoma; however, the findings exhibit variability. Additionally, prolonged exposure to low levels of radiation may result in impairments in T-cell-mediated immune responses (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExposure to radiation in anatomical regions, such as the thorax, can result in radiation-induced damage, including reversible pneumonitis and irreversible pulmonary fibrosis, which may ultimately result in fatality (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). On the contrary, cell types like progenitor cells and bone marrow stem cells, which exhibit a rapid rate of division, appear to display increased sensitivity to radiation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In addition, these cells express pro-apoptotic genes. This makes them vulnerable to radiation and rapidly induces apoptosis (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). A reduction in the number of circulating blood cells, including red blood cells, white blood cells and platelets, is one of the important side effects of IR (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegardless of whether the organs are directly in the IR field, close by or far away, they can still suffer side effects. This is because radiation can cause both local and systemic reactions (bystander and abscopal effects) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Some examples of radiation-related normal tissue complications include damage to the optic and auditory nerves (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), bone necrosis (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), damage to the endothelium of blood vessels, which can lead to cardiovascular and respiratory complications (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), decreased secretion of exocrine glands (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), and decreased renal filtration rate (GFR) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). A comprehensive understanding of the mechanisms of IR-induced damage is crucial for the management of its adverse effects and the development of radiation protection approaches. Knowledge of the effective radiation doses to induce organ damage and the molecular pathways involved is essential for the design of preventive measures. In addition, the use of IR for therapeutic purposes, such as cancer treatment, underlines the need for effective preventive and protective strategies. Therefore, a comprehensive understanding of the health effects of IR and the development of targeted protective measures are essential to minimize the risks associated with radiation exposure. IR exerts its effects on the living biological environment in two main ways. The first and probably most important pathway is the production of reactive free radicals based on oxygen or nitrogen species (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Such chemically reactive molecules interact with macromolecules such as DNA and proteins to produce their damaging effects (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The lifetime of reactive oxygen species (ROS) is no more than a fraction of a second, but the adverse effects persist for much longer (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNuclear factor-kappa beta (NF-κB) is a transcription factor that controls the expression of a number of genes associated with IR (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), including an increase in ROS production via the mitochondrial pathway (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). NF-κβ induces a chronic increase in intracellular ROS by increasing the expression of NADPH oxidase (NOX) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). NOX is known to be one of the major pro-oxidant enzymes that generate superoxide species via NADPH (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The most important member of the NOX family is NOX4, which acts as a major producer of intracellular ROS, particularly H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The expression of NOX4 increases the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced in turn leads to the activation of NF-κβ, and this cycle continues so that the damaging effects of radiation are amplified each time (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, the production of ROS, in particular through the activation of NOX4, is a major factor in the damage to healthy tissues following radiation exposure (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Considering the major role of NOX4 among all the possible harmful mediators of IR, the level of NOX4 could be a crucial marker in assessing the adverse effects of IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this systematic review and meta-analysis, the effects of IR on the expression of NOX4 (protein and mRNA) in various organs of animals and different cell lines, as well as the parameters of radiation (dose, source, and method), follow-up time, and evaluation tests, were investigated.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eFive databases were searched to identify relevant studies including PubMed, Scopus, Web of Science, Embase, and Google Scholar up to December 20, 2023. No language restrictions were applied. Given that the same search strategy does not work for different databases, a separate strategy was written for each database. In addition to the systematic search, a manual search was performed to obtain additional articles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor instance, a search in Google Scholar was done based on keywords related to the subject. A\u0026nbsp;combination\u0026nbsp;of the following search words\u0026nbsp;was used for the relevant literature: (\u0026ldquo;NADPH oxidase 4\u0026rdquo;, \u0026ldquo;NOX4\u0026nbsp;protein\u0026rdquo;, \u0026ldquo;Lenox NAD(P)H Oxidase \u0026ldquo;, \u0026ldquo;renal NAD(P)H Oxidase\u0026rdquo;, \u0026ldquo;NOX4\u0026rdquo;, \u0026ldquo;radiotherapy\u0026rdquo;, \u0026ldquo;radiation therapy\u0026rdquo;, \u0026ldquo;radiotherapies\u0026rdquo;, \u0026ldquo;rradiation\u0026nbsp;treatment\u0026rdquo;, \u0026ldquo;targeted radiotherapy\u0026rdquo;). The detailed search strategy for PubMed is provided in the\u0026nbsp;Appendix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion Criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inclusion criteria were applied: peer-reviewed studies based on NOX4 measurement after IR, peer-reviewed in-vitro studies that evaluated NOX4 expression after IR, studies that used a healthy or control group in addition to the IR group, and IR exposure carried out by a standard device.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion Criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe following articles\u003c/strong\u003e were excluded; review articles, articles not discussing NOX4 expression after IR, studies that did not use appropriate NOX4 evaluation methods, studies without a control group, case reports, letters to the editor, short reports, and congress abstracts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods of Article Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformation gathering was performed based on a web checklist in conformance with PRISMA rules\u0026nbsp;(32). At first, two co-authors independently screened the\u0026nbsp;articles\u0026nbsp;based on the title and\u0026nbsp;abstract, and then on the full text of the article. Where there was any disagreement\u0026nbsp;a discussion took place, and then a third researcher was recruited to resolve the disagreement. Finally, an Excel spreadsheet was designed to enter the required information, including the c, outcome, and possible biases. Data such as Mean, SEM, and SD were extracted by plot digitizer software and added to the Excel table\u0026nbsp;(33-35).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRisk of Bias Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SYRCLE Risk of Bias tool was employed for the quality assessment of preclinical\u0026nbsp;animal\u0026nbsp;studies and evaluated by two independent researchers\u0026nbsp;(36). This scale comprises 10 items covering 5 categories (Table 1). The scale was modified to change the quantitative risk of bias into a subjective appraisal: \u0026lt;50% (poor), 50%-69% (reasonable), 70%-79% (good), and 80%-100% (exceptionally good). The CONSORT statement was updated for in-vitro studies\u0026nbsp;(37)\u0026nbsp;(Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were presented as mean and standard deviation (SD). Comprehensive meta-analysis Software (3.3.070; USA) was used for data analysis. The effect size with a 95% confidence interval (95% CI) was calculated. The random-effect model was applied, and if the heterogeneity was less than 50%, the fixed-effect model was used. The presence of publication bias was examined by Egger\u0026apos;s precision-weighted linear regression method and the results were presented as a funnel plot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeave-one out sensitivity analyses: Sensitivity analyses were conducted by omitting one study at a time to assess the influence of potential bias on the results of the meta-analysis and to gauge the strength of the findings in light of significant heterogeneity, guaranteeing the dependability of the meta-analysis outcomes. The I\u0026sup2; statistic was utilized to assess heterogeneity, while subgroup analyses were carried out to probe the sources of variation among the studies included.\u003c/p\u003e\n\u003cp\u003eIn both animal and in-vitro\u0026nbsp;studies, the\u0026nbsp;quantification\u0026nbsp;of the NOX4 expression (both mRNA and protein), was analyzed after and without\u0026nbsp;IR\u0026nbsp;exposure. Subgroup analysis in animal studies was done based on evaluation test, organ, radiation dose, radiation source, irradiation method, and follow-up time. Subgroup analysis in in-vitro\u0026nbsp;normal cell\u0026nbsp;studies were done based on cell type, radiation dose, radiation source, and follow-up time. The findings were reported as standardized mean difference and 95% CI, and in all analyses, p \u0026lt; 0.05 was considered as a significant level.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the analysis of NOX4 expression in different articles, it was found that differing follow-up times were used to assess NOX4. To avoid double counting and ensure accurate analysis, the data from different time points were averaged. However, for subgroup analysis based on follow-up time, the data from each time point were classified into different groups, so averaging was not necessary. This approach was taken to ensure that the data were accurately analyzed and that the results were not skewed by double counting.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e519 articles were extracted based on our search strategy, of which 266 were from PubMed, 170 from Embase, 52 from Scopus, and 31 from Web of Science. After removing duplicate articles by Endnote software, 217 studies remained and were selected for initial screening. Finally, 43 in-vivo (animal) and 15 in-vitro studies were included in the qualitative and quantitative analysis. The results of the different stages of screening are shown in the PRISM flowchart in\u0026nbsp;Figure 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristics of Articles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe characteristics of the articles included in the study are listed in\u0026nbsp;Table 3, divided into in-vivo animal and in vitro sections.\u003c/p\u003e\n\u003cp\u003eTable 3-A\u0026nbsp;includes 43 in-vivo (animal) studies, including 28 studies measuring NOX4 protein and 16 studies measuring NOX4 mRNA. Protein measurement was conducted using\u0026nbsp;Western blot\u0026nbsp;(WB) tests in 15 studies,\u0026nbsp;immunohistochemistry\u0026nbsp;(IHC) in 8 studies,\u0026nbsp;flow cytometry\u0026nbsp;(FC) in 6 studies, and\u0026nbsp;enzyme-linked immunosorbent assay (ELISA) in one study. In addition, mRNA measurement was conducted using\u0026nbsp;real-time PCR\u0026nbsp;(qRT PCR) in 16 studies.\u0026nbsp;NOX4 expression was measured in various organs, including the lungs in 14 studies, bone marrow in 11 studies, cardiovascular system in 7 studies, kidney in 3 studies, salivary gland in 2 studies, and 8 other organs each in one study, comprising the brain, liver, small intestine, spleen, thymus, prostate, parotid gland, and ovarian. The studies employed administered doses of\u0026nbsp;IR\u0026nbsp;that varied from 1 to 75 Gy. Out of the total, 18 studies focused on whole-body irradiation, while 21 studies investigated localized radiation treatment. The radiation sources utilized were: proton beam in one study, electron beam in one study, X-ray (KV) in 12 studies, X-ray (MV) in 9 studies, γ-ray (60Co) in 5 studies, and γ-ray (137Cs) in 15 studies. The time intervals\u0026nbsp;post-irradiation\u0026nbsp;employed for evaluating NOX4\u0026nbsp;expression\u0026nbsp;varied from 24 hours to 22 months.\u003c/p\u003e\n\u003cp\u003eTable 3-B\u0026nbsp;includes 15 in-vitro studies, with 10 measuring NOX4 protein and 10 measuring NOX4\u0026nbsp;mRNA, with some\u0026nbsp;studies\u0026nbsp;measuring both. The measurement of protein used WB tests in 8 studies and FC in 2 studies. For mRNA assessment, qRT-PCR was the method used in all 10 studies. NOX4 expression was evaluated in various cell types, including human cells in 11 studies, rat cells in 3 studies, and mouse cells in 2 studies. The doses of\u0026nbsp;IR\u0026nbsp;used in these studies ranged from 3 cGy to 16 Gy, with X-ray used in 10 studies and γ-ray in 5 studies. The follow-up period for NOX4 analysis post-irradiation varied from 15 minutes to 5 days. Of these, 13\u0026nbsp;studies utilized normal cells, while 2 studies employed cancer\u0026nbsp;cells.\u003c/p\u003e\n\u003cp\u003eThe following studies were excluded from the meta-analysis; an in vivo study from Jiyoung Park 2022\u0026nbsp;(38), and\u0026nbsp;in vitro studies from Jiwon Choi 2022\u0026nbsp;(39), Urbain Weyemi 2015\u0026nbsp;(40), Yingchun Zhang 2012\u0026nbsp;(92), \u0026nbsp;Yingchun Zhou\u0026nbsp;2022 (93), \u0026nbsp; Sung Hyo Park\u0026nbsp;2020 (48), Sarah Park-2010 (96), Eun Joo Chung 2019 (97), and XiaoHong Yang 2017 (98) because they did not contain complete analysis, the results were not clearly stated, or were not in line with the\u0026nbsp;inclusion criteria.\u0026nbsp;Based on the search results, there were no clinical studies specifically investigating the effect of IR on NOX4 expression in humans.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeta-analysis (Animal studies)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunnel plot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the funnel plot analysis and Egger’s test on the\u0026nbsp;animal studies\u0026nbsp;are\u0026nbsp;reported in\u0026nbsp;Figure 2, indicating possible publication bias for the outcome measures including mRNA expression (Egger's p \u0026lt; 0.05) (Figure 3-A), and protein expression (Egger's p \u0026lt; 0.05) (Figure 3-B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverall Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was found that 16 studies (containing 19\u0026nbsp;separate\u0026nbsp;experiments) reported a noteworthy increase in NOX4\u0026nbsp;mRNA expression in\u0026nbsp;the irradiated animals, with a SMD of 5.070 and CI95% ranging from 3.374 to 6.766;(p \u0026lt; 0.001; I\u003csup\u003e2\u003c/sup\u003e=91.8%). Furthermore, 28 studies (containing\u0026nbsp;separate\u0026nbsp;38 experiments) reported a significant increase in NOX4\u0026nbsp;protein expression post-irradiation,\u0026nbsp;with an SMD of 3.452, CI95% of 2.807 to 4.097, (p \u0026lt; 0.001, and; I\u003csup\u003e2\u003c/sup\u003e=74.5%) (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensitivity Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leave-one-out sensitivity analysis indicated that no single study had a significant effect on the overall findings of NOX4 mRNA and protein expression after IR, suggesting that the results were robust and not unduly influenced by any individual study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubgroup Analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubgroup analysis was conducted based on various factors including the type of tests used to measure NOX4 expression, specific organs studied, dose of IR administered, source of radiation, and the follow-up time after exposure to IR.\u003c/p\u003e\n\u003cp\u003eSubgroup analysis of tests measuring NOX4 expression post-irradiation show varying results. The use of WB (SMD=2.886; P \u0026lt; 0.001), IHC (SMD=6.165; P \u0026lt; 0.001), FC (SMD=2.862; P \u0026lt; 0.001), and qRT-PCR (SMD=5.070; P \u0026lt; 0.001) all provided significant results, while ELISA was only utilized in a single study (Table 5-a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEight organs (brain, liver, ovaries, parotid gland, prostate, small intestine, spleen, and thymus) were limited to a single study (Table 5-b).\u003c/p\u003e\n\u003cp\u003eSubgroup analysis focusing on different organs where NOX4 expression was assessed showed notable increases following\u0026nbsp;IR\u0026nbsp;exposure in specific organs. \u0026nbsp;Increased NOX4 protein expression was observed in the lungs (SMD=4.391; P \u0026lt; 0.001), bone marrow (SMD=2.862; P \u0026lt; 0.001), cardiovascular system (SMD=2.206; P \u0026lt; 0.01), salivary gland (SMD=3.714; P \u0026lt; 0.001), and kidney (SMD=1.502; P \u0026lt; 0.01) post-irradiation. Moreover, increased NOX4 mRNA expression was observed in the bone marrow (SMD=9.371; P \u0026lt; 0.01) and cardiovascular system (SMD=2.716; P \u0026lt; 0.01) post-irradiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubgroup analysis based on the dose of\u0026nbsp;IR\u0026nbsp;showed a substantial increase in NOX4 protein expression across different dose\u0026nbsp;levels: 3-6 Gy (SMD=2.631; P \u0026lt; 0.001); 6-12 Gy (SMD=4.236; P \u0026lt; 0.001); 12-24 Gy (SMD=2.941; P \u0026lt; 0.001); and 75 Gy (SMD=8.303; P \u0026lt; 0.05) (Table 5c). Subgroup analysis based on the dose of\u0026nbsp;IR\u0026nbsp;showed also a substantial increase in NOX4 mRNA expression across different dose\u0026nbsp;levels: 1-3 Gy (SMD=13.904; P \u0026lt; 0.05); 3-6 Gy (SMD=11.505; P \u0026lt; 0.05); 6-12 Gy (SMD=2.706; P \u0026lt; 0.01); and 12-24 Gy (SMD=4.405; P \u0026lt; 0.01) (Table 5-c).\u003c/p\u003e\n\u003cp\u003eMoreover, subgroup analysis based on the irradiation method revealed that both whole body irradiation (SMD=3.907; P \u0026lt; 0.001) as well as local irradiation (SMD=3.581; P \u0026lt; 0.001) had a significant effect on increasing NOX4 protein expression (Table 5d). The same results were observed for NOX4 mRNA expression: whole body irradiation (SMD=6.784; P \u0026lt; 0.001) and local irradiation (SMD=2.736; P \u0026lt; 0.01) (Table 5-d).\u003c/p\u003e\n\u003cp\u003eSubgroup analysis based on the radiation source, showed that various radiation sources, including X-ray (KV) (SMD=2.725; P value \u0026lt; 0.001); X-ray (MV) (SMD=3.162; P value \u0026lt; 0.001); γ-ray (137Cs) (SMD=3.724; P value \u0026lt; 0.001); γ-ray (60Co) (SMD=16.507; P value \u0026lt; 0.05); and electron beam (SMD=1.997; P value \u0026lt; 0.05) all had a significant effect on increasing NOX4\u0026nbsp;protein expression (Table 5-e). Similarly, for NOX4\u0026nbsp;mRNA expression\u0026nbsp;similar results were observed: X-ray (KV) (SMD=1.288; P value \u0026lt; 0.01); X-ray (MV) (SMD=17.133; P value \u0026lt; 0.05); γ-ray (137Cs) (SMD=4.823; P value \u0026lt; 0.001); γ-ray (60Co) (SMD=9.211; P value \u0026lt; 0.05); and proton beam (SMD=15.014; P value \u0026lt; 0.01) (Table 5e). Proton beam and electron beam were each only used in one study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, subgroup analysis based on the follow-up time after IR exposure showed a consistent increase in NOX4 protein expression across all follow-up times. This included times below one week (SMD=2.455; P \u0026lt; 0.001), between one and two weeks (SMD=3.588; P \u0026lt; 0.001), between two weeks and one month (SMD=3.169; P \u0026lt; 0.001), and more than one month (SMD=4.653; P \u0026lt; 0.001) (Table 5f). Similarly, for NOX4\u0026nbsp;mRNA expression\u0026nbsp;similar results were observed: below one week (SMD=8.110; P \u0026lt; 0.001), between one and two weeks (SMD=9.347; P \u0026lt; 0.01), between two weeks and one month (SMD=5.861; P \u0026lt; 0.01), and more than one month (SMD=3.916; P \u0026lt; 0.001) (Table 5-f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeta-analysis (in-vitro studies)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunnel plot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the funnel plot analysis and Egger’s test of the\u0026nbsp;in-vitro studies are\u0026nbsp;reported in (Figure 5), indicating possible publication bias for the outcome measure of\u0026nbsp;mRNA expression\u0026nbsp;in normal cells (Egger's p \u0026lt; 0.05) (Figure 5-A) and protein expression in normal cells (Egger's p \u0026lt; 0.05) (Figure 5-B), but showed no evidence of publication bias for the outcome measure of\u0026nbsp;protein expression\u0026nbsp;in cancer\u0026nbsp;cells (Egger's p \u0026gt; 0.05) (Figure 5-C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of normal and cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGeneral analysis of the\u0026nbsp;normal cell\u0026nbsp;studies revealed that 9\u0026nbsp;studies (10 independent experiments) had a significant increase in NOX4\u0026nbsp;(mRNA) expression in\u0026nbsp;the irradiated cells (SMD = 3.563; CI95% 2.297 to 4.829; P \u0026lt; 0.001; I\u003csup\u003e2\u003c/sup\u003e=69 %). Additionally,\u0026nbsp;8 studies\u0026nbsp;reported a significant increase in NOX4\u0026nbsp;protein\u0026nbsp;expression after IR (SMD = 2.689; CI95% 1.314 to 4.064; P \u0026lt; 0.001; I\u003csup\u003e2\u003c/sup\u003e=79.5 %)\u0026nbsp;(Figure 6-A). These findings are in agreement with the meta-analysis of the\u0026nbsp;animal studies. In addition, the studies included in Table 3B utilized various methods to assess the effects of\u0026nbsp;IR\u0026nbsp;on NOX4 expression, with the majority of studies showing a significant increase in NOX4\u0026nbsp;expression\u0026nbsp;in normal cells after irradiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe general analysis of the cancer cell\u0026nbsp;studies revealed that 1 study observed a significant increase in NOX4 (mRNA) expression in irradiated cells (SMD = 3.280; CI 95% 1.865 to 4.694; P \u0026lt; 0.001). In addition,\u0026nbsp;2 studies\u0026nbsp;(3 independent experiments) reported a significant increase in NOX4 (protein) expression after IR (SMD = 2.159; CI 95% 0.202 to 4.116; P \u0026lt; 0.05; I\u003csup\u003e2\u003c/sup\u003e=81.5 %) (Figure 6-B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensitivity Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leave-one-out sensitivity analysis indicated that no single study had a significant effect on the results of NOX4\u0026nbsp;mRNA and protein\u0026nbsp;expression\u0026nbsp;in normal cells after IR, suggesting that the results were robust and not heavily influenced by any individual study.\u003c/p\u003e\n\u003cp\u003eThe estimated pooled SMD was significant in the original analysis of NOX4 protein expression in cancer cells (P \u0026lt; 0.05). However, the leave-one-out sensitivity analysis revealed that the statistical significance was changed by the removal of the experiments included in Jiyoung Park 2022-3 study\u0026nbsp;(38): 4T1-Luc mouse breast cancer cells independent experiment (SMD = 2.064; CI95% (-1.638 to 5.766); P \u0026gt; 0.05) or MDA-MB-231 human breast cancer cells independent experiment (SMD = 1.319; CI95% (-0.786 to 3.424); P \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubgroup Analysis of Normal Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubgroup analysis of normal cells was performed based on the tests that measured NOX4 expression after radiation, including WB (SMD=1.945; P value \u0026lt; 0.01), FC (SMD=4.193; P \u0026lt; 0.001), and\u0026nbsp;qRT-PCR (SMD=3.563; P \u0026lt; 0.001) (Table 6-a).\u003c/p\u003e\n\u003cp\u003eFollowing the initial analysis, further investigation was carried out focusing on the source of the normal cells in which NOX4\u0026nbsp;expression\u0026nbsp;was assessed. The findings indicated a significant rise in NOX4 protein expression levels after\u0026nbsp;IR\u0026nbsp;in human cells (SMD=2.491; P \u0026lt; 0.01) (Table 6-b). Also, the findings indicated a significant rise in NOX4 mRNA expression levels after\u0026nbsp;IR\u0026nbsp;in human cells (SMD=3.412; P \u0026lt; 0.01), rat cells (SMD=5.652; P \u0026lt; 0.001), and mouse cells (SMD=2.878; P \u0026lt; 0.01) (Table 6-b).\u003c/p\u003e\n\u003cp\u003eAdditionally, subgroup analysis was undertaken based on the radiation dose, radiation source, and duration of follow-up. The subgroup analysis concerning the dose of\u0026nbsp;IR\u0026nbsp;revealed a noteworthy impact on the\u0026nbsp;upregulation\u0026nbsp;of NOX4\u0026nbsp;protein expression\u0026nbsp;in normal cells at two dose levels; 3-50 cGy (SMD=4.193; P \u0026lt; 0.001) and 8-16 Gy (SMD=2.516; P \u0026lt; 0.05) (Table 6c). NOX4\u0026nbsp;mRNA expression\u0026nbsp;in normal cells was upregulated at all dose levels; 3-50 cGy (SMD=4.149; P \u0026lt; 0.001), 2-6 Gy (SMD=3.173; P \u0026lt; 0.001), and 8-16 Gy (SMD=2.987; P \u0026lt; 0.05) (Table 6-c).\u003c/p\u003e\n\u003cp\u003eMoreover, the subgroup analysis, focusing on the radiation source, showed that both X-ray (SMD=2.601; P \u0026lt; 0.01) and γ-ray (SMD=3.531; P \u0026lt; 0.01) sources had a significant impact on increasing NOX4\u0026nbsp;protein expression\u0026nbsp;in normal cells\u0026nbsp;(Table 6d). Similarly, for NOX4\u0026nbsp;mRNA expression\u0026nbsp;in normal cells, the same results were observed: X-ray (SMD=3.020; P \u0026lt; 0.001) and γ-ray (SMD=4.224; P \u0026lt; 0.001) (Table 6-d).\u003c/p\u003e\n\u003cp\u003eLastly, the subgroup analysis based on the follow-up time post-IR exposure indicated an increase in NOX4 protein expression in normal cells at all follow-up time points: 1 hour (SMD=3.601; P \u0026lt; 0.05), 2-24 hours (SMD=2.590; P \u0026lt; 0.01), and 2-5 days (SMD=2.306; P \u0026lt; 0.001) (Table 6-e). Similarly, for NOX4 mRNA expression in normal cells, the same results were observed: 1 hour (SMD=4.112; P \u0026lt; 0.001), 2-24 hours (SMD=3.843; P \u0026lt; 0.001), and 2-5 days (SMD=2.612; P \u0026lt; 0.01) (Table 6-e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAll animal studies were conducted in normal animals, while a significant number of in vitro studies were conducted in normal cells, with only a limited number of studies focusing on cancer cells. In both animal and in vitro studies, both mRNA and protein levels of NOX4 were found to be increased after IR. In addition, the results of in vitro and in vivo analyses indicated that the effect of IR on NOX4 mRNA was more pronounced than its effect on NOX4 protein levels at various times after exposure. Furthermore, based on the in vitro results, it was found that NOX4 mRNA protein and mRNA expression increased in both normal and cancer cells, with a more pronounced increase observed in normal cells. However, this finding must be interpreted with caution due to the limited number of studies using cancer cells.\u003c/p\u003e \u003cp\u003eThe subgroup analysis suggested that the lungs, cardiovascular system, salivary glands and bone marrow were highly sensitive to radiation exposure. The lungs showed the highest sensitivity to increases in NOX4 protein, followed by the salivary glands, bone marrow and cardiovascular system. Conversely, the kidney showed the lowest increase in NOX expression levels. In addition, the highest mRNA up-regulation was found in the bone marrow.\u003c/p\u003e \u003cp\u003eAs mentioned above, the kidneys showed the lowest increase in NOX4 protein expression after radiation exposure. However, it is important to note that only three studies were carried out on the kidney, highlighting the need for further research on this organ. However, this finding may be due to the high levels of NOX4 expression found in the distal tubular cells of the normal human kidney (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). Conversely, studies have suggested that NOX enzymes are involved in facilitating radiation-induced damage in various organs, such as the kidney. In particular, NOX4 has been identified as a contributor to the development of cisplatin-induced acute kidney injury through the induction of ROS-mediated programmed cell death and inflammation (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e). Therefore, further research into the role of NOXs in the kidney and the potential for NOX-specific drugs to reduce radiation-induced kidney injury is warranted.\u003c/p\u003e \u003cp\u003eIn vitro analysis of normal cells showed that increasing dose within the study range (3 cGy to 16 Gy) led to a reduction in the up-regulation of NOX4 mRNA expression levels. Notably, the highest dose group (6\u0026ndash;16 Gy) showed no significant changes in NOX4 mRNA expression. Similarly, analysis of NOX4 protein levels showed that increasing the dose from 3 cGy to 6 Gy resulted in a decrease in the upregulation of NOX4 protein expression, with the 2\u0026ndash;6 Gy dose group showing no significant changes in NOX4 protein expression. However, when the dose was further increased to 6\u0026ndash;16 Gy, NOX4 protein expression was increased, although to a lesser extent than in the cGy group. Interestingly, both NOX4 mRNA and protein analyses in the in vitro normal cell group suggested that the cGy doses were more effective than the Gy range doses in modulating NOX4 expression levels.\u003c/p\u003e \u003cp\u003eOn the other hand, the in vivo results showed that NOX4 mRNA changes were most pronounced at the lowest IR dose (1 to 3 Gy). Within the dose range of 1 to 12 Gy, dose escalation initially resulted in a reduced effect; however, after 12 Gy, there was a resurgence of the effect on mRNA expression. In essence, dose escalation resulted in an initial decrease in effect, followed by a subsequent increase in effect at even higher doses. The subgroup analysis of NOX4 protein showed that a lower dose of radiation resulted in higher NOX4 expression, but 75 Gy, which was the highest dose, had the greatest effect in increasing NOX4.\u003c/p\u003e \u003cp\u003eThese findings are supported by various studies, such as the induction of NOX4 expression following a dose-dependent pattern and a significantly higher expression observed after 4 Gy of heavy ion irradiation (\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e). In addition, research has shown that both low (\u0026lt;\u0026thinsp;100 mGy) and high doses (10 Gy) of IR can lead to the up-regulation of NADPH oxidase, contributing to the increased expression of NOX4 (\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e). These collective findings highlight the need for a comprehensive understanding of the effect of radiation dose on NOX4 expression and the implications for radiation-induced damage. Although the exact mechanism for the observed increase in NOX4 expression at lower radiation doses is not explicitly explained in the search results, it is evident that NOX4 expression is closely linked to IR dose and the resulting production of ROS. Further research may be required to fully elucidate the specific mechanisms underlying the dose-dependent effects of radiation on NOX4 expression.\u003c/p\u003e \u003cp\u003eBased on the results of the in vitro normal cells, the expression levels of both NOX4 protein and mRNA decreased over time after irradiation. Therefore, the highest levels of protein and mRNA were recorded during the first hour after irradiation. While the expression of mRNA decreased with time after IR exposure, the expression of NOX4 protein increased in in vivo studies. After mRNA is transcribed from DNA, it serves as a template for protein synthesis, so mRNA levels can decrease over time as the mRNA molecules are translated into protein. As proteins are synthesised and fulfil their functions, the mRNA molecules may be degraded or have a short half-life compared to proteins. This can lead to a decrease in mRNA levels over time as protein levels increase. This illustrates the complex relationship between mRNA and protein levels, which is influenced by factors such as protein turnover rates and post-translational regulatory mechanisms (\u003cspan additionalcitationids=\"CR101\" citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile a slight decrease was observed at follow-ups between two weeks and one month, it returned to a higher level than at the one-week follow-up at one month. This pattern may be an important reason for the spread of damage following radiation exposure. Over time, NOX4 expression may lead to the sustained production of excessive ROS, resulting in oxidative stress and damage to various cellular components such as nucleic acids, proteins and lipids. This could lead to various pathological conditions such as hyperglycemia, inflammation, cancer and vascular abnormalities (\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhole-body irradiation results in a higher production of NOX4 protein and mRNA compared to local irradiation, as suggested by the results. Studies have shown that whole body irradiation causes residual bone marrow injury by inducing excessive NOX activity, which leads to increased ROS production.\u003c/p\u003e \u003cp\u003eThis underlines the importance of immediate regulation or inhibition of NOX4 activity in individuals who are accidentally exposed to radiation, such as in nuclear accidents, mishandling of radioactive sources, cosmic and solar radiation, terrestrial IR emitted from the earth or from building materials (\u003cspan additionalcitationids=\"CR106\" citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe changes in NOX4 expression were influenced by the type of radiation source used. Specifically, X-rays (MV) and γ-rays (60Co) showed the most pronounced effects on mRNA expression, while X-rays (KV) had the least effect on mRNA expression. On the other hand, γ-rays (60Co) had the greatest effect on NOX4 protein expression. The observed significant effect of X-rays (MV) on mRNA should be interpreted with caution due to the high heterogeneity and limited number of articles included in the analysis. In in vitro studies, due to the limited number of articles available, a subgroup analysis was performed based only on the radiation source and not on the energy level. In in vitro normal cell studies, γ-rays showed a more pronounced effect on increasing NOX4 protein and mRNA expression compared to X-rays.\u003c/p\u003e \u003cp\u003eThese results, which link the effect of radiation sources on NOX4 expression to clinical outcomes and treatment strategies, may provide valuable insights to improve the practice of personalised medicine in radiation oncology. By optimising treatment approaches and minimising side effects, these findings have the potential to improve patient outcomes. NOX4 expression may also serve as an indicator for assessing treatment efficacy, predicting poorer outcomes or determining increased risk of metastasis. In addition, regulating NOX4 activity may be a promising way to enhance treatment efficacy or sensitize cancer cells to radiotherapy.\u003c/p\u003e \u003cp\u003eIn the in-vitro studies, NOX4 levels were increased in all three types of animal cells. However, the rise in NOX4 protein levels in rat cells was not statistically significant, likely due to limited data and notable heterogeneity within the samples.\u003c/p\u003e \u003cp\u003eSome reports have suggested that the increase in NOX4 after radiotherapy in glioblastoma may play an important role in the radioresistance produced by the tumor microenvironment (\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e). In pelvic cancer, NOX4 causes radiation nephrotoxicity (\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e). However, one study suggested that increased expression of NOX4 may lead to less radiation-induced damage to the hematopoietic system (\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNOX4 plays a role in mediating radiation-induced damage in various organs, including the kidneys, lungs, and heart\u003cstrong\u003e.\u003c/strong\u003e The increase in NOX4 expression in various organs depends on the radiation dose\u0026nbsp;radiation, source, and time after exposure, NOX4 protein expression increases over time, in line with the spread of damage. Additionally, the potential role of NOX4 as a therapeutic target for enhancing the sensitivity of cancer cells to ionizing radiation warrants further exploration. The implications of NOX4 expression in the context of biodosimetry and the development of radiation resistance need to be elucidated to advance our understanding of the biological effects of ionizing radiation exposure. Overall, ionizing radiation exposure triggers NOX4 expression, which contributes to cellular senescence, DNA damage, tissue inflammation, oxidative stress and fibrotic responses, highlighting the significance of NOX4 in the biological effects of radiation exposure. Further research is needed to fully elucidate the mechanisms underlying the expression of NOX4 over time in various organs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical impact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings suggest the ongoing development of NOX4 inhibitors for clinical radiotherapy applications should be continued, particularly in the context of protecting against vascular, lung, bone marrow, and other radiation-induced injuries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests that may have influenced the research, results, or conclusions presented in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proposal with ethical code (IR.IUMS.REC.1401.705) was approved by the IACUC of the Iran University of Medical Science, Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of the article in the journal “BMC Molecular and Cell Biology”.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by [IR.IUMS.REC.1401.705]. The funding sources had no involvement in the study design, data collection, analysis, interpretation, or writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their appreciation to the Radiation Biology Research Center (RBRC), Physiology Research Center, and Systematic Review Network under the Vice-Chancellor for Research and Technology at Iran University of Medical Sciences for their assistance and support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLaurier D, Marsh J, Rage E, Tomasek LJAotI. Miner studies and radiological protection against radon. 2020;49(1_suppl):57-67.\u003c/li\u003e\n\u003cli\u003eTimins JKJHp. Communication of benefits and risks of medical radiation: a historical perspective. 2011;101(5):562-5.\u003c/li\u003e\n\u003cli\u003eHendee WR, O\u0026rsquo;Connor MKJR. 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Mitigation of radiation‐induced hematopoietic system injury by melatonin. 2020;35(8):815-21.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section\u003c/p\u003e"},{"header":"Appendix","content":"\u003cp\u003eAppendix is not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ionizing Radiation, NOX4, Radiotherapy, Meta-analysis, Systematic Review","lastPublishedDoi":"10.21203/rs.3.rs-4854221/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4854221/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Patients exposed to ionizing radiation (IR) from various sources experience several side effects. Understanding the mechanisms through which IR induces these effects could minimize their consequences. This study investigates the activation pathway of NADPH oxidase 4 (NOX4) after exposure to IR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The analysis incorporated studies that assessed NOX4 expression as an outcome variable. The study involved searches across various databases. A total of 58 articles were included in the meta-analysis, and data extracted from these studies were analyzed using Comprehensive Meta-Analysis Software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Analysis of the impact of IR on NOX4 expression, demonstrated a notable increase in protein expression in animals (SMD=3.452; p\u0026lt;0.001), in normal cells in vitro (SMD=2.689; p\u0026lt;0.001), and in cancer cells (SMD=2.159; p\u0026lt;0.05). Furthermore, there was a significant increase in NOX4 mRNA expression in animals (SMD=5.070; p\u0026lt;0.001), in normal cells in vitro (SMD=3.563; p\u0026lt;0.001), and in cancer cells (SMD=3.280; p\u0026lt;0.001). Subgroup analysis was conducted based on the tests utilized to measure NOX4 expression, various organs, IR parameters, and follow-up time after IR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e NOX4 plays a crucial role in mediating radiation-induced damage in many organs. The upregulation of NOX4 expression in these organs is influenced by factors such as the radiation dose and source. Additionally, there was a further increase in NOX4 protein expression over time, highlighting its potential role in the progression of radiation-induced damage in vital organs.\u003c/p\u003e","manuscriptTitle":"Effect of Ionizing Radiation Exposure on NOX4 Expression in-Vitro and in-Vivo Studies: A Systematic Review and Meta-analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-11 12:54:35","doi":"10.21203/rs.3.rs-4854221/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c1fd88b-977b-4862-a7d7-6ed219e1bd86","owner":[],"postedDate":"September 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-14T11:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-11 12:54:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4854221","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4854221","identity":"rs-4854221","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}