ABSTRACT

Objective

Ischemia-reperfusion (IR) of the aorta is a significant contributor to the development of postoperative acute lung damage after abdominal aortic surgery. The aim of the present study was to examine the effect of alpha B-crystallin, a small heat shock protein (known as HspB5), on lung injury induced by abdominal aortic IR in rats.

Methods

Male Sprague-Dawley rats were divided into three groups: control, ischemia-reperfusion (IR, 90 min ischemia and 180 min reperfusion), and alpha B-crystallin +IR. Alpha B-crystallin (50 µg/100 g) was intraperitoneally administered 1 h before IR. Lung tissue samples were obtained for histological and biochemical analyses of oxidative stress and cytokine and apoptosis parameters in plasma, lung tissues, and bronchoalveolar lavage (BAL) fluid.

Results

The levels of malondialdehyde, reactive oxygen species, total oxidant status, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), nuclear factor kappa B (NFKβ), caspase-9 (CASP-9), 8-hydroxy-2’-deoxyguanosine, total antioxidant status, superoxide dismutase, and interleukin-10 levels in lung tissues, plasma, and BAL fluid (p<0.05 versus control) increased in Aortic IR. However, alpha B-crystallin significantly reduced the lung tissue levels of oxidative, inflamatuvar, and apoptotic parameters in the plasma, lung tissues, and BAL fluid (p<0.05 versus aortic IR). Histopathological results showed that alpha B-crystallin ameliorated the morphological changes related to lung injury (p<0.001).

Conclusion

Alpha B-crystallin substantially restored disrupted the redox balance, inflammation, and apoptotic parameters in rats exposed to IR. The cytoprotective effect of alpha B-crystallin on redox balance might be attributed to improved lung injury.

Keywords:

Abdominal surgery, cytokines, inflammation, immunology, ischemia-reperfusion

INTRODUCTION

Infrarenal abdominal aortic clamping and declamping after coronary artery surgery cause ischemia-reperfusion^{1, 2}. Distant organ damage is often associated with ischemia-reperfusion (IR) injury. The lungs are one of the distant organs that are damaged. IR inflammation exacerbates ischemic trauma and necessitates the maintenance of blood supply to prevent cell death³. Although the exact pathophysiological mechanisms of distant organ injury remain unclear, damage is attributed to oxidative, inflammatuvar, and apoptotic mediators released after IR^{2, 4}.

Oxidative stress is an early and important symptom of acute lung injury. After reoxygenation of the lungs, a number of intricate events may take place, including oxidative damage to deoxyribonucleic acid (DNA) and proteins, peroxidation of membrane lipids, release of intracellular enzymes, increase in intracellular calcium, and neutrophil buildup in ischemic tissue^{5, 6}. Clinical and experimental studies have shown that IR in the lungs induces proinflammatory cytokines and apoptosis. In this condition, lung tissue are affected by oxidative, inflammatory, and apoptotic pathways^{7, 8}. The cause of lung injury as a result of IR following aortic surgery can be attributed to the release of cytokines, immune cells, various mediators secreted by immune cells, and their accumulation in the lungs⁹. Additionally, reactive oxygen species that cause DNA damage and apoptosis may lead to increased levels of proinflammatory cytokines and decreased expression of anti-inflammatory cytokines.

Heat shock proteins comprise a group of proteins including subfamilies. Their expressions are elevated under stress conditions. The subfamily includes small heat shock proteins. Alpha B-crystallin (known as HspB5) is one of the main lens proteins in vertebrates and a member of the small heat shock protein family¹⁰. The roles of alpha B-crystallin have been reported in different IR models. A retinal rat IR model showed that alpha B-crystallin decreased ischemic retinal damage, oxidative stress, iNOS, and nuclear factor kappa B (NFKβ) levels, and improved retinal function¹¹. In addition, an experimental anterior ischemic optic neuropathy model in mice showed that both short-term treatment with alpha B-crystallin decreased inflammation and long-term treatment improved optic neuropathy function¹². Kirbach et al.¹³ demonstrated that HspB5 was upregulated in a rat cerebral ischemia model. In this study, HspB5 was one of the most significant HspBs in the neuronal stress response to IR injury. There are no articles in the literature about the expression of alpha B-crystallin after aortic IR-induced acute lung injury or its effect in a lung aortic IR model.

In addition, alpha B-crystallin has been used therapeutically for stroke and spinal cord contusion of mice studies^{14, 15}. In the first study, HspB5 decreased both the volume of stroke and inflammatory cytokines related to stroke pathology. In the second study, mice treated with HspB5 showed improvement of locomotor capabilities and a reduction in secondary tissue injury. In another study, the administration of alpha B-crystallin chronically improved cardiac function in a mouse myocardial infarction model. Mice treated with HspB5 exhibited increased left ventricular ejection fraction¹⁶. Different studies have shown that alpha B-crystalline decreases plasma interleukin (IL)-6¹⁷, increases IL-10¹⁸, inhibits NF-κB activation, suppress tumor necrosis factor-alpha (TNF-α) induced apoptosis¹⁹, and reduces lipid peroxidation and the protective role of reactive oxygen against lung injury through its role in immune, inflammatory, and ischemic processes.

To our knowledge, no research has been conducted on the antioxidant, anti-inflammatory, and anti-apoptotic regulatory effects of alpha B-crystalline on aortic IR-induced lung damage in vivo. In the present study, we demonstrated that exogenous administration of alpha B-crystalline reduced lung tissue oxidative stress and cellular injury by inhibiting oxidative and proinflammatory responses, including cytokines.

MATERIALS and METHODS

Animals

Adult 24 male Sprague-Dawley rats (12 weeks-average weight: 320 grams) were housed in separate cages in a standardized temperature and light-dark cycle controlled environment. Animals had free access to standard feed and water. The animal studies were conducted after receiving approval from the Institutional Animal Care and Istanbul University Animal Experiments Local Ethics Committee (decision no: 2014/62 date: 29.05.2014).

Sprague-Dawley rats were divided into three groups. 1) Control (C; n=8) group: the control group underwent midline laparotomy and dissection of infrarenal abdominal aorta (IAA) without occlusion. 10 mL physiological serum was administered to this group into the peritoneal cavity during the procedure. IR procedure was not applied. 2) Ischemia-Reperfusion (IR; n=8) group: 10 mL physiological serum was administered into the peritoneal cavity during the procedure. In addition, 90 min of ischemia and 180 min of reperfusion were performed. 3) Alpha B-crystallin+Ischemia-Reperfusion (Alpha B-crystallin+IR; n=8) group: 10 mL physiological serum was administered during the procedure. Intraperitoneal alpha B-crystallin administration (50 µg /100 g) were performed for 1 h before ischemia, 90 min of ischemia, and 180 min of reperfusion. We used the dosage of alpha B-crystallin according to its nontoxic effects^{18, 20}.

Ischemia-reperfusion Procedure

All animals were anesthetized intraperitoneally with thiopental sodium (60 mg/kg). Tracheotomy was performed after anesthesia; trachea was intubated with a polyvinylchloride cannula. Animals were placed on a heating plate to maintain body temperature at 37 ^ᵒC during experiment. A 22-gauge catheter was inserted to draw blood from the carotid artery and monitor blood pressure. After the skin of rats was prepared aseptically, a midline laparotomy was performed, and the intestines were pulled to the left with wet gauze. Heparin was mixed with physiological serum 50 U/kg (500 mL volume), (Nevpar’s; MN Pharmaceutical, Istanbul, Türkiye) before the ischemia protocol to prevent clotting. The area called the IAA just below the right and left kidney arteries was isolated, and dissection of IAA was performed in the IR and alpha B-crystallin+IR groups. A non-traumatic microvascular clamp (vascu-statts II, midi straight 1001-532; Scanlan Int, St Paul, MN) was placed to perform the 90 min ischemia protocol. 10 mL of physiological serum adjusted to body temperature was administered to the peritoneal cavity. After suturing the incision area with surgical thread, the area was covered with moist gauze to minimize fluid and heat loss. After 90 min of ischemia, the incision was reopened, and the microvascular clamp from the infrarenal aortic area was removed²¹. These changes were also confirmed using a computerized system (PowerLab, 16 SP, ADInstruments, Castle Hill, Australia) with systemic arterial blood pressure records from the carotid artery. At the end of 180 min of reperfusion, a high dose of intravenous thiopental sodium (150 mg/kg) was administered for euthanasia after collecting bronchoalveolar lavage (BAL) fluid samples from the lungs and blood from the carotid artery. The lung was removed together with the trachea, and the lower right lung lobe was stored in the formol for histological examination. The lung, BAL fluid, and plasma samples were stored at -80 ^°C for biochemical analyses.

Bronchoalveolar Lavage (BAL) Procedure

The left main bronchus was cannulated and secured. Saline (5 mL) was injected into three quick aliquots (5 mL) each, with each aliquot withdrawn slowly. The BAL specimen was collected with a fluid recovery of 90% or greater²². The fluid was centrifuged at 1500 rpm for 8 minutes at +4 ^°C. The supernatant was then stored at -80 ^°C until biochemical analysis.

Lung Tissue, Plasma, and BAL Fluid Biochemical Analysis

Lung tissue samples were washed in physiological serum and phosphate buffer and then crushed by freezing in liquid nitrogen. After thawing, phosphate buffer was added, and the tissues were homogenized. Homogenates were centrifuged (3000 rpm, 20 min, +4 ˚C), and the resulting supernatants were used for biochemical analysis. BAL fluid and plasma samples were collected after reperfusion. Levels of malondialdehyde (MDA), reactive oxygen species (ROS), total oxidant status (TOS), total antioxidant status (TAS), superoxide dismutase (SOD), IL-10, TNF-α, IL-1β, NF-κB, CASP-9, and 8-OHdG were quantified using Sunred ELISA kits.

Histological Evaluation

The middle lobe of the right lung was fixed in 10% neutral formalin, embedded in paraffin, and sectioned into 5 µm slices. After deparaffinization and staining with hematoxylin and eosin, the sections were evaluated histologically by two independent pathologists using a light microscope (Olympus BX61, Japan). Histologic injury scores were determined based on five parameters: alveolar wall thickness, intra-alveolar edema-infiltration, intra-alveolar hemorrhage, capillary blood collection, and alveolar damage, each scored on a scale from 0 to 4 (0: absent and normal appearance, 1: mild, 2: moderate, 3: severe, 4: intense). Additionally, the total lung injury score was calculated for each animal group²³.

Statistical Analysis

The data was processed by the statistical analysis software GraphPad Prism version 5.0 for Windows. The Bonferroni test was used to assess significant variations between groups using one-way analysis of variance (ANOVA). Statistical significance was set as p<0.05. The histological data was processed by the statistical analysis software Sigma Stat for Windows, version 3.0 (Jandel Scientific, San Rafael, CA) and one-way ANOVA. The Holm-Sidak test was used to assess group variations. Statistical significance was set as p≤0.001. All the data are shown as the mean values±standard deviation.

RESULTS

Oxidative Stress Parameters

When the ELISA results for oxidative stress markers were analyzed in all animal groups, lung tissue, BAL, and plasma results were similar. ROS, TOS, and MDA levels increased in the IR group compared with the control group. Alpha B-crystallin application decreased these parameters in alpha B-crystallin+IR group compared to IR group. Table 1 shows the p-values, which are the statistically significant differences between means of groups. SOD and TAS values increased in the alpha B-crystallin+IR group compared with the IR group (Figures 1 and 2).

Inflammation Parameters

When inflammation parameters in all animal groups were compared, TNF-α, IL-1β and NF-κB levels differed between the IR and control groups. TNF-α, IL-1β and NF-κB were higher in the IR group. Compared with the IR group, the levels of these parameters in the alpha B-crystallin+IR group decreased. Table 2 shows the means of groups and p-values. When we looked at the IL-10 levels, IL-10 was significantly decreased in the IR group. However, when the IR and alpha B-crystallin+IR groups were compared, IL-10 levels increased significantly in the alpha B-crystallin+IR group (Figure 3).

Apoptotic Parameters

When we analyzed apoptotic parameters in the rat lung and plasma of all groups, CASP-9 and 8-OHdG levels were significantly higher in the IR group than in the control group. Alpha B-crystallin application reduced these parameters in alpha B-crystallin+IR group compared to IR group. Figures 4 and 5 show the CASP-9 and 8-OHdG parameters in the specimens.

Histopathologic Results

Histopathological examination of the right lung lobe was performed in all animal groups. Total lung injury was the least in the control group and the most severe IR group. The lung injury score was significantly higher in the IR group (9.0±0.8 p<0.001) than in the control group (2.286±0.714 p<0.001). Lung injury parameters in the IR group included intra-alveolar macrophage, neutrophil infiltration, alveolar damage, and alveolar wall thickening. Application of alpha B-crystallin before IR significantly reduced IR damage, and the total lung injury score decreased significantly in the alpha B-crystallin+IR group [5.56±0.56 p<0.001] compared with the IR group (9.0±0.8 p<0.001). Lung injury parameters observed in the alpha B-crystallin+IR group were less evident (Figure 6).

DISCUSSION

Our study showed that 90 min of infrarenal aortic occlusion followed by 180 min of reperfusion resulted in acute lung damage, including destruction of the alveolar architecture and inflammatory cell infiltration. The levels of MDA, ROS, TOS, TNF-α, IL-1β, NF-κB,, CASP-9, and 8-OHdG increased, whereas the levels of SOD, TAS, and IL-10 decreased in plasma, BAL fluid, and lung tissue samples. Interestingly, pre-administration of alpha B-crystallin 1 h before ischemia mitigated these effects, improving oxidative, inflammatory, and apoptotic parameters in plasma, lung tissue, and BAL fluid samples and enhancing cellular structure.

Lung injury, IR, and systemic inflammation are clinically interrelated. Physiopathological mechanisms describing the interaction of these three titles are not fully understood. Various mediators are released by immune cells after IR injury, and these mediators seem to be important in the development of pulmonary damage²⁴. Increase in biochemically triggered cytokine levels after aortic aneurysm treatment causes death with fever, tissue destruction, and even septic shock postoperatively².

Aortic IR injury causes pulmonary dysfunction²¹. IR injury is the result of a sequence of biochemical processes in which unbalanced ROS scavenging²⁵. Increased ROS levels can lead to protein, DNA, and lipid peroxidation, as well as mitochondria-induced cell death, which can contribute to IR injury²⁶. One of the related therapies for IR injury is antioxidant therapy. Antioxidants have been shown to reduce cellular and tissue damage by lowering intracellular ROS levels and reducing oxidative stress²⁷.

Tural et al.²⁸ determined that aortic occlusion contributes to the increase in lung tissue MDA levels. Similarly, we observed that aortic occlusion increased pulmonary tissue MDA levels. There was a significant positive correlation between reactive oxygen species and MDA, a marker of lipid peroxidation. Further analysis showed that the application of alpha B-crystallin before IR decreased the production of reactive oxygen species and lipid peroxidation. It was also found that the SOD and TAS parameters increased. This implies that alpha B-crystallin systematically exerts an antioxidant effect.

Changes in the processes following IR can cause pulmonary apoptosis. Morphologically, apoptosis can also be characterized by DNA fragmentation²⁹. Caspase-9 and 8-OhDG analyses were performed to demonstrate apoptosis and DNA damage. According to our results, the value of these parameters increased significantly not only in the lungs but also in plasma and BAL fluid in the IR group. 8-OHdG is the dominant form of oxidative damage caused by free radical species in nuclear and mitochondrial DNA and is used as a biomarker for oxidative stress³⁰. Increased 8-OHdG levels of oxidative stress in the aortic IR-induced lung injury group indicates nuclear or mitochondrial DNA damage occurred. A significant reduction in 8-OHdG was detected in all samples from the alpha B-crystallin-treated group compared with the IR group. In addition, a significant decrease in caspase-9 expression was observed following alpha B-crystallin treatment. Initial studies have shown that alpha B-crystallin is effective against proapoptotic proteins and stops the translocation of Bax and Bcl-Xs³¹. Another anti-apoptotic role of alpha B-crystallin is its ability to prevent caspase-3 activation regardless of intrinsic or extrinsic pathway^{32, 33}. In light of this information, we can state that alpha B-crystallin affects apoptosis through different mechanisms and prevents apoptosis by inhibiting caspases. The effect of alpha B-crystallin on caspase-9 levels probably occurs via two mechanisms. One of the possible explanations for this is that alpha B-crystallin connects Bax and prevents the transfer of Bax from mitochondrial membrane from the cytosol³². Kamradt et al.³⁴ confirmed that alpha B-crystallin disrupts the proteolytic activation of caspase-3. In light of this information, another possible explanation is that alpha B-crystallin prevents the transition of procaspase-9 from an inactive to an active form, caspase-9. The secondary pathway can be more possible due to the proteolytic activation of caspase 9.

Histopathological examination of lung tissues in the control group showed less severe disease than that in the IR group. Intra-alveolar macrophages, neutrophil infiltration, alveolar damage, and alveolar wall thickening were observed in the lung tissues of the IR group. These injuries were less evident in rats administered alpha B-crystallin than in the IR group. Our analysis found that in rats treated with alpha B-crystallin, total histological injury was substantially reduced. Different studies showed that TNF-α and IL-1β play an important role in IR damage. These proinflammatory cytokines damage vascular beds, activate neutrophils, and migrate to the alveolar area³⁵. Activated neutrophils release free radicals, proteolytic enzymes and peroxidase. Activated neutrophils can also lead to acute lung injury by increasing pulmonary vascular permeability³⁶.

Our experiments showed that TNF-α and IL-1β levels increased, whereas IL-10 levels decreased, in rats exposed to IR. This decrease may have resulted from the suppression of activity of IL-10 by TNF-α and IL-1β ³⁷. The transcription factor NF-κB, which promotes the expression of proinflammatory cytokines such as TNF-α and IL-1β, is also linked to neutrophil infiltration into the lungs and lipid peroxidation^{38, 39}. In our study, the increase of TNF-α and IL-1β together with NF-κβ suggests that inflammation in acute lung injury is increased by NF-κβ-mediated proinflammatory cytokines. Based on our results of decreasing TNF-α and IL-1β levels and increasing IL-10 levels in rats, we conclude that alpha B-crystallin reduces acute inflammation. This effect of alpha B-crystallin may inhibit the activity of proinflammatory cytokines by inhibiting the suppression of IL-10. Levels of NF-κβ decreased in all alpha B-crystallin+IR groups compared to IR group. Lentsch et al.⁴⁰ showed that the inflammatory-suppressing effect of IL-10 occurs by inhibiting the translocation of NF-κβ from cytoplasm to the nucleus in rat lung tissues. However, this study has some limitations. First, the unavailability of resources for immunohistochemical staining may limit the representation of apoptotic DNA fragmentation in TUNEL assays. Second, western blotting could be used to identify proteins, but insufficient resources limits this.

CONCLUSION

Based on our findings, alpha B-crystallin has the potential to prevent acute lung injury caused by aortic IR, mainly because of its antioxidant and anti-inflammatory properties. It also appears to mitigate DNA damage and cell apoptosis associated with IR. Preadministration of alpha B-crystallin before aortic surgery could potentially mitigate IR injury. The present study underscores the potential of alpha B-crystallin as a novel treatment for IR-induced lung injury, marking its debut in this therapeutic context.

References

Rossi M, Delbauve S, Roumeguère T, et al. HO-1 mitigates acute kidney injury and subsequent kidney-lung cross-talk. Free Radic Res. 2019;53:1035-43.

Erbel R, Aboyans V, Boileau C, et al. 2014 ESC Guidelines on the diagnosis and treatment of aortic diseases: Document covering acute and chronic aortic diseases of the thoracic and abdominal aorta of the adult. The Task Force for the Diagnosis and Treatment of Aortic Diseases of the European Society of Cardiology (ESC). Eur Heart J. 2014;35:2873-926.

Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol. 2012;298:229-317.

Nieuwenhuijs-Moeke GJ, Pischke SE, Berger SP, et al. Ischemia and Reperfusion Injury in Kidney Transplantation: Relevant Mechanisms in Injury and Repair. J Clin Med. 2020;9:253.

de Perrot M, Liu M, Waddell TK, Keshavjee S. Ischemia-reperfusion-induced lung injury. Am J Respir Crit Care Med. 2003;167:490-511.

Bezerra FS, Lanzetti M, Nesi RT, et al. Oxidative Stress and Inflammation in Acute and Chronic Lung Injuries. Antioxidants (Basel). 2023;12:548.

Fischer S, Cassivi SD, Xavier AM, et al. Cell death in human lung transplantation: apoptosis induction in human lungs during ischemia and after transplantation. Ann Surg. 2000;231:424-31.

Forgiarini Junior LA, Holand AR, Forgiarini LF, et al. Endobronchial perfluorocarbon reduces inflammatory activity before and after lung transplantation in an animal experimental model. Mediators Inflamm. 2013;2013:193484.

Fadanni GP, Calixto JB. Recent progress and prospects for anti-cytokine therapy in preclinical and clinical acute lung injury. Cytokine Growth Factor Rev. 2023;71-72:13-25.

Bakthisaran R, Tangirala R, Rao ChM. Small heat shock proteins: Role in cellular functions and pathology. Biochim Biophys Acta. 2015;1854:291-319.

Yan H, Peng Y, Huang W, Gong L, Li L. The Protective Effects of αB-Crystallin on Ischemia-Reperfusion Injury in the Rat Retina. J Ophthalmol. 2017;2017:7205408.

Pangratz-Fuehrer S, Kaur K, Ousman SS, Steinman L, Liao YJ. Functional rescue of experimental ischemic optic neuropathy with αB-crystallin. Eye (Lond). 2011;25:809-17.

Bartelt-Kirbach B, Slowik A, Beyer C, Golenhofen N. Upregulation and phosphorylation of HspB1/Hsp25 and HspB5/αB-crystallin after transient middle cerebral artery occlusion in rats. Cell Stress Chaperones. 2017;22:653-63.

Arac A, Brownell SE, Rothbard JB, et al. Systemic augmentation of alphaB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation. Proc Natl Acad Sci U S A. 2011;108:13287-92.

Klopstein A, Santos-Nogueira E, Francos-Quijorna I, et al. Beneficial effects of αB-crystallin in spinal cord contusion injury. J Neurosci. 2012;32:14478-88.

Velotta JB, Kimura N, Chang SH, et al. αB-crystallin improves murine cardiac function and attenuates apoptosis in human endothelial cells exposed to ischemia-reperfusion. Ann Thorac Surg. 2011;91:1907-13.

Rothbard JB, Kurnellas MP, Brownell S, et al. Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins. J Biol Chem. 2012;287:9708-21.

Masilamoni JG, Vignesh S, Kirubagaran R, Jesudason EP, Jayakumar R. The neuroprotective efficacy of alpha-crystallin against acute inflammation in mice. Brain Res Bull. 2005;67:235-41.

Adhikari AS, Singh BN, Rao KS, Rao ChM. αB-crystallin, a small heat shock protein, modulates NF-κB activity in a phosphorylation-dependent manner and protects muscle myoblasts from TNF-α induced cytotoxicity. Biochim Biophys Acta. 2011;1813:1532-42.

Masilamoni JG, Jesudason EP, Baben B, Jebaraj CE, Dhandayuthapani S, Jayakumar R. Molecular chaperone alpha-crystallin prevents detrimental effects of neuroinflammation. Biochim Biophys Acta. 2006;1762:284-93.

Guner I, Yaman MO, Aksu U, et al. The effect of fluoxetine on ischemia-reperfusion after aortic surgery in a rat model. J Surg Res. 2014;189:96-105.

Yaman OM, Guner I, Guntas G, et al. Protective Effect of Thymosin β4 against Abdominal Aortic Ischemia-Reperfusion-Induced Acute Lung Injury in Rats. Medicina (Kaunas). 2019;55:187.

Murakami K, Katahira J, McGuire R, et al. Heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep. Shock. 2002;18:236-41.

Ferrari RS, Andrade CF. Oxidative Stress and Lung Ischemia-Reperfusion Injury. Oxid Med Cell Longev. 2015;2015:590987.

Naito H, Nojima T, Fujisaki N, et al. Therapeutic strategies for ischemia reperfusion injury in emergency medicine. Acute Med Surg. 2020;7:e501.

Auten RL, Davis JM. Oxygen toxicity and reactive oxygen species: the devil is in the details. Pediatr Res. 2009;66:121-7.

Zhou T, Prather ER, Garrison DE, Zuo L. Interplay between ROS and Antioxidants during Ischemia-Reperfusion Injuries in Cardiac and Skeletal Muscle. Int J Mol Sci. 2018;19:417.

Tural K, Ozden O, Bilgi Z, et al. The protective effect of betanin and copper on heart and lung in end‑organ ischemia reperfusion injury. Bratisl Lek Listy. 2020;121:211-7.

Liu J, Wang J, Xiong A, et al. Mitochondrial quality control in lung diseases: current research and future directions. Front Physiol. 2023;14:1236651.

Valavanidis A, Vlachogianni T, Fiotakis C. 8-hydroxy-2’ -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2009;27:120-39.

Mao YW, Liu JP, Xiang H, Li DW. Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis. Cell Death Differ. 2004;11:512-26.

Hu W-F, Gong L, Cao Z, et al. αA- and αB-crystallins interact with caspase-3 and Bax to guard mouse lens development. Curr Mol Med. 2012;12:177-87.

Ikeda R, Yoshida K, Ushiyama M, et al. The small heat shock protein alphaB-crystallin inhibits differentiation-induced caspase 3 activation and myogenic differentiation. Biol Pharm Bull. 2006;29:1815-9.

Kamradt MC, Lu M, Werner ME, et al. The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3. J Biol Chem. 2005;280:11059-66.

Khimenko PL, Bagby GJ, Fuseler J, Taylor AE. Tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs. J Appl Physiol (1985). 1998;85:2005-11.

Pararajasingam R, Weight SC, Bell PR, Nicholson ML, Sayers RD. Endogenous renal nitric oxide metabolism following experimental infrarenal aortic cross-clamp-induced ischaemia-reperfusion injury. Br J Surg. 1999;86:795-9.

Akdis CA, Blaser K. Mechanisms of interleukin-10-mediated immune suppression. Immunology. 2001;103:131-6.

Lingappan K. NF-κB in Oxidative Stress. Curr Opin Toxicol. 2018;7:81-6.

Miskolci V, Rollins J, Vu HY, Ghosh CC, Davidson D, Vancurova I. NFkappaB is persistently activated in continuously stimulated human neutrophils. Mol Med. 2007;13:134-42.

Lentsch AB, Shanley TP, Sarma V, Ward PA. In vivo suppression of NF-kappa B and preservation of I kappa B alpha by interleukin-10 and interleukin-13. J Clin Invest. 1997;100:2443-8.

Alpha B-crystallin Ameliorates Imbalance of Redox Homeostasis, Inflammation and Apoptosis in an Acute Lung Injury Model with Rats