Dexpanthenol ameliorates doxorubicin-induced lung injury by regulating endoplasmic reticulum stress and apoptosis

Doxorubicin (DOX), which is used as a chemotherapeutic agent in the treatment of tumors, has limited use due to its toxicity in various organs and tissues. One of the organs where DOX has a toxic effect is the lung. DOX shows this effect by increasing oxidative stress, inflammation, and apoptosis. Dexpanthenol (DEX), a homologue of pantothenic acid, has anti-inflammatory, antioxidant, and anti-apoptotic properties. Therefore, the purpose of our investigation was to explore how DEX could counteract the harmful effects of DOX on the lungs. Thirty-two rats were used in the study, and 4 groups were formed (control, DOX, DOX + DEX, and DEX). In these groups, parameters of inflammation, ER stress, apoptosis, and oxidative stress were evaluated by immunohistochemistry, RT-qPCR, and spectrophotometric methods. In addition, lung tissue was evaluated histopathologically in the groups. While CHOP/GADD153, caspase-12, caspase-9, and Bax gene expressions increased in the DOX group, Bcl-2 gene expression levels significantly decreased. In addition, changes in Bax and Bcl-2 were supported immunohistochemically. There was a significant increase in oxidative stress parameters and a significant decrease in antioxidant levels. In addition, an increase in inflammatory marker (TNF-α and IL-10) levels was determined. There was a decrease in CHOP/GADD153, caspase-12, caspase-9, and Bax gene expressions and an increase in Bcl-2 gene expression in the DEX-treated group. In addition, it was determined that there was a decrease in oxidative stress levels and inflammatory findings. The curative effect of DEX was supported by histopathological findings. As a result, it was experimentally determined that DEX has a healing effect on oxidative stress, ER stress, inflammation, and apoptosis in lung damage caused by DOX toxicity.


Introduction
Doxorubicin (DOX) is an anthracycline group antibiotic widely used in the treatment of various tumors as a chemotherapeutic agent. Many of the chemotherapeutic agents, including DOX, cannot differentiate between normal and tumor cells and accumulate in various tissues, causing toxicity. The predominant toxic effect of DOX is seen in the heart, but it also causes testicular, liver, and lung damage, which limits its clinical use (Qi et al. 2020;Owumi et al. 2021).
Previous studies have revealed that DOX increases oxidative stress, inhibits cell division by suppressing topoisomerase type II, and ultimately causes cell death by apoptosis (Sauter et al. 2010). Although DOX has a direct effect on damage to organs and tissues as a result of inflammation, apoptosis, DNA damage, calcium metabolism disorder, and excessive free radical production, the specific mechanisms of its toxicity on organs have not yet been fully determined (Gharanei et al. 2014). Bioactivation of DOX causes the release of reactive oxygen species and the reduction of antioxidant enzymes, which cause oxidative stress. Oxidative stress causes oxidative damage to lipids, proteins, and DNA, leading the cell to apoptosis. DOX also increases the synthesis of proinflammatory cytokines like tumor necrosis 1 3 factor-alpha (TNF-α), interleukin-1, and interleukin-6 (Kuzu et al. 2018;Hu et al. 2020). While DOX causes an increase in proapoptotic Bax levels, it causes a decrease in anti-apoptotic Bcl-2 levels (Elblehi et al. 2021). In addition to these changes, at the organelle level, DOX also causes an enlargement in the endoplasmic reticulum (ER) (Singal and Iliskovic 1998). The ER plays an important role in protein folding and calcium homeostasis (Shakeri et al. 2019). ER dysfunction induced by DOX leads to accumulation of unfolded proteins and calcium derangement (Wang et al. 2018). CHOP/ GADD153 is a gene associated with growth arrest and DNA damage and is involved in the ER stress-mediated apoptotic pathway. Under normal physiological conditions, CHOP in the cytoplasm is activated as a result of ER stress and accumulates in the nucleus. It has been reported that DOX induces ER stress-mediated apoptosis by overexpressing CHOP, causing a decrease in anti-apoptotic Bcl-2 and an increase in pro-apoptotic Bax levels (Oyadomari and Mori 2004). Bax is normally found in the cytoplasm; upon initiation of apoptotic signaling, Bax becomes associated with the mitochondrial membrane to induce apoptosis. Bcl-2 is localized to the outer membrane of the mitochondria, where it plays an important role in promoting cellular survival and inhibiting the actions of pro-apoptotic (Czabotar et al. 2014). Caspase-12 from the caspase family, which plays a central role in the mechanism of apoptosis, is localized in the ER and is activated especially in cells exposed to ER stress (Nakagawa et al. 2000). Activated caspase-12 activates caspase-9. Caspase-9 is also activated by the apoptosome resulting from mitochondrial damage. Activated caspase-9 also activates caspase-3 and causes apoptosis (Morishima et al. 2002). In addition, DOX-induced oxidative stress may increase ER stress, which may induce apoptosis via CHOP, caspase-12, caspase-9, and caspase-3 (Luo et al. 2019).
It has been determined by previous studies that dexpanthenol (DEX), an analogue of pantothenic acid (PA) and the component of coenzyme A (CoA), induces glutathione (GSH) synthesis, especially mitochondrial CoA, and adenosine 5′-triphosphate (ATP). PA supports antioxidant enzyme activity by increasing glutathione, glutathione peroxidase (GPx), catalase (CAT), and SOD activity (Slyshenkov et al. 2001). DEX has an important role in oxidative stress, cell damage, and the inflammatory response (Wojtczak and Slyshenkov 2003). In studies investigating the effect of DEX on various organs and tissues, it was determined that it has an ameliorating effect on pulmonary fibrosis, neuronal damage, hepatotoxicity, inflammation, and cardiovascular toxicity (Karakuyu and Özmen 2022;Ermis et al. 2013;Bilgic et al. 2018;Kose et al. 2020;Erdogan et al. 2021).
In our study, we aimed to evaluate the protective effect of DEX through these mechanisms, while evaluating the effect of DOX on lung toxicity through ER stress-mediated apoptosis, inflammation, and oxidative stress.

Experimental animal protocol
Thirty-two male rats (weight 300 g) were randomly divided into 4 groups, with 8 in each group. An ad libitum feeding regime was applied by keeping the rats in a 12-h light and 12-h dark cycle between 21 and 22 °C using Euro type 4 cages.
Group I (control group): Rats in this group were administered 0.8 ml of intraperitoneal (i.p.) saline solution once a day for 14 days in order to experience the same stress as the rats in other groups. Group II (DOX): 2.5 mg/kg DOX was given as intraperitoneally 3 times a week for a total of 6 times a cumulative dose of 15 mg/kg (Sheibani et al. 2020). Group III (DEX + DOX group): 500 mg/kg DEX was applied to the left inguinal regions of the rats as i.p. every day for 14 days, and 2.5 mg/kg DOX was applied to the right inguinal regions 3 times a week. The total dose of DOX given was determined as 15 mg/kg. Group IV (DEX group): 500 mg/kg DEX i.p. was administered daily for 14 days (Korkmaz et al. 2020;Karahan et al. 2021).
Animals were slaughtered while under anesthesia consisting of xylazine (8-10 mg/kg) and ketamine (90 mg/kg) 24 h following the last medication treatment. Parts of the lung tissues were placed in formaldehyde (10%) for histopathological and immunohistochemical examinations. The remaining were transferred to Eppendorf tubes for biochemical and genetic analyses and stored at − 80 °C.

Histopathological evaluation
After being fixed in paraffin wax for 2 days, lung tissue samples were routinely processed using a fully automated tissue processor (Leica ASP300S; Leica Microsystem, Nussloch, Germany). Using a fully automated Leica RM 2155 rotary microtome (Leica Microsystem, Nussloch, Germany), 5-µm sections were cut from paraffin blocks. After, sections were processed for hematoxylin-eosin (HE) staining and a light microscope was used to examine the samples.

Immunohistochemical examination
Using the streptavidin-biotin method per the manufacturer's instructions, three series of sections scraped from each paraffin block and drawn on poly-l-lysine-coated slides were stained immunohistochemically for the expression of Bcl-2 (Bcl-2 antibody, A11434), Bax (Bax antibody, A11427), IL-10 (IL-10 antibody, A16445), and TNF-α (TNF-α2 antibody, A10303). All primary antibodies were purchased from AFG Bioscience (USA) and used at 1/100 dilution. The sections were incubated with primary antibodies at 4 °C overnight and immunohistochemistry was performed using streptavidin-alkaline phosphatase conjugate and biotinylated EXPOSE Mouse and Rabbit Specific HRP/ DAB Detection IHC kit (ab80436) (Abcam, Cambridge, UK) secondary antibody. The chromogen utilized was diaminobenzidine (DAB). An antigen dilution solution, but not the primary antibody, was used for negative controls. Each test was performed on blinded samples by an experienced pathologist.
Sections were examined for each antibody separately. Semiquantitative analysis was conducted using a grading scale from (0) to (3) to assess the severity of the immunohistochemical reactivity of cells to the markers: Negative values are represented by (0), focal weak staining (1), diffuse weak staining (2), and diffuse strong staining (3). In each slice, 10 distinct locations were inspected under 40 × objective magnification for evaluation (Ozer et al. 2020). The Olympus cellSens standard software was used for morphometric analysis and microphotography (Olympus Corporation, Tokyo, Japan).

Analysis of biochemical parameters
Lung tissues of rats (about 180 mg) were homogenized with Ultra Turrax Janke & Kunkel T-25 Stirrer, Homogenizer mixer (IKA® Werke, Germany) with 1:9 (w/v) phosphatebuffered saline (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4). Then, the homogenates were centrifuged for 10 min at a speed of 10,000 rpm. The supernatants were used to conduct analyses on glutathione peroxidase (GPx) (Cat No: RS504) (Randox Laboratories, UK), superoxide dismutase (SOD) (Cat No:SD125) (Randox Laboratories, UK), total antioxidant status (TAS) (Cat No: RL0017) (Mega Medicine, Turkey), and total oxidant status (TOS) (Cat No: RL0024) (Mega Medicine, Turkey) levels. GPx was studied according to the Paglia and Valentine method, and results are reported as units per milligram of protein (Paglia and Valentine 1967). SOD activity was measured using the Sun et al. technique and represented as units per milligram of protein (Sun et al. 1988). The protein content of the supernatant was measured spectrophotometrically in a Beckman Coulter autoanalyzer (Beckman Coulter, USA).

The reverse transcription-polymerase chain reaction (RT-qPCR)
Using the GeneAll RiboEx (TM) RNA Isolation Kit (Cat No: 301-001) and the manufacturer's instructions, RNA was extracted from homogenized lung tissues (GeneAll Biotechnology, Seoul, Korea). A BioSpec-nano NanoDrop UV-Vis spectrophotometer (UV-2600, Shimadzu Ltd. Kyoto, Japan) instrument was used to measure the quantity and purity of the RNAs that were collected. cDNA synthesis was performed using the A.B.T.™ cDNA Synthesis Kit (Cat No: C03-01-05) from Atlas Biotechnology in Turkey according to the instructions. One microgram of RNA was used for cDNA synthesis. Using the Primer-BLAST tool, NCBI website, specific mRNA sequences were found, and potential primer sequences were then tested. In Table 1, the primers' sequences used, accession numbers, and product size of the genes are given. A.B.T.™ SYBR Master Mix (Atlas Biotechnology, Turkey) (Cat No: Q04-01-05) was used to quantify the expression levels of genes in a Biorad CFX96 realtime PCR equipment (CA, USA). In the study, the GAPDH gene was used as a housekeeping gene. The reaction mixture was prepared according to the manufacturer's protocol to a final volume of 20 µL. The resultant reaction mixture was put into a real-time qPCR equipment with thermal cycling setup in accordance with the manufacturer's protocol for the kit, and each sample was examined in three replications. The PCR protocol was applied as 1 cycle with an initial denaturation at 95 °C for 300 s and 40 cycles with denaturation at 95 °C for 15 s, annealing/extension at 60 °C for 30 s. Relative mRNA levels were calculated by applying the 2 −ΔΔCt formula to the normalize the results (Livak and Schmittgen 2001). The fold increases of the genes were shown graphically. The entire study was performed following the MIQE protocol (Bustin et al. 2009). .00 (SPSS, USA) package program was used for statistical analysis. Data normality was controlled with Shapiro-Wilk's test, and group comparisons were performed with a one-way analysis of variance (ANOVA). Bonferroni's test was applied to determine inter-group differences. The level of significance was considered as p < 0.05.

Histopathological findings
The histology of the lungs in the control and DEX groups revealed normal tissue organization. The DOX group demonstrated emphysema, significant hyperemia, increased septal tissue thickness, and inflammatory cell infiltrations. The DOX + DEX group's DEX treatment reduced the abnormal results. As a result of DOX applied after DEX application, there was a decrease in histopathological effects (Fig. 1).

Immunohistochemical findings
In the C group, there was no or very little Bax, IL-10, or TNF-α expression, but there was marked Bcl-2 expression. The Bax, IL-10, and TNF-α expressions were significantly increased in the DOX group while Bcl-2 expression was significantly decreased. Additionally, following DEX treatment, the expressions of Bax, IL-10, TNF-α, and Bcl-2 were increased. In comparison to the control group with markers, the DEX group displayed similar expressions (Fig. 2). Expressions typically occurred in alveolar macrophages, alveolar epithelial cells, and inflammatory cells. Table 2 summarizes the semiquantitative analysis of the IHC markers here used and respective statistical study.
The findings of this study demonstrated that DEX protected against lung damage brought on by DOX while DOX caused acute pathological abnormalities in the lungs.

Biochemical analysis findings
When we compared the control and DOX groups, there was a significant decrease in total antioxidant status (TAS), GPx, and SOD values, while total oxidant status (TOS) and oxidative stress index (OSI) increased in the DOX group. In the DOX + DEX group, an improvement was observed in oxidant and antioxidant parameters close to those of the control group (Table 3). This suggests that DEX may contribute to regulate the oxidative stress created by DOX in the lung tissue.

mRNA expression analysis findings
Expression levels of genes associated with ER stress and apoptosis were compared between groups. There was an increase in the expressions of the Bax, CHOP, Cas 12, Cas 9, and Cas 3 genes in the DOX group compared to the control group and a significant decrease in the expression levels of the Bcl-2 gene. It was determined that there was a significant change in these genes in the DOX + DEX group treated with DEX, similar to the control group (Fig. 3). According to these results, it was determined that DEX decreased the ER stress created by DOX in the lung tissue.

Discussion
DOX, which has been involved in the treatment of tumors, is limited in its use due to its side effects in various organs and tissues. Various methods are being tried to reduce these side effects and minimize toxicity (Owumi et al. 2021). For this purpose, we evaluated whether DEX, which is thought to have antioxidant, anti-inflammatory, and anti-apoptotic properties, has ameliorative effect on lung toxicity caused by DOX through ER stress-mediated apoptosis oxidative stress and inflammation. It has been reported that the main cause of DOXinduced toxicity is increased oxidative stress. It is also known that oxidative stress is closely related to inflammation, ER stress, and apoptosis (Chaudhari et al. 2014). Lai et al. reported that DOX increases oxidative stress and apoptosis by decreasing SOD and GPx activities (Lai et al. 2011). Liao et al. reported that DOX decreased catalase (CAT), glutathione reductase (GR), and GPx activities, and increased the inflammatory markers (IL-1 β, TNF-α, iNOS, and IL-6) and ER stress markers (CHOP and caspase-12) in a DOX-induced neuroinflammation model in rats (Liao et al. 2018). Owumi et al. reported that DOX causes toxicity by increasing anti-inflammatory cytokines (IL-10), oxidant molecules (MDA, xanthine oxidase), proinflammatory cytokines (IL-1β and TNF-α), and caspase-3 in the lung (Owumi et al. 2021). In addition, in the review report published by Yarmohammadi et al. 2021, it was stated that ER stress, inflammation, autophagy, and apoptosis are important pathways in the cardiotoxicity of DOX (Yarmohammadi et al. 2021). It has also been reported that DOX causes an excessive increase in the expression of CHOP, causing a decrease in Bcl-2 and an increase in Bax, and increasing ER-mediated apoptosis (Oyadomari and Mori 2004). In addition, it was determined that DOX activates the ER stress-related Cas 12 pathway and increases apoptosis through the activation of Cas 9 and Cas 3 (Jang et al. 2004). Das et al. reported that DOX administration increased caspase-9, caspase-3, caspase-8, and caspase-12 and oxidative stress, and it was supported that DOX played a role in ER-mediated apoptosis. In our study, DOX caused changes in oxidant and antioxidant parameters in lung tissues. DOX increased oxidative stress by increasing TOS and OSI levels and decreasing TAS, GPx, and SOD levels. When we looked at how DOX affected the inflammatory response, we discovered that when compared to the control group, there was an increase in TNF-α and IL-10 levels. TNF-α increased in the DOX group, which was consistent with previous research as a proinflammatory cytokine. However, considering the anti-inflammatory effects of IL-10, the increase in IL-10 expression in the DOX group was in contrast with the literature. The pleiotropic role that IL-10 plays during the immune response may limit damage in some contexts but can cause tissue injury in others. These conflicting effects have been reported to depend on the cell type and tissue involved, as well as on the local environment (Saraiva et al. 2020). In a study by Alves et al. in 2021, elevated levels of plasma IL-10 were  (Alves et al. 2021). Therefore, the increased IL-10 level we found in the DOX group suggested that it may be related to the prolongation of the inflammatory response in the lung tissue. When we investigated the effect of DOX on genes involved in the ERrelated apoptotic process, we determined that pro-apoptotic Bax increased and anti-apoptotic Bcl-2 decreased due to CHOP increase. In addition, when we investigated ER-mediated apoptosis through a different pathway related to ER stress, we determined that DOX was effective by increasing Cas 12, Cas 9, and Cas 3. The damage of DOX on the lung was also determined histopathologically. We determined that these findings we obtained on the effect of DOX on lung tissues were compatible with the literature. Previous studies have revealed that DEX reduces tissue damage through its antioxidant, anti-apoptotic, and antiinflammatory properties. Zhao et al. evaluated the efficacy of DEX on liver and kidney in their sepsis model and determined that it reduced pathological damage, inflammation, and cell apoptosis (Zhao et al. 2022). Li-Mei et al. reported that DEX prevents oxidative stress by decreasing myeloperoxidase (MPO) and malondialdehyde (MDA) and increasing superoxide dismutase (SOD) and glutathione (GSH) activities in the lung. It was also reported in this report that it inhibits inflammation by reducing TNF-α and IL-6 (Li-Mei et al. 2016). DEX, according to Ozdemir et al., reduces the severity of lung injury by increasing catalase and GSH levels while decreasing TNF-α and IL-1B levels (Ozdemir et al. 2016). Köse et al. reported that DEX reduces oxidative stress and apoptosis in the heart and lungs (Kose et al. 2020). Bilgiç et al. reported that DEX has healing properties by regulating oxidative stress (MDA, CAT, GSH, GSH-Px, TOS, TAS, OSI, total nitrite), inflammation (IL-1β, IL-6, and TNF-α), and apoptosis (caspase-3) in the liver (Bilgic et al. 2018). Karakuyu et al. reported that DEX has antioxidant, anti-inflammatory, and anti-apoptotic effects in lung tissue by reducing TOS, OSI, caspase-3, and VCAM-1 levels (Karakuyu and Özmen 2022). Although the positive effects of DEX on oxidative stress and inflammation and apoptotic process have been investigated, we could not find any study on its effect on ER-mediated apoptosis in our literature research. We also determined that DEX increased bcl-2 while decreasing CHOP and Bax in the ER-mediated apoptotic process. In addition, we have shown that Cas 12, Cas 9, and Cas 3 levels, which are different pathways of the ERmediated apoptotic process, can be reduced, resulting in a positive improvement in the apoptosis process. The positive efficacy of DEX in the apoptotic process is also associated with mitochondrial pathways. We determined this activity Fig. 3 Graph and statistical analysis of relative mRNA expression fold increase of genes in all groups. Error bars are given as standard deviation (SD). *p < 0.05, **p < 0.001 through Bax, Bcl-2, and Cas 9, which are involved in both ER stress-mediated and mitochondrial apoptotic processes.
However, our study has some limitations. The TUNEL test was not included in the study in the evaluation of apoptosis. We also think that the inclusion of the ER sensor, the GRP78, will contribute to the evaluation.

Conclusion
When we searched the relevant literature, we could not find any studies on the usability of DEX to prevent organ and tissue toxicities caused by DOX. Our results revealed that DEX may have a curative effect on lung toxicity caused by DOX. However, we suggest that more studies be done before it can be used clinically.
Authors contributions MYT and ES conducted genetic analysis, prepared the article design, designed figures and tables, and compiled literature resources. HIB made biochemical analyzes and article editing. ÖÖ and ŞT performed histopathological and immunohistochemical experiments and analysis of the results. The authors declare that all data is produced in-house and is paper mill free.
Data availability It can be obtained from the corresponding author upon request.

Ethical approval
The experiment was carried out in accordance with the guidelines for the treatment and experimentation of animals provided in the pertinent European Communities Council Directive (86/609/EEC), and it was given the go-ahead by the Suleyman Demirel University Committee on Animal Research (Approval No. 01.26.2023/01-122).

Competing interests
The authors declare no competing interests.