A Systematic Review of the Potential Chemoprotective Effects of Resveratrol on Doxorubicin-Induced Cardiotoxicity: Focus on the Antioxidant, Antiapoptotic, and Anti-Inflammatory Activities

Purpose Although doxorubicin chemotherapeutic drug is commonly used to treat various solid and hematological tumors, its clinical use is restricted because of its adverse effects on the normal cells/tissues, especially cardiotoxicity. The use of resveratrol may mitigate the doxorubicin-induced cardiotoxic effects. For this aim, we systematically reviewed the potential chemoprotective effects of resveratrol against the doxorubicin-induced cardiotoxicity. Methods In the current study, a systematic search was performed based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline for the identification of all relevant studies on “the role of resveratrol on doxorubicin-induced cardiotoxicity” in the electronic databases of Web of Science, PubMed, and Scopus up to March 2021 using search terms in their titles and abstracts. Two hundred and eighteen articles were screened in accordance with a predefined set of inclusion and exclusion criteria. Finally, 33 eligible articles were included in this systematic review. Results The in vitro and in vivo findings demonstrated a decreased cell survival, increased mortality, decreased heart weight, and increased ascites in the doxorubicin-treated groups compared to the control groups. The combined treatment of resveratrol and doxorubicin showed an opposite pattern than the doxorubicin-treated groups alone. Furthermore, this chemotherapeutic agent induced the biochemical and histopathological changes on the cardiac cells/tissue; however, the results (for most of the cases) revealed that these alterations induced by doxorubicin were reversed near to normal levels (control groups) by resveratrol coadministration. Conclusion The results of this systematic review stated that coadministration of resveratrol alleviates the doxorubicin-induced cardiotoxicity. Resveratrol exerts these chemoprotective effects through several main mechanisms of antioxidant, antiapoptosis, and anti-inflammatory.


Introduction
Cancer, the uncontrolled growth of cells, is the second leading cause of death in the world [1][2][3], and also, its incidence and mortality are rapidly growing [4]. According to a recent report, an estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths happened worldwide in 2020 [5]. The conventional modalities for cancer treatment are surgery, chemotherapy, and radiotherapy [6][7][8][9]. Although chemotherapy is effectively used for systemic treatment of different cancers, it suffers from several restrictions, such as nonspecificity and various adverse effects on normal cells and tissues; hence, its clinical utility is limited [10,11].
Doxorubicin (Adriamycin) belongs to the anthracycline class medication which is commonly applied since the late 1960s for treatment of various tumors, including Hodgkin lymphoma, non-Hodgkin lymphoma, breast cancer, lung cancer, testicular cancer, thyroid cancer, and ovarian cancer [12][13][14]. Some immediate side effects have been reported for this chemotherapeutic drug, which are reversible or clinically manageable, such as arrhythmia, myelosuppression, vomiting, and nausea [13]. However, doxorubicin-induced cardiotoxicity is a serious adverse effect and can reduce quality of life and sometimes fatalities, leading to restriction of the clinical use of this chemotherapy drug [15]. In this regard, the use of chemoprotective agents during doxorubicin treatment has been suggested which may mitigate the adverse effects and improve patient survival.
According to the published studies, it can be mentioned that using the herbal and natural products or their derivatives to alleviate the chemotherapy-induced adverse effects (chemoprotectors) or increase the sensitivity of cancer cells to chemotherapy drugs (chemo/radiosensitizers) has attracted much attention over the past several decades. Resveratrol (3,5,4′-trihydroxy-trans-stilbene, Figure 1), as a natural polyphenol, is found in more than 70 plant species. The main sources reported for this herbal agent are grapes, red wine, soy, and peanuts [16][17][18][19]. Naturally, resveratrol can protect the herbs against fungal, ultraviolet rays, and other stresses [20]. Moreover, it is reported that this herbal agent has potent antioxidant and anticlastogenic activities, helping to protect against genomic instability and carcinogenesis [21,22]. Resveratrol also has some abilities to kill tumoral cells and sensitize tumor cells to therapeutic modalities such as chemotherapy and radiotherapy [21], as its anticancer activity has been assessed in many tumor types, such as colorectal cancer, prostate cancer, lung cancer, liver cancer, and breast cancer [23][24][25][26]. Additionally, it has been shown that resveratrol not only acts as a chemosensitizer agent but also has chemopreventive activities which are linked to its antiproliferative, antioxidant, antiapoptosis, and anti-inflammatory activities [27,28]. Other biological activities of resveratrol such as neuroprotective, radioprotective, and cardioprotective properties have also been reported [19,29]. It has also recently been reported that resveratrol can be effective in the management of acute pancreatitis [30].
To the best of our knowledge, this study is the first systematic review on the cardioprotective role of resveratrol, as an adjuvant, during doxorubicin treatment. Additionally, it was tried to discuss the following questions. (1) How does doxorubicin chemotherapeutic agent lead to cardiotoxicity? (2) What are the underlying mechanisms of doxorubicininduced cardiotoxicity? (3) What is the role of resveratrol on the doxorubicin-induced cardiotoxicity? (4) What are the cardioprotective mechanisms of resveratrol against doxorubicin-induced cardiotoxicity?

Methods
The present systematic review was performed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [31] (see Table 1). A PICO framework was also applied to structure the review process [31] that was composed of participants (P): doxorubicin-damaged cardiac cells (in vitro studies) and/or patients/animals with doxorubicin-induced cardiotoxicity (clinical/in vivo studies); intervention (I): chemotherapies with doxorubicin-based regimens; comparison (C): patients/animals/cells treated with resveratrol and doxorubicin; and outcomes (O): we selected two critical outcomes as follows: (i) changes in the cardiac cells/tissue following doxorubicin administration compared to control or untreated groups and (ii) changes in the cardiac cells/tissue following combined treatment of resveratrol and doxorubicin compared to doxorubicin treatment alone. Of note, the review protocol was not registered.

Search Strategy.
A comprehensive systematic search was performed to obtain all relevant studies on "the role of resveratrol on doxorubicin-induced cardiotoxicity" in both medical subject heading (MeSH) or advance in the electronic databases of Web of Science, PubMed, and Scopus up to Mach 2021 using the keywords "Resveratrol" AND "Doxorubicin" OR "Adriamycin" AND "Heart" OR "myocardium" OR "Myocardial" OR "Cardiac Toxicity" OR "Cardiac Toxicities" "Cardiomyopathy" OR "Myocyte" OR "Cardiopathic" OR "Cardiopathy" OR "cardiotoxicity" OR "Cardiotoxicities" OR "Cardiomyocyte" OR "Arrhythmias" OR "Cardiac" in title, abstract, or keywords.

Study Selection Process.
The inclusion criteria considered in the current study were full-text articles with (a) English language, (b) our per-defined purpose on the role of resveratrol on doxorubicin-induced cardiotoxicity (based on the aforementioned keywords), (c) adequate data, (d) no restriction in publications with clinical, in vivo, or in vitro studies, and (e) no restriction on publication year. The exclusion criteria considered for this study were (a) hemodynamic data, (b) not related studies, (c) review articles, (d) case reports, (e) editorials, (f) letters to the editors, (g) oral presentations, (h) posters, and (i) book chapters.

Data Extraction Process.
Each eligible study was assessed by two researchers and the following information were then extracted: (1) author name and year of publication, (2) models (clinical, in vivo, or/and in vitro), (3) doxorubicin dosage, protocol of usage, and type of HO OH OH Figure 1: Chemical structure of resveratrol. 2 Oxidative Medicine and Cellular Longevity Specify study characteristics (e.g., PICOS and length of follow-up) and report characteristics (e.g., years considered, language, and publication status) used as criteria for eligibility, giving rationale.

and 6
Information sources 7 Describe all information sources (e.g., databases with dates of coverage and contact with study authors to identify additional studies) in the search and date last searched. 5 Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 5 Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). 6 Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. 6

Data items 11
List and define all variables for which data were sought (e.g., PICOS and funding sources) and any assumptions and simplifications made. 6

Risk of bias in individual studies 12
Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level) and how this information is to be used in any data synthesis.

Study selection 17
Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. 6-7 Figure 1 Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, and follow-up period) and provide the citations. Table 2 Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). N/A

Results of individual studies 20
For all outcomes considered (benefits or harms), present for each study (a) simple summary data for each intervention group and (b) effect estimates and confidence intervals, ideally with a forest plot. Table 2 Synthesis of results

Results
3.1. Literature Search and Screening. In Figure 2, the process of study selection is shown.  Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses and metaregression (see item 16)). Discussion

Summary of evidence 24
Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers). 10-16

Limitations 25
Discuss limitations at study and outcome level (e.g., risk of bias) and at review level (e.g., incomplete retrieval of identified research and reporting bias). 16

Conclusions 26
Provide a general interpretation of the results in the context of other evidence and implications for future research. 16

Funding 27
Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. 17 4 Oxidative Medicine and Cellular Longevity     Oxidative Medicine and Cellular Longevity 9 Oxidative Medicine and Cellular Longevity Two hundred and eighteen articles were obtained by a systematic search on the above-mentioned electronic databases up to March 2021. After removing the duplicated articles (n = 95), the remaining ones (n = 123) were screened in their titles and abstracts, and 64 of them were excluded. Fifty-nine articles were qualified for evaluation of their full texts. Thirty-three articles were finally included in the current study based on the inclusion and exclusion criteria. A summary of the data extracted from the eligible articles are listed in Table 2.
According to the in vivo results, the mortality of mice/rats treated with doxorubicin was significantly higher than that of the untreated group. The use of resveratrol significantly decreased doxorubicin-induced mortality [38,44,45]. For instance, Angelis et al. reported that the mortality rate of 67% observed in doxorubicin-treated animals was reduced to 33% in the group cotreated with resveratrol and doxorubicin [38]. In other study by Cappetta et al., the mortality rate of 40% observed in doxorubicin-treated rats was declined to 12% in the resveratrol plus doxorubicin group [44]. Furthermore, it was found that resveratrol cotreatment delayed the doxorubicin-induced mortality in mice/rats to a much greater extent than the chemotherapy group alone [38,44,45].

Body Weight, Heart Weight, and Ascites Changes.
The results demonstrated that the body weight and heart weight of mice/rats were reduced in the doxorubicin groups than in the control groups [38,43,[45][46][47][48][49][50][51][52]. Additionally, it was observed that the ratio of heart to the body weight as well as the ratio of heart weight to tibia length of animals was decreased following doxorubicin administration [43,47,48,50]. Moreover, there was a significant accumulation of ascites in the doxorubicin-treated rats compared to the untreated rats. Of note, the survival rate in the rats with high ascites was significantly more than the other rats [38].
Coadministration of resveratrol and doxorubicin to the mice/rats increased the body weight, heart weight, ratio of heart to body weight, and ratio of heart weight to tibia length compared to the doxorubicin-treated groups alone [38, 43, 45-49, 51, 52]. The increased ascites values of doxorubicintreated rats were significantly decreased by resveratrol cotreatment [38].

10
Oxidative Medicine and Cellular Longevity

Histological Changes.
A series of histological changes in cardiac tissue of animals have been reported following doxorubicin treatment, including congestion of blood vessels, cytoplasmic vacuolization, increments in edema, necrosis and inflammatory infiltration, hyalinization of muscle fiber, chromatin margination of nuclei and pyknotic nuclei, massive fragmentation and lysis of myofibrils, complete loss of cristae, interruption of Z lines, myocyte wavy degeneration, interstitial fibrosis, massive deposition of collagen, widely spaced deep acidophilic fibers, dense collagen fibers among thin fibers, massive interstitial hemorrhage and interfibrillar congestion, and so on (the other histological changes are represented in Table 2) [45, 46, 49, 51, 53-55, 62, 63].

Discussion
In the present systematic review, we aimed to assess the adverse effects of doxorubicin chemotherapeutic drug on the cardiac cells/tissue. The coadministration effects of resveratrol on this disorder were also investigated. Table 2 represents a summary of these findings. Furthermore, some of the important alterations on the cardiac cell following doxorubicin treatment as well as the effects of resveratrol on these changes are depicted in Figure 3.
Anthracyclines, as a class of chemotherapy drugs, can lead to chronic toxicity to the heart. The cardiotoxicity is more common in patients receiving doxorubicin, the most familiar anthracycline [64]. This chemotherapy agent has been widely applied for over forty years in case of different hematological and solid cancers [65]. In the cancerous cells, doxorubicin is capable of intercalation into DNA and disruption of DNA repair mediated by topoisomerase-II generation as well as production of free radicals and their damage to proteins, DNA, and cellular membranes [66]. Although doxorubicin has been standardized as an antitumoral drug, its potential life-threatening cardiomyopathy and congestive cardiac failure should be taken into consideration [67]. It has been shown that doxorubicin exerts toxic effects through oxidative stress, apoptosis, inflammation activities, etc. [68]. To mitigate the doxorubicin-induced cardiotoxicity, various studies have shown that the use of chemoprotective agents can provide acceptable results. In this regard, the combination treatment of doxorubicin with less toxic substances derived from plants, such as resveratrol, has received more attention in recent years.
The antitumoral activity of resveratrol has been reported in some cancers [69][70][71][72]. Resveratrol can also affect the expression of different genes [73,74]. Additionally, it has been shown that resveratrol cotreatment not only can alleviate chemotherapy-induced adverse effects but can also decrease drug resistance (synergistic effect) [75,76]. Resveratrol exerts its chemoprotective effects via several mechanisms, including antioxidant, antiapoptotic, and antiinflammatory activities. In the following, the mechanistic effects of doxorubicin chemotherapeutic drug on the cardiac cellular pathway and also the chemoprotective effects of resveratrol and its underlying mechanisms on the doxorubicininduced cardiotoxicity are discussed.

Antioxidant Actions.
Although oxygen molecules are necessary to survive aerobic cells, their forms of free radicals are dangerous to the cells [77]. On the other hand, the free radicals are commonly generated in the cells, but they are neutralized by several defense mechanisms [78]. In oxidative stress conditions, such as chemotherapy, the amount of free radicals increases and an imbalance is created between the generated free radicals and the antioxidant defense system [79,80]. It has been reported that the use of doxorubicin increases the ROS level of cardiac cells, and subsequently, they are able to attack the cell macromolecules, leading to malfunction of the heart tissue [35,38,50,59]. Upon mitochondrial injury, the generation of free radicals in the cells elevates [81]; in this regard, doxorubicin through impairment of mitochondrial malfunction can increase the generated free radicals [35]. This chemotherapeutic agent also increases lipid peroxidation (LPO) markers of MDA and TBARS in the heart tissue, leading to the cell membrane devastation and malfunction [41, 46, 47, 49, 53-55, 58, 61]. Furthermore, it has been reported that doxorubicin administration declines GSH, catalase, GPx, and MnSOD levels [41, 46, 49, 50, 53-55, 58, 61]. Of note, H 2 O 2 (as a nonradical ROS) through the activity of GPx enzyme and consuming GSH produces 2H 2 O [82]. The catalase enzyme also decomposes H 2 O 2 to H 2 O and O 2 [83]. Additionally, MnSOD is the mitochondrial SOD and acts as the primary line of defense system against oxidant stress [84]. These findings represent that doxorubicin impairs scavenging capacity of intracellular antioxidant enzymes.
According to the obtained results, it was found that resveratrol via the antioxidant actions could decrease doxorubicin-elevated ROS level of cardiac cells [35,38,50,59]. This antioxidant agent is also able to polarize mitochondria, thereby inhibiting doxorubicin-induced ROS generation [35]. Furthermore, it has been reported that increased MnSOD activity following resveratrol cotreatment may play a main role in the reduction of ROS because the mitochondrion is the site of doxorubicin accumulation and the associated ROS generation [35,50,85]. Moreover, this herbal agent could upregulate GSH, catalase, and GPx expressions [38,41,44,46,49,53,54,58,61] and downregulate MDA and TBARS levels in the cardiac cells [41,46,47,49,53,54,58].

4.3.
Anti-Inflammatory Actions. The inflammatory process can occur following tissue injuries induced from various harmful stimuli, including chemotherapy, radiotherapy, microbial pathogen infection, and/or wounding [116][117][118][119]. The inflammation plays a vital role in tumor resistance and is responsible for the incidence of different adverse effects following chemotherapy [120]. It has been reported that doxorubicin can induce heart inflammation during cancer chemotherapy [53,55]. Moreover, doxorubicin-induced oxidative stress can affect LPO and activate lysosomal enzymes, leading to promote the inflammation in heart tissue [15]. It has also been reported that the doxorubicin administration upregulates proinflammatory mediators of TNF-α, intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), TGF-β, nuclear factor-kappa B (NF-κB), MPO, IL-1β, and IL-6 levels in the cardiac cells [44,46,49,53,121]. ICAM-1 is a surface protein that is able to infiltrate leucocytes to the damaged regions of heart tissue [122]. COX-2 is a proinflammatory enzyme, and it is overexpressed at the inflammatory site of cancer [123]. TGF-β, as a profibrogenic cytokine, mediates several aspects of the fibrotic process. In this regard, it induces fibroblast proliferation and transformation to myofibroblasts which leads to the deposition of collagen and extracellular matrix protein [124,125]. TGF-β is also able to modulate cell proliferation, differentiation, apoptosis, and migration [126]. Furthermore, doxorubicin could decline HO-1 level in the cardiac cells [48]. HO-1 is a nuclear factor-erythroid factor 2related factor 2-(Nrf2-) regulated gene that has a vital role in the prevention of vascular inflammation [127].
The anti-inflammatory effects of resveratrol on various normal/tumoral tissues have been reported [22,[128][129][130][131][132]. Resveratrol, through its anti-inflammatory activities, can protect the normal cells and also decrease the resistance of cancer cells to chemotherapy drugs. The findings represented in the current systematic review demonstrated that resveratrol cotreatment alleviates the doxorubicin-induced cardiac inflammation. In this regard, it was found that combined treatment of resveratrol and doxorubicin declines the elevated levels of TNF-α, TGF-β1, MPO, and IL-6 and also increased HO-1 level in the heart tissue of the doxorubicin-treated rats [44,46,49,53,57]. Moreover, the histological findings showed that doxorubicin-induced inflammation is mitigated by resveratrol administration [46,49,53,55,58].

Perspective of Future Research
Doxorubicin, as a chemotherapy drug, is commonly used for the treatment of various cancers. Nevertheless, the clinical use of doxorubicin may be restricted because of its adverse effects on the normal cells/tissues, especially cardiotoxicity. Researchers reported that the use of chemoprotective agents, such as resveratrol, can alleviate the doxorubicin-induced cardiotoxicity. This herbal agent exerts its chemoprotective effects through several main mechanisms of antioxidant, antiapoptosis, and anti-inflammatory. In addition to its chemoprotective effect, resveratrol can sensitize cancer cells to chemotherapy drugs (chemosensitizer effects) [133][134][135][136]. Despite its remarkable protective effects, unfavorable pharmacokinetics/pharmacodynamics profile of resveratrol such as poor bioavailability has restricted its applications [137,138]. To solve this problem, some studies have introduced novel derivatives and analogues for this chemoprotective agent [139][140][141][142][143][144][145][146][147]. Additionally, it has been proposed that nanostrategies for delivery of resveratrol can overcome these limitations [20,[148][149][150][151].
It should be mentioned that the data represented in the current systematic review are based on in vitro and in vivo models. Therefore, suggesting the use of resveratrol as a chemoprotector agent combined to doxorubicin in cancer patients requires further studies, because sometimes results are different between the in vitro and in vivo models and clinical studies. 13 Oxidative Medicine and Cellular Longevity

Limitations
Some limitations should be addressed. Firstly, significant heterogeneity was encountered perhaps because of different regimens, doses, duration, center settings, populations enrolled, and so on, calling for cautious interpretation of the findings. Secondly, many of the studies suffer from significant sources of bias. Thirdly, the effect in many occasions was evaluated by very few studies; therefore, the evidence to support it is low.

Conclusion
The findings showed that doxorubicin chemotherapeutic agent can induce the biochemical and histological changes on the cardiac cells/tissue. However, using resveratrol alleviates the doxorubicin-induced adverse effects on the cardiac cells/tissue. Mechanically, resveratrol exerts its chemoprotective effects through several main mechanisms of antioxidant, antiapoptosis, and anti-inflammatory.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflict of interest.

Authors' Contributions
L-F H and H-R L performed the literature search, wrote the manuscript, and drafted the figures. X-M L and K-T J gave the idea, edited the manuscript, and supervised the whole study. All authors read and approved the manuscript. Li-Feng Hu and Huan-Rong Lan contributed equally to this work.