Impacts of Curcumin Treatment on Experimental Sepsis: A Systematic Review

Background and Aims Sepsis is defined as a life-threatening organ dysfunction due to a dysregulated host immune response to an infection. Curcumin is a yellow polyphenol derived from the rhizome of Curcuma longa with anti-inflammatory and antioxidant properties scientifically proven, a condition that allowed its use as a tool in the treatment of sepsis. Thus, the purpose of this article was to systematically review the evidence on the impact of curcumin's anti-inflammatory effect on experimental sepsis. Methods For this, the PubMed, MEDLINE, EMBASE, Scopus, Web of Science, and LILACS databases were used, and the research was not limited to a specific publication period. Only original articles in English using in vivo experimental models (rats or mice) of sepsis induction performed by administration of lipopolysaccharide (LPS) or cecal ligation and perforation surgery (CLP) were included in the study. Studies using curcumin in dry extract or with a high degree of purity were included. At initial screening, 546 articles were selected, and of these, 223 were eligible for primary evaluation. Finally, 12 articles with full text met all inclusion criteria. Our results showed that curcumin may inhibit sepsis-induced complications such as brain, heart, liver, lungs, and kidney damage. Curcumin can inhibit inflammatory factors, prevent oxidative stress, and regulate immune responses in sepsis. Additionally, curcumin increased significantly the survival rates after experimental sepsis in several studies. The modulation of the immune response and mortality by curcumin reinforces its protective effect on sepsis and indicates a potential therapeutic tool for the treatment of sepsis.


Introduction
According to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), sepsis is defined as lifethreatening organ dysfunction due to a host's unregulated immune response to infection [1][2][3]. It is characterized by a set of hemostatic, biochemical, immunological, and metabolic changes that can lead to multiple organ dysfunctions and progress to septic shock (a subset of sepsis with critical circulatory, cellular, and metabolic abnormalities that increase the risk of mortality) and even death [1]. It is associated with high mortality rates, and the pathogenesis remains unclear despite numerous medical advances, making it difficult to develop new therapies and effective approaches [4]. Sepsis has a unique and time-sensitive clinical evolution, and its incidence has been increasing every year [5]. In 2017, the estimate of incident cases of sepsis worldwide was 48.9 million, with 11.0 million sepsisrelated deaths reported, which represents about 19.7% of all global deaths [6]. In addition to the increasing incidence and deaths related to sepsis worldwide, its management is very expensive for health services, costing about $24 billion annually for the USA [7,8]. For this reason, sepsis is considered a serious global challenge [9].
Sepsis develops by exacerbating the inflammatory response and suppressing immunity, with organ dysfunction being a key factor in its development [4,10]. Its pathophysiology seems to be involved with a series of disorders in the organism such as dysregulation of the immune system, hemostasis, immunosuppression, cellular, tissue, and organ dysfunction [11,12].
Despite scientific and technological advances aimed at the development of new drugs to combat sepsis, mortality rates remain high [13]. From this perspective, the search for new natural or synthetic compounds/drugs with antiinflammatory effects against sepsis is of great importance. Curcumin is a yellow polyphenol derived from the rhizome of Curcuma longa with anti-inflammatory, antioxidant, and immunomodulatory activities [14,15], frequently studied in the treatment of endocrine, respiratory, and liver disorders [16]. This compound has also been described for the treatment of several inflammatory diseases, such as inflammatory bowel disease [16] and rheumatoid arthritis [17].
Taking into account the understanding effects of curcumin in the treatment of sepsis and the fact that few systematic reviews are showing the anti-inflammatory effect of curcumin, the purpose of this study was to conduct a systematic review of studies in animals using the cecal ligation and puncture (CLP) sepsis induction models (considered the gold standard) and the endotoxemia model, induced by intraperitoneal injection of lipopolysaccharide (LPS) to describe the anti-inflammatory effects of curcumin on sepsis and its possible mechanism of action.

Methodology
2.1. Protocol and Registration. This systematic review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis checklist [18] under the protocol number CDR42019146945 in the International Prospective Register of Systematic Reviews (https://www.crd .york.ac.uk/prospero/display_record.php?RecordID= 146945). Additionally, as supplementary material (available here), we present the 27-item PRISMA 2020 checklist, used to design and report a robust protocol for this systematic review.
2.2. Eligibility Criteria 2.2.1. Inclusion Criteria. For this review, the minimum degree to be included (in accordance with PRISMA 2020) in the text was only articles in English available in full in scientific databases (PubMed, MEDLINE, EMBASE, Scopus, Web of Science, and LILACS). In vivo experiments (rats or mice) with a sepsis model induced by LPS administration or CLP were included using a dried extract of curcumin or with a high degree of purity.

Exclusion Criteria.
Studies in which the text was not available in full form or published in a language other than English were discarded. Additionally, experiments performed in vivo, but not in rats or mice, using a "two-hit" bacterial bolus-induced sepsis model or any other model were also rejected. In vitro studies, review articles, conference proceedings or abstracts, and dissertations were also not accepted.

Research Strategies.
Search strategies were carried out by three pairs of researchers (PR) in the following electronic bibliographic databases: PubMed (public full-text archive of biomedical and life sciences journal literature), MEDLINE (National Library of Medicine (NLM) journal citation database), EMBASE (medical literature database), Scopus (Elsevier's database with a broad representation of scientific production in Latin America), Web of Science (independent global citation database), and LILACS (Latin American and Caribbean Literature in Health Sciences database). The search strategy used was "sepsis OR septic shock AND curcumin" (MeSH term -Medical Subject Headings to index citations). The research included all articles published on or before July 8, 2019, with no publication time restrictions.
In the first phase, the three pairs of researchers (PR1: MAFF and BMV; PR2: MTC and JFO; and PR3: FSA and FDT) performed the initial analysis based on the titles and abstracts of the manuscripts. Articles that did not meet the minimum inclusion criteria were excluded, and those that did were included in the second phase. In the case of disagreement, a third researcher (MRNC) intervened. In the second phase, the articles were independently read by two researchers (MAFF and BMV) to select acceptable articles in agreement with the inclusion criteria. Articles that did not fit the inclusion criteria were excluded, and each article's reason for exclusion was recorded. The third researcher (MRNC) intervened in any disagreement.

Data Collection.
One researcher (MAFF) collected the necessary information from the articles selected for the study, and a second researcher (MTC) checked the data extracted by the first author. Any disagreements were resolved by discussion and, where necessary, a third researcher (MRNC) was involved.

Risk of Bias
Analysis. The quality of information and the risk of studies were systematically performed using the SYR-CLE tool (SYRCLE's risk of bias) for in vivo studies [19]. Two researchers (MAFF and MTC) classify each item judged as "yes," "no," or "uncertain" for each article. When there were discrepancies, a third researcher (MRNC) made the final decision.

Results
not administering curcumin in the form of dry extract, or with the same level of purity), and 31 articles remained. The remaining 31 studies were submitted for evaluation, and 19 articles were excluded after a critical analysis of the title, abstracts, and full texts. Finally, only 12 articles were selected ( Figure 1).
3.3. Summary of Results. The protective effect of curcumin on the survival rate of septic animals has been demonstrated in the studies. Survival rates were analyzed using the Kaplan-Meier survival curve and compared using the log-rank test [21,22,26,27,29]. Pretreatment of mice with curcumin before induction of sepsis showed significant improvement in survival compared to untreated septic mice [30,31]. Mice treated with curcumin after sepsis induction also showed improved survival. Curcumin improved the survival rate of rats with CLP-induced acute lung injury (ALI) by 40%-50 [27]. The survival rate was significantly improved by up to 60% in curcumin-treated mice compared to 20% in untreated mice [21]. Improvement in survival was also observed when different concentrations of curcumin were used. Septic rats treated with curcumin showed increased survival rates, approximately 80% when treated with 50 mg/kg curcumin and 90% when treated with 200 mg/kg curcumin compared to septic untreated animals, which showed 40% of survival, suggesting that the survival rate may be related to the administered dose [22]. Furthermore, animals treated with curcumin before LPS injection had reduced lethality, an improvement directly related to curcumin dosage [26].
Pulmonary findings showed that curcumin inhibited the production of inflammatory cytokines [27,30], expression of proteins associated with oxidative stress [23], and alveolar exudation and modulation of platelet adhesion leading to a reduction in degeneration and cell death by necrosis [24,29], consequently reducing the pulmonary edema and injury, in addition to promoting an improvement in survival between 40 and 50% [27]. In the liver, curcumin promoted the reduction of inflammatory processes through the reduction of cytokine production [20,28] and decreased expression of proteins associated with oxidative stress and normalization of liver enzyme levels, such as alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase [20,23]; in addition, decreased cell degeneration and necrosis [28] with reduced damage to hepatocytes were related [20,29]. Reduction of histological damage with the improvement of inflammatory lesions and reduction of degeneration and necrosis of glomeruli and renal tubules were observed in curcumin-treated septic animals [20,21,28,29]. Protective effects of curcumin were observed on cardiac function, with improvement in contractility by increasing ejection fraction and fractional shortening in septic rats [25]. Additionally, curcumin alleviates myocardial inflammation and reduces the structural damage of cardiomyocyte cells in sepsis [25]. Moreover, curcumin improved the blood-brain barrier integrity by attenuating 3 Oxidative Medicine and Cellular Longevity brain edema, decreasing apoptosis, and reducing mitochondrial dysfunction in septic mice [22,31].

Risk of Bias Analysis.
All 12 studies [20][21][22][23][24][25][26][27][28][29][30][31] included were considered with unclear risk of bias according to SYR-CLE's risk of bias analysis (Table 2). Information about randomization, allocation, and blinding process was not clearly described in articles, which are required for SYRCLE's assessment. • Language restrictions (n = 10); • Conference abstracts, review and personal opinion (n = 4); • Curcumin was compared or associated to another compound (n = 2); • Sepsis was not properly induced (n = 1); • Full copy was not available (n = 2).  Oxidative Medicine and Cellular Longevity  Oxidative Medicine and Cellular Longevity    10 Oxidative Medicine and Cellular Longevity remains associated with high mortality, and most survivors present long-term cognitive dysfunction [32]. The lack of data limits the prediction of global cases; however, there are estimates of about 5.3 million deaths per year [33]. In 2020, Fleischmann-Struzek et al. [34] performed a metaanalysis study with data from 22 countries. In this study, they adverted the high incidence of sepsis in all studied regions and reported that one in four patients with sepsis did not survive hospitalization [34]. During the literature search, we reviewed several articles that explain the high therapeutic value and very low toxicity of curcumin in different diseases. A significant improvement in survival rate was observed in animals treated with curcumin before and after sepsis induction by the CLP or LPS model [21,27]. This enhancement of survival was also reported when the studies used different concentrations of curcumin showing survival rates of 80% (50 mg/kg) and 90% (200 mg/kg), whereas control rat groups had a 40% survival rate, showing the efficiency of curcumin in different doses [21,23,27].
If we look at sepsis and septic shock from the perspective of a critically ill patient, there are a few questions to be asked: What is the best route for curcumin administration? How many doses are necessary to induce protection? How could maintaining curcumin's chemical and physical properties increase its bioavailability in the organism? All of these questions are interrelated, and the answers will be more easily addressed if we continue to understand the basic mechanisms implicated.

Curcumin: Anti-inflammatory Effects and Tissue
Damage Reduction. For centuries, curcumin has been used for its anti-inflammatory properties and, in recent decades, it has become the target of many studies that have tried to elucidate its anti-inflammatory mechanism of action. In animal models, curcumin prevents the formation of ethanolinduced liver damage [41] as well as experimental alcoholic and nonalcoholic pancreatitis [36]. According to Gaddipati et al. [37], pretreatment of animals with curcumin attenuates the increased production of proinflammatory cytokines induced by hemorrhage and inhibits the activation of nuclear factor B (NF-κB) and activated protein-1 (AP-1), shown to subdue inflammation by different mechanisms. Curcumin led to a significant reduction in TNF-α and IL-8 in bronchoalveolar lavage and a reduction in TNF-α in serum [21,26,30]. In addition, iNOS mRNA was suppressed with the use of curcumin [20]. Serum levels of IL-1β and IL-6 also significantly decreased with the use of curcumin in the treatment of sepsis, while IL-10 showed increased plasma levels [27,28]. Treatment with curcumin led to the regulation of protein expression of IKKβ, IκBα, p-NF-κB, TNF-α, IL-1β, and IL-18 stimulated by sepsis [23]. Transforming growth factor-β1 gene expression significantly decreased with treatment, and its plasma levels were also regulated with the use of curcumin [24,30]. Curcumin has a cytoprotective action and is also effective in suppressing the hepatic microvascular inflammatory response in endotoxemia, as demonstrated by its inhibitory effects on Kupffer cell activation, neutrophil adhesion, and endothelial cell edema [38][39][40].
Curcumin can be useful in the therapy of organ dysfunction associated with sepsis, shock, and other diseases related to local or systemic inflammation [28]. Analyzed studies show that curcumin promoted neuroprotective properties in a clinically relevant model of sepsis [22]; significantly reduced the proportion of wet/dry lung weight in acute lung injury induced by sepsis in rats [27]; significantly reduced the total cell count, neutrophil infiltration, and lymphocyte count after the administration of curcumin for 45 days [26]; and significantly reduced the number of inflammatory cells in the bronchoalveolar fluid after intraperitoneal administration of 20 mg/kg in the LPS sepsis model [30].
Among the histopathological benefits of curcumin is the attenuation of hydropic degeneration of hepatocytes, reduction of necrosis in the liver lobes, and diminution of inflammatory infiltration in the portal areas [28]. Dilation and congestion of hepatic sinusoids [29] were reduced and associated with improved collagen deposition and reduced fibrosis [30]. In lung tissue, curcumin treatment reduced fibrin exudation in the alveolar space and cell necrosis, as well as resulted in the absence of red cell exudation/hemorrhage [21,26,43], decreased inflammation, bronchoconstriction, and mucus secretion in the airways, and improved collagen deposition induced by LPS [30]. Considered a vital organ during the sepsis infection, the disordered progression of the disease could culminate in acute lung injury and acute respiratory distress syndrome (ARDS) [30], a common cause of respiratory failure in critically ill patients.
In kidneys, a reduction of glomerular and tubular damage [29] attenuation of acute tubular epithelial necrosis [20,23], a reduction in inflammatory cell infiltration [29], and an improvement in tubular dilation and inflammatory infiltrate [21] were reported. Chen et al. [21] suggested that the protective effects of curcumin are associated with the modulation of the NF-κB signaling pathway and consequent reduction in the expression of IL-1β, IL-6, IL-8, and TNF-α that prevent the formation of kidney inflammatory lesions. Curcumin also prevented renal tubular oxidative damage by reducing ROS production. Furthermore, curcumin restored mitochondrial homeostasis by regulating OPA1, modulating DRP1 expression, and inhibiting caspase-3 activation [44]. Together, all results show that even with incomplete knowledge of the mechanism of action of curcumin for the modulation of inflammation, we can predict its beneficial effects on the outcome of mortality in sepsis.

Curcumin Effects on Molecular and Biochemical
Parameters. Kothari et al. [45] demonstrated that during sepsis, the exacerbated activation of neutrophils in peripheral blood and tissues increases the myeloperoxidase (MPO) release into both the phagolysosomal compartment and the extracellular environment, leading to a strong oxidative activity of MPO. However, curcumin also exhibits antioxidant properties upon MPO activity. Different studies showed that MPO activity was blocked by the curcumin treatment in the lung [26], liver [23], and hepatic and renal tissues [29]. Additionally, malondialdehyde (MDA) was also significantly attenuated by treatment with curcumin in the lung [30] and liver and kidney tissues [29], and a decrease in MDA was also observed in the plasma [28]. Doses of 50 and 100 mg/kg of curcumin also significantly reduced lipid peroxidation by 25% and 39.28%, respectively [26]. With the administration of curcumin, superoxide dismutase (SOD) activity increased or there was a small reduction in its plasma levels [23]. The improvement in the liver SOD activity was observed with different concentrations of curcumin (20,40, and 80 mg/kg) [27], and a significant increase in SOD activity was observed with 50 and 100 mg/kg curcumin treatments [26].
The administration of curcumin normalizes the mRNA expression of the PI3K/AKT signaling pathway, which increases with sepsis induction and is related to the progression of apoptosis [23]. Additionally, the suppression of other proteins that contribute to the onset of apoptosis (Bad, Bcl-xL, Cyto-c, Apaf1, and cleaved Caspase-3/6/9) was observed, in association with the inhibited expression of mRNA related to LPS-induced apoptosis in the liver [23,41]. Na +/K+-ATPase activity increased significantly with the use of curcumin [29]. Its use also reduced hepatic and serum levels of ALT, AST, and ALP, related to liver injury, with significant differences between doses [10,23]. Restoration of CAT activity with treatment and reduction of O 2 -, H 2 O 2 , and NO were also observed [23]. Curcumin significantly increased catalase activity at doses of 50 and 100 mg/kg [26], reduced F4/80+ CD11c+ cells in the spleen of rats [20], and, in addition, decreased the expression of FOXP3 and the proliferation activity of CD4+ CD25+ splenic Treg cells in a dose-dependent manner in septic mice [21]. Curcumin in sepsis reduced cerebral mitochondrial dysfunction (MMP, ROS, and mitochondrial complex activity I), apoptosis in neurons, and expression of BAX and increased the expression of BCL-2 [22]. P-selectin expression was reduced with the use of curcumin in the brain, liver, and kidney tissues [10]. Glutathione (GSH) was elevated in the kidney [29] and liver [23,29] in animals treated with curcumin, and GSH-px was also recovered [23]. Cardiac troponin I (cTnI), which increases after sepsis induction, is significantly lower with the administration of curcumin [25].
Herein, the results revealed that in sepsis, treatment with curcumin, a bioactive compound with proven immunoregulatory and antioxidant properties, has contributed to the growing interest in understanding its effects, especially its anti-inflammatory action, with decreased release of inflammatory mediators and consequent reduction of damage during sepsis and also of its antioxidant effect by regulating the production of free radicals and increasing levels of antioxidant enzymes at different doses [21,46,47].
In addition, several experimental studies use different curcumin concentrations, forms of administration, and treatment periods [48][49][50]. According to the American reg-ulatory agency Food and Drug Administration (FDA), its intake is also considered safe for humans, even in larger amounts, for example, in the Indian diet (daily dose of 60 to 100 mg of curcumin) [51][52][53][54].

Limitations
The main limitations observed during the evaluation of the studies were related to the induction of sepsis, administration routes, doses, and administration intervals of curcumin.
The induction of sepsis using the cecal ligation and puncture (CLP) model can generate limitations because, according to Hubbard et al. [55], the gauge of the needle used to puncture the animals' intestine changes the intensity of the sepsis stimulus. Furthermore, the LPS concentrations used in the different studies were distinct. Together, these conditions could lead to different biochemical, molecular, and tissue responses in experimental sepsis.
Heterogeneous doses and different routes of administration for treatment with curcumin, lack of standardization for the intervals for the administration of curcumin, and also the lack of important information to assess the risk of bias in the selected articles were the important limitations found in carrying out this study.

Conclusions
Studies analyzed pointed to the positive effects of curcumin, demonstrating its ability to inhibit oxidative and inflammatory factors through the modulation of the immune response. However, further studies involving the mechanisms by which curcumin acts in the regulation and neutralization of antioxidant and inflammatory compounds in addition to tissue protection in several stages of the pathophysiology of sepsis and its complications are necessary to investigate its effect and possible mechanisms of action in humans.

Data Availability
The authors confirm that data supporting the findings of this study are available in the article.